DUAL VARIABLE DOMAIN IMMUNOGLOBULINS AND USES
THEREOF
Cross-Reference to Related Applications
This application claims priority to U.S. Provisional Patent Application No. 61/229,586,
filed July 29, 2009, and U.S. Provisional Patent Application No. 61/252,790, filed October 19,
2009, which are hereby expressly incorporated herein by reference in their entirety for any purpose.
Field of the Invention
The present invention relates to multivalent and multispecific binding proteins that bind
NKGD2, methods of making, and specifically to their uses in the, diagnosis, prevention and/or
treatment of acute and chronic inflammatory diseases, cancer, and other diseases.
Background of the Invention
Engineered proteins, such as multispecific antibodies that bind to two or more antigens
are known in the art. Such multispecific binding proteins can be generated using cell fusion,
chemical conjugation, or recombinant DNA techniques.
Bispecific antibodies have been produced using quadroma technology (see Milstein, C.
and Cuello, A.C. (1983) Nature 305(5934):537-40) based on the somatic fusion of two different
hybridoma cell lines expressing murine monoclonal antibodies (mAbs) with the desired
specificities of the bispecific antibody. Because of the random pairing of two different
immunoglobulin (Ig) heavy and light chains within the resulting hybrid-hybridoma (or quadroma)
cell line, up to ten different Ig species are generated, of which only one is the functional bispecific
antibody. The presence of mis-paired by-products, and significantly reduced production yields,
means sophisticated purification procedures are required.
Bispecific antibodies can also be produced by chemical conjugation of two different
mAbs (see Staerz, U.D., et al (1985) Nature 314(6012): 628-31). This approach does not yield
homogeneous preparation. Other approaches have used chemical conjugation of two different
mAbs or smaller antibody fragments (see Brennan, M., et al. (1985) Science 229(4708): 81-3).
Another method used to produce bispecific antibodies is the coupling of two parental
antibodies with a hetero-bifunctional crosslinker, but the resulting bispecific antibodies suffer
from significant molecular heterogeneity because reaction of the crosslinker with the parental
antibodies is not site-directed. To obtain more homogeneous preparations of bispecific antibodies
two different Fab fragments have been chemically crosslinked at their hinge cysteine residues in a
site-directed manner (see Glennie, M.J., et al. (1987) J. Immunol. 139(7): 2367-75). But this
method results in Fab'2 fragments, not a full IgG molecule.
A wide variety of other recombinant bispecific antibody formats have been developed
(see Kriangkum, J., et al. (2001) Biomol. Engin. 18(2): 31-40). Amongst them tandem singlechain
Fv molecules and diabodies, and various derivatives thereof, are the most widely used.
Routinely, construction of these molecules starts from two single-chain Fv (scFv) fragments that
recognize different antigens (see Economides, A.N., et al. (2003) Nat. Med. 9(1): 47-52).
Tandem scFv molecules (taFv) represent a straightforward format simply connecting the two scFv
molecules with an additional peptide linker. The two scFv fragments present in these tandem
scFv molecules form separate folding entities. Various linkers can be used to connect the two
scFv fragments and linkers with a length of up to 63 residues (see Nakanishi, K., et al. (2001)
Ann. Rev. Immunol. 19: 423-74). Although the parental scFv fragments can normally be
expressed in soluble form in bacteria, it is, however, often observed that tandem scFv molecules
form insoluble aggregates in bacteria. Hence, refolding protocols or the use of mammalian
expression systems are routinely applied to produce soluble tandem scFv molecules. In a recent
study, in vivo expression by transgenic rabbits and cattle of a tandem scFv directed against CD28
and a melanoma-associated proteoglycan was reported (see Grade, J.A., et al. (1999) J. Clin.
Invest. 104(10): 1393-401). In this construct, the two scFv molecules were connected by a CHI
linker and serum concentrations of up to 100 mg/L of the bispecific antibody were found.
Various strategies including variations of the domain order or using middle linkers with varying
length or flexibility were employed to allow soluble expression in bacteria. A few studies have
now reported expression of soluble tandem scFv molecules in bacteria (see Leung, B.P., et al.
(2000) J. Immunol. 164(12): 6495-502; Ito, A., etal. (2003) J. Immunol. 170(9): 4802-9; Kami,
A., et al. (2002) J. Neuroimmunol. 125(1-2): 134-40) using either a very short Ala3 linker or long
glycine/serine-rich linkers. In a recent study, phage display of a tandem scFv repertoire
containing randomized middle linkers with a length of 3 or 6 residues was employed to enrich for
those molecules that are produced in soluble and active form in bacteria. This approach resulted
in the isolation of a tandem scFv molecule with a 6 amino acid residue linker (see Arndt, M. and
Krauss, J. (2003) Methods Mol. Biol. 207: 305-21). It is unclear whether this linker sequence
represents a general solution to the soluble expression of tandem scFv molecules. Nevertheless,
this study demonstrated that phage display of tandem scFv molecules in combination with
directed mutagenesis is a powerful tool to enrich for these molecules, which can be expressed in
•bacteria in an active form.
Bispecific diabodies (Db) utilize the diabody format for expression. Diabodies are
produced from scFv fragments by reducing the length of the linker connecting the VH and VL
domain to approximately 5 residues (see Peipp, M. and Valerius, T. (2002) Biochem. Soc. Trans.
30(4): 507-11). This reduction of linker size facilitates dimerization of two polypeptide chains by
crossover pairing of the VH and VL domains. Bispecific diabodies are produced by expressing,
two polypeptide chains with, either the structure VHA-VLB and VHB-VLA (VH-VL
configuration), or VLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. A
large variety of different bispecific diabodies have been produced in the past and most of them are
expressed in soluble form in bacteria. However, a recent comparative study demonstrates that the
orientation of the variable domains can influence expression and formation of active binding sites
(see Mack, M. et al.(1995) Proc. Natl. Acad. Sci. USA 92(15): 7021-5). Nevertheless, soluble
expression in bacteria represents an important advantage over tandem scFv molecules. However,
since two different polypeptide chains are expressed within a single cell inactive homodimers can
be produced together with active heterodimers. This necessitates the implementation of additional
purification steps in order to obtain homogenous preparations of bispecific diabodies. One
approach to force the generation of bispecific diabodies is the production of knob-into-hole
diabodies (see Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90(14): 6444-8.18). This was
demonstrated for a bispecific diabody directed against HER2 and CD3. A large knob was
introduced in the VH domain by exchanging Val37 with Phe and Leu45 with Trp and a
complementary hole was produced in the VL domain by mutating Phe98 to Met and Tyr87 to Ala,
either in the anti- HER2 or the anti-CD3 variable domains. By using this approach the production
of bispecific diabodies could be increased from 72% by the parental diabody to over 90% by the
knob-into-hole diabody. Importantly, production yields did only slightly decrease as a result of
these mutations. However, a reduction in antigen-binding activity was observed for several
analyzed constructs. Thus, this rather elaborate approach requires the analysis of various
constructs in order to identify those mutations that produce heterodimeric molecule with unaltered
binding activity. In addition, such approach requires mutational modification of the
immunoglobulin sequence at the constant region, thus creating non-native and non-natural form
of the antibody sequence, which may result in increased immunogenicity, poor in vivo stability, as
well as undesirable pharmacokinetics.
Single-chain diabodies (scDb) represent an alternative strategy for improving the
formation of bispecific diabody-like molecules (see Holliger, P. and Winter, G. (1997) Cancer
Immunol. Immunother. 45(3^): 128-30; Wu, A.M., et al. (1996) Immunotechnology 2(1): p. 21-
36). Bispecific single-chain diabodies are produced by connecting the two diabody-forming
polypeptide chains with an additional middle linker with a length of approximately 15 amino acid
residues. Consequently, all molecules with a molecular weight corresponding to monomeric
single-chain diabodies (50-60 kDa) are bispecific. Several studies have demonstrated that
bispecific single chain diabodies are expressed in bacteria in soluble and active form with the
majority of purified molecules present as monomers (see Holliger, P. and Winter, G. (1997)
Cancer Immunol. Immunother. 45(3-4): 128-30; Wu, A.M., et al. (1996) Immunotechnol. 2(1):
21-36; Pluckthun, A. and Pack, P. (1997) Immunotechnol. 3(2): 83-105; Ridgway, J.B., et al.
(1996) Protein Engin. 9(7): 617-21). Thus, single-chain diabodies combine the advantages of
tandem scFvs (all monomers are bispecific) and diabodies (soluble expression in bacteria).
More recently diabodies have been fused to Fc to generate more Ig-like molecules, named
di-diabodies (see Lu, D., et al. (2004) J. Biol. Chem. 279(4): 2856-65). In addition, multivalent
antibody constructs comprising two Fab repeats in the heavy chain of an IgG and that bind four
antigen molecules have been described (see WO 0177342A1, and Miller, K., et al. (2003) J.
Immunol. 170(9): 4854-61).
There is a need in the art for improved multivalent binding proteins that bind two or more
antigens. U.S. Patent No. 7,612,181 provides a novel family of binding proteins that bind two or
more antigens with high affinity, and which are called dual variable domain immunoglobulins
(DVD-Ig™). The present disclosure provides further novel binding proteins that bind two or
more antigens.
Summary of the Invention
This invention pertains to multivalent binding proteins that bind two or more antigens.
The present invention provides a novel family of binding proteins capable of binding two or more
antigens with high affinity.
In one embodiment the invention provides a binding protein comprising a polypeptide
chain, wherein the polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein VD1 is a first
variable domain, VD2 is a second variable domain, C is a constant domain, XI represents an
amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1. In an embodiment the VD1
and VD2 in the binding protein are heavy chain variable domains. In another embodiment, the
heavy chain variable domain is selected from the group consisting of a murine heavy chain
variable domain, a human heavy chain variable domain, a CDR grafted heavy chain variable
domain, and a humanized heavy chain variable domain. In yet another, embodiment VD1 and
VD2 bind the same antigen. In another embodiment VD1 and VD2 bind different antigens. In
still another embodiment, C is a heavy chain constant domain. For example, XI is a linker with
the proviso that XI is not CHI. For example, XI is a linker selected from the group consisting of
AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2);
AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5);
RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO:
8); RADAAAA(G4S)4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP
(SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13);
TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID
NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID
NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21);
ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23);
GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25);
GHEAAAVMQVQYPAS (SEQ ID NO: 26); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO:
27); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28). In an embodiment, X2 is an
Fc region. In another embodiment, X2 is a variant Fc region.
In an embodiment the binding protein disclosed herein comprises a polypeptide chain,
wherein the polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein VD1 is a first heavy
chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant
domain, XI is a linker with the proviso that it is not CH1, and X2 is an Fc region.
In an embodiment, VD1 and VD2 in the binding protein are light chain variable domains. In an
embodiment, the light chain variable domain is selected from the group consisting of a murine
light chain variable domain, a human light chain variable domain, a CDR grafted light chain
variable domain, and a humanized light chain variable domain. In one embodiment VD1 and
VD2 bind the same antigen. In another embodiment VD1 and VD2 bind different antigens. In an
embodiment, C is a light chain constant domain. In another embodiment, X1 is a linker with the
proviso that X1 is not CL1. In an embodiment, X1 is a linker selected from the group consisting
of AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2);
AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5);
RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO:
8); RADAAAA(G4S)4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP
(SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13);
TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID
NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID
NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21);
ASTKGPSVFPLAP (SEQ ID NO: 22) GGGGSGGGGSGGGGS (SEQ ID NO: 23);
GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25);
GHEAAAVMQVQYPAS (SEQ ID NO: 26); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO:
27); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28. In an embodiment, the
binding protein does not comprise X2.
In an embodiment, both the variable heavy and variable light chain comprise the same
linker. In another embodiment, the variable heavy and variable light chain comprise different
linkers. In another embodiment, both the variable heavy and variable light chain comprise a short
(about 6 amino acids) linker. In another embodiment, both the variable heavy and variable light
chain comprise a long (greater than 6 amino acids) linker. In another embodiment, the variable
heavy chain comprises a short linker and the variable light chain comprises a long linker. In
another embodiment, the variable heavy chain comprises a long linker and the variable light chain
comprises a short linker.
In an embodiment the binding protein disclosed herein comprises a polypeptide chain,
wherein said polypeptide chain comprises VD1-(Xl)n-VD2-C-(X2)n, wherein VD1 is a first light
chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant
domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region.
In another embodiment the invention provides a binding protein comprising two
polypeptide chains, wherein said first polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n,
wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable
domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1,
and X2 is an Fc region; and said second polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n,
wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain,
C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 does
not comprise an Fc region. In a particular embodiment, the Dual Variable Domain (DVD)
binding protein comprises four polypeptide chains wherein the first two polypeptide chains
comprises VD1-(X1)n-VD2-C-(X2)n, respectively wherein VD1 is a first heavy chain variable
domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is
a linker with the proviso that it is not CH1, and X2 is an Fc region; and the second two
polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n respectively, wherein VD1 is a first light
chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant
domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region.
Such a Dual Variable Domain (DVD) protein has four antigen binding sites.
In another embodiment the binding proteins disclosed herein bind one or more targets. In
an embodiment, the target is selected from the group consisting of cytokines, cell surface proteins,
enzymes and receptors. In another embodiment, the binding protein modulates a biological
function of one or more targets. In another embodiment, the binding protein neutralizes one or
more targets. The binding protein of the invention binds cytokines selected from the group
consisting of lymphokines, monokines, polypeptide hormones, receptors, and tumor markers. For
example, the DVD-Ig of the invention is capable of binding two or more of the following: CD-20,
CD-19, human epidermal growth factor receptor 2 (HER-2), epidermal growth factor receptor
(EGFR), insulin-like growth factor receptor (IGF1R), and NKG2D (see also Table 2). In a
specific embodiment the binding protein binds pairs of targets selected from the group consisting
of NKG2D and CD-20; NKG2D and CD-19 (seq. 1); NKG2D and EGFR (seq. 1); NKG2D and
EGFR (seq. 2); NKG2D and HER-2 (seq. 1); NKG2D and IGF1R (seq. 1); and NKG2D and
EGFR (seq. 3).
In another embodiment, the binding protein capable of binding NKG2D and CD-20
comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID
NO. 50 and SEQ ID NO. 52; and a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 51 and SEQ ID NO. 53. In an embodiment, the binding protein
capable of binding NKG2D and CD-20 comprises a DVD heavy chain amino acid sequence of
SEQ ID NO.50 and a DVD light chain amino acid sequence of SEQ ID NO: 51. In another
embodiment, the binding protein capable of binding NKG2D and CD-20 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 52 and a DVD light
chain amino acid sequence of SEQ ID NO: 53.
In another embodiment, the binding protein capable of binding NKG2D and CD-19 (seq.
1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ
ID NO. 54 and SEQ ID NO. 56; and a DVD light chain amino acid sequence selected from the
group consisting of SEQ ID NO. 55 and SEQ ID NO. 57. In an embodiment, the binding protein
capable of binding NKG2D and CD-19 (seq. 1) comprises a DVD heavy chain amino acid
sequence of SEQ ID NO. 54 and a DVD light chain amino acid sequence of SEQ ID NO: 55. In
another embodiment, the binding protein capable of binding NKG2D and CD-19 (seq. 1) has a
reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 56
and a DVD light chain amino acid sequence of SEQ ID NO: 57.
In another embodiment, the binding protein capable of binding NKG2D and EGFR (seq.
2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ
ID NO. 58 and SEQ ID NO. 60; and a DVD light chain amino acid sequence selected from the
group consisting of SEQ ID NO. 59 and SEQ ID NO. 61. In an embodiment, the binding protein
capable of binding NKG2D and EGFR (seq. 2) comprises a DVD heavy chain amino acid
sequence of SEQ ID NO. 58 and a DVD light chain amino acid sequence of SEQ ID NO: 59. In
another embodiment, the binding protein capable of binding NKG2D and EGFR (seq. 2) has a
reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 60
and a DVD light chain amino acid sequence of SEQ ID NO: 61.
In another embodiment, the binding protein capable of binding NKG2D and EGFR (seq.
1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ
ID NO. 62 and SEQ ID NO. 64; and a DVD light chain amino acid sequence selected from the
group consisting of SEQ ID NO. 63 and SEQ ID NO. 65. In an embodiment, the binding protein
capable of binding NKG2D and EGFR (seq. 1) comprises a DVD heavy chain amino acid
sequence of SEQ ID NO. 62 and a DVD light chain amino acid sequence of SEQ ID NO: 63. In
another embodiment, the binding protein capable of binding NKG2D and EGFR (seq. 1) has a
reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 64
and a DVD light chain amino acid sequence of SEQ ID NO: 65.
In another embodiment, the binding protein capable of binding NKG2D and HER-2 (seq.
1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ
ID NO. 66 and SEQ ID NO. 68; and a DVD light chain amino acid sequence selected from the
group consisting of SEQ ID NO. 67 and SEQ ID NO. 69. In an embodiment, the binding protein
capable of binding NKG2D and HER-2 (seq. 1) comprises a DVD heavy chain amino acid
sequence of SEQ ID NO. 66 and a DVD light chain amino acid sequence of SEQ ID NO: 67. In
another embodiment, the binding protein capable of binding NKG2D and HER-2 (seq. 1) has a
reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 68
and a DVD light chain amino acid sequence of SEQ ID NO: 69.
In another embodiment, the binding protein capable of binding NKG2D and IGFR1 (seq.
1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ
ID NO. 70 and SEQ ID NO. 72; and a DVD light chain amino acid sequence selected from the
group consisting of SEQ ID NO. 71 and SEQ ID NO. 73. In an embodiment, the binding protein
capable of binding NKG2D and IGFR1 (seq. 1) comprises a DVD heavy chain amino acid
sequence of SEQ ID NO. 70 and a DVD light chain amino acid sequence of SEQ ID NO: 71. In
another embodiment, the binding protein capable of binding NKG2D and IGFR1 (seq. 1) has a
reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 72
and a DVD light chain amino acid sequence of SEQ ID NO: 73.
In another embodiment, the binding protein capable of binding NKG2D and EGFR (seq.
3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ
ID NO. 74 and SEQ ID NO. 76; and a DVD light chain amino acid sequence selected from the
group consisting of SEQ ID NO. 75 and SEQ ID NO. 77. In an embodiment, the binding protein
capable of binding NKG2D and EGFR (seq. 3) comprises a DVD heavy chain amino acid
sequence of SEQ ID NO. 74 and a DVD light chain amino acid sequence of SEQ ID NO: 75. In
another embodiment, the binding protein capable of binding NKG2D and EGFR (seq. 3) has a
reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 76
and a DVD light chain amino acid sequence of SEQ ID NO: 77.
In another embodiment the invention provides a binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein; VD1 is a
first heavy chain variable domain obtained from a first parent antibody, or antigen binding portion
thereof; VD2 is a second heavy chain variable domain obtained from a second parent antibody, or
antigen binding portion thereof, which can be the same or different from the first parent antibody;
C is a heavy chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein
said (X1)n is either present or absent; and (X2)n is an Fc region, wherein said (X2)n is either
present or absent. In an embodiment, the Fc region is absent from the binding protein.
In another embodiment, the invention provides a binding protein comprising a
polypeptide chain, wherein said polypeptide chain comprises VD1-(Xl)n-VD2-C-(X2)n, wherein,
VD1 is a first light chain variable domain obtained from a first parent antibody or antigen binding
portion thereof; VD2 is a second light chain variable domain obtained from a second parent
antibody or antigen binding portion thereof, which can be the same or different from the first
parent antibody; C is a light chain constant domain; (X1)n is a linker with the proviso that it is not
CH1, wherein said (X1)n is either present or absent; and (X2)n does not comprise an Fc region,
wherein said (X2)n is either present or absent. In an embodiment, (X2)n is absent from the
binding protein.
In another embodiment the binding protein of the invention comprises first and second
polypeptide chains, wherein said first polypeptide chain comprises a first VDl-(Xl)n-VD2-C-
(X2)n, wherein VD1 is a first heavy chain variable domain obtained from a first parent antibody
or antigen binding portion thereof; VD2 is a second heavy chain variable domain obtained from a
second parent antibody or antigen binding portion thereof, which can be the same or different
from the first parent antibody; C is a heavy chain constant domain; (Xl)n is a linker with the
proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n is an Fc
region, wherein said (X2)n is either present or absent; and wherein said second polypeptide chain
comprises a second VD1-(Xl)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable domain
obtained from a first parent antibody or antigen binding portion thereof; VD2 is a second light
chain variable domain obtained from a second parent antibody or antigen binding portion thereof,
which can be the same or different from the first parent antibody; C is a light chain constant
domain; (Xl)n is a linker with the proviso that it is not CH1, wherein said (Xl)n is either present
or absent; and (X2)n does not comprise an Fc region, wherein said (X2)n is either present or
absent. In another embodiment, the binding protein comprises two first polypeptide chains and
two second polypeptide chains. In yet another embodiment, (X2)n is absent from the second
polypeptide. In still another embodiment, the Fc region, if present in the first polypeptide is
selected from the group consisting of native sequence Fc region and a variant sequence Fc region.
In still another embodiment, the Fc region is selected from the group consisting of an Fc region
from an IgG 1, an Fc region from an IgG2, an Fc region from an IgG3, an Fc region from an IgG4,
an Fc region from an IgA, an Fc region from an IgM, an Fc region from an IgE, and an Fc region
from an IgD.
In another embodiment the binding protein of the invention is a DVD-Ig that binds two
antigens comprising four polypeptide chains, wherein, each of the first and third polypeptide
chains comprises VDl-(Xl)n-VD2-C-(X2)n, wherein,VDl is a first heavy chain variable domain
obtained from a first parent antibody, or antigen binding portion thereof; VD2 is a second heavy
chain variable domain obtained from a second parent antibody, or antigen binding portion thereof,
which can be the same as or different from the first parent antibody; C is a heavy chain constant
domain; (X1 )n is a linker with the proviso that it is not CH1, wherein said (X1 )n is either present
or absent; and (X2)n is an Fc region, wherein said (X2)n is either present or absent; and wherein
each of the second and fourth polypeptide chains comprises VD1-(X1)n-VD2-C-(X2)n, wherein
VD1 is a first light chain variable domain obtained from a first parent antibody or antigen binding
portion thereof; VD2 is a second light chain variable domain obtained from a second parent
antibody, or antigen binding portion thereof, which can be the same as or different from the first
parent antibody; C is a light chain constant domain; (X1)n is a linker with the proviso that it is not
CH1, wherein said (X1)n is either present or absent; and (X2)n does not comprise an Fc region,
wherein said (X2)n is either present or absent.
The invention provides a method of making a DVD-Ig binding protein by preselecting the
parent antibodies. In an embodiment, the method of making a Dual Variable Domain
Immunoglobulin that binds two antigens comprises the steps of a) obtaining a first parent
antibody, or antigen binding portion thereof, that binds a first antigen; b) obtaining a second
parent antibody or antigen binding portion thereof, that binds a second antigen; c) constructing
first and third polypeptide chains, each of which comprises VD1-(X1)n-VD2-C-(X2)n, wherein,
VD1 is a first heavy chain variable domain obtained from said first parent antibody, or antigen
binding portion thereof; VD2 is a second heavy chain variable domain obtained from said second
parent antibody or antigen binding portion thereof, which can be the same as or different from the
first parent antibody; C is a heavy chain constant domain; (X1)n is a linker with the proviso that it
is not CH1, wherein said (X1)n is either present or absent; and (X2)n is an Fc region, wherein said
(X2)n is either present or absent; d) constructing second and fourth polypeptide chains each of
which comprises VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first light chain variable domain
obtained from said first parent antibody, or antigen binding portion thereof; VD2 is a second light
chain variable domain obtained from said second parent antibody, or antigen binding thereof,
which can be the same as or different from the first parent antibody; C is a light chain constant
domain; (X1 )n is a linker with the proviso that it is not CH 1, wherein said (X1 )n is either present
or absent; and (X2)n does not comprise an Fc region, wherein said (X2)n is either present or
absent; and e) expressing said first, second, third and fourth polypeptide chains; such that a DVDIg
binds said first antigen and said second antigen is generated.
In still another embodiment, the invention provides a method of generating a DVD-Ig that
binds two antigens with desired properties comprising the steps of a) obtaining a first parent
antibody, or antigen binding portion thereof, that binds a first antigen and possessing at least one
desired property exhibited by the DVD-Ig; b) obtaining a second parent antibody, or antigen
binding portion thereof, which can be the same as or different from the first parent antibody, can
bind to a second antigen and possesses at least one desired property exhibited by the Dual
Variable Domain Immunoglobulin; c) constructing first and third polypeptide chains comprising
VD1-(Xl)n-VD2-C-(X2)n, wherein; VD1 is a first heavy chain variable domain obtained from
said first parent antibody, or antigen binding portion thereof; VD2 is a second heavy chain
variable domain obtained from said second parent antibody, or antigen binding portion thereof; C
is a heavy chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein
said (X1)n is either present or absent; and (X2)n is an Fc region, wherein said (X2)n is either
present or absent; d) constructing second and fourth polypeptide chains comprising VD1-(X1)n-
VD2-C-(X2)n, wherein; VD1 is a first light chain variable domain obtained from said first parent
antibody, or antigen binding portion thereof; VD2 is a second light chain variable domain
obtained from said second parent antibody, or antigen binding portion thereof), which can be the
same as or different from the first parent antibody; C is a light chain constant domain; (Xl)n is a
linker with the proviso that it is not CH1, wherein said (X1 )n is either present or absent; and
(X2)n does not comprise an Fc region, wherein said (X2)n is either present or absent; e)
expressing said first, second, third and fourth polypeptide chains; such that a Dual Variable
Domain Immunoglobulin capable of binding said first and said second antigen with desired
properties is generated.
In one embodiment, the VDI of the first and second polypeptide chains disclosed herein
are obtained from the same parent antibody or antigen binding portion thereof. In another
embodiment, the VDI of the first and second polypeptide chains disclosed herein are obtained
from different parent antibodies or antigen binding portions thereof. In another embodiment, the
VD2 of the first and second polypeptide chains disclosed herein are obtained from the same
parent antibody or antigen binding portion thereof. In another embodiment, the VD2 of the first
and second polypeptide chains disclosed herein are obtained from different parent antibodies or
antigen binding portions thereof.
In one embodiment the first parent antibody or antigen binding portion thereof, and the
second parent antibody or antigen binding portion thereof, are the same antibody. In another
embodiment the first parent antibody or antigen binding portion thereof, and the second parent
antibody or antigen binding portion thereof, are different antibodies.
In one embodiment the first parent antibody or antigen binding portion thereof, binds a
first antigen and the second parent antibody or antigen binding portion thereof, binds a second
antigen. In a particular embodiment, the first and second antigens are the same antigen. In
another embodiment, the parent antibodies bind different epitopes on the same antigen. In
another embodiment the first and second antigens are different antigens. In another embodiment,
the first parent antibody or antigen binding portion thereof, binds the first antigen with a potency
different from the potency with which the second parent antibody or antigen binding portion
thereof, binds the second antigen. In yet another embodiment, the first parent antibody or antigen
binding portion thereof, binds the first antigen with an affinity different from the affinity with
which the second parent antibody or antigen binding portion thereof, binds the second antigen.
In another embodiment the first parent antibody or antigen binding portion thereof, and
the second parent antibody or antigen binding portion thereof, are selected from the group
consisting of, human antibody, CDR grafted antibody, and humanized antibody. In an
embodiment, the antigen binding portions are selected from the group consisting of a Fab
fragment, a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv
fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment,
an isolated complementarity determining region (CDR), a single chain antibody, and diabodies.
In another embodiment the binding protein of the invention possesses at least one desired
property exhibited by the first parent antibody or antigen binding portion thereof, or the second
parent antibody or antigen binding portion thereof. Alternatively, the first parent antibody or
antigen binding portion thereof and the second parent antibody or antigen binding portion thereof
possess at least one desired property exhibited by the Dual Variable Domain Immunoglobulin. In
an embodiment, the desired property is selected from one or more antibody parameters. In
another embodiment, the antibody parameters are selected from the group consisting of antigen
specificity, affinity to antigen, potency, biological function, epitope recognition, stability,
solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross
reactivity, and orthologous antigen binding.In an embodiment the binding protein is multivalent.
In another embodiment, the binding protein is multispecific. The multivalent and or multispecific
binding proteins described herein have desirable properties particularly from a therapeutic
standpoint. For instance, the multivalent and or multispecific binding protein may (1) be
internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to
which the antibodies bind; (2) be an agonist antibody; and/or (3) induce cell death and/or
apoptosis of a cell expressing an antigen to which the multivalent antibody binds. The "parent
antibody," which provides at least one antigen binding specificity of the multivalent and/ or
multispecific binding proteins, may be one which is internalized (and/or catabolized) by a cell
expressing an antigen to which the antibody binds; and/or may be an agonist, cell death-inducing,
and/or apoptosis-inducing antibody, and the multivalent and or multispecific binding protein as
described herein may display improvement(s) in one or more of these properties. Moreover, the
parent antibody may lack any one or more of these properties, but may be endowed with them
when constructed as a multivalent binding protein as described herein.
In another embodiment the binding protein of the invention has an on rate constant (Kon)
to one or more targets selected from the group consisting of: at least about 102M-1s-1; at least about
103M-1s-1; at least about 104M-1s-1; at least about 105M-1S-1; and at least about 106M-1s-1, as
measured by surface plasmon resonance. In an embodiment, the binding protein of the invention
has an on rate constant (Kon) to one or more targets between about 102M-1s-1 and about 103M-1s-1;
between about 103M-1s-1 and about 104M-1s-1; between about 104M-1s-1 and about 105M-1s-1; or
between about 105M-1s-1 and about 106M-1s-1, as measured by surface plasmon resonance.
In another embodiment the binding protein has an off rate constant (Koff) for one or
more targets selected from the group consisting of: at most about 103s-1; at most about 10-4s-1; at
most about 10-5s-1; and at most about 10-6s-1, as measured by surface plasmon resonance. In an
embodiment, the binding protein of the invention has an off rate constant (Koff) to one or more
targets of from about 10-3s-1 to about 10-4s-1; of from about 10-4s-1 to about 10-5s-1; or of from about
10-5s-1 to about 10-6s-1, as measured by surface plasmon resonance.
In another embodiment the binding protein has a dissociation constant (KD) to one or
more targets selected from the group consisting of: at most about 10-7 M; at most about 10-8 M; at
most about 10'9 M; at most about 10-10 M; at most about 10-11 M; at most about 10-12 M; and at
most about 10"13 M. In an embodiment, the binding protein of the invention has a dissociation
constant (KD) to its targets of from about 10"7 M to about 10~8 M; of from about 10-8 M to about
10-9 M; of from about 10-9 M to about 10-10 M; of fromabout 10-10 to about 10-11 M; of from about
10-11 M to about 10-12 M; or of from about 10-12 M to about 10-13 M.
In another embodiment, the binding protein described herein is a conjugate further
comprising an agent selected from the group consisting of an immunoadhesion molecule, an
imaging agent, a therapeutic agent, and a cytotoxic agent. In an embodiment, the imaging agent is
selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent
label, a bioluminescent label, a magnetic label, and biotin. In another embodiment, the imaging
agent is a radiolabel selected from the group consisting of: 3H, 14C 35S, 90Y, 99Tc, 111In, 1251,131I,
l77Lu, 166Ho, and 153Sm. In yet another embodiment, the therapeutic or cytotoxic agent is selected
from the group consisting of an anti-metabolite, an alkylating agent, an antibiotic, a growth factor,
a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an
apoptotic agent.
In another embodiment, the binding protein described herein is a crystallized binding
protein and exists as a crystal. In an embodiment, the crystal is a carrier-free pharmaceutical
controlled release crystal. In yet another embodiment, the crystallized binding protein has a
greater half life in vivo than the soluble counterpart of said binding protein. In still another
embodiment, the crystallized binding protein retains biological activity.
In another embodiment, the binding protein described herein is glycosylated. For
example, the glycosylation is a human glycosylation pattern.
One aspect of the invention pertains to an isolated nucleic acid encoding any one of the
binding proteins disclosed herein. A further embodiment provides a vector comprising the
isolated nucleic acid disclosed herein wherein said vector is selected from the group consisting of
pcDNA; pTT (Durocher et al. (2002) Nucl. Acids Res. 30: 2); pTT3 (pTT with additional multiple
cloning site; pEFBOS (Mizushima, S. andNagata, S. (1990) Nucl. Acids Res. 18: 17); pBV; pJV;
pcDNA3.1 TOPO; pEF6 TOPO; and pBJ. In an embodiment, the vector is a vector disclosed in
U.S. Patent Publication No. 2009/0239259.
In another aspect a host cell is transformed with the vector disclosed herein. In an
embodiment, the host cell is a prokaryotic cell. In another embodiment, the host cell is E.coli. In
a related embodiment the host cell is a eukaryotic cell. In another embodiment, the eukaryotic
cell is selected from the group consisting of a protist cell, an animal cell, a plant cell and a fungal
cell. In yet another embodiment, the host cell is a mammalian cell including, but not limited to,
CHO, COS; NS0, SP2, PER.C6 or a fungal cell, such as Saccharomyces cerevisiae; or an insect
cell such as Sf9.
In an embodiment, two or more DVD-Igs, e.g., with different specificities, are produced
in a single recombinant host cell. For example, the expression of a mixture of antibodies has been
called Oligoclonics™ (Merus B.V., The Netherlands); U.S. Patent Nos. 7,262,028; 7,429,486.
Another aspect of the invention provides a method of producing a binding protein
disclosed herein comprising culturing any one of the host cells also disclosed herein in a culture
medium under conditions sufficient to produce the binding protein. In an embodiment, 50%-75%
of the binding protein produced by this method is a dual specific tetravalent binding protein. In a
particular embodiment, 75%-90% of the binding protein produced by this method is a dual
specific tetravalent binding protein. In a particular embodiment, 90%-95% of the binding protein
produced is a dual specific tetravalent binding protein.
One embodiment provides a composition for the release of a binding protein wherein the
composition comprises a formulation that in turn comprises a crystallized binding protein, as
disclosed herein, and an ingredient, and at least one polymeric carrier. For example, the
polymeric carrier comprises one or more polymers selected from the group consisting of poly
(acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide),
poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate),
poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl)
methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly
(vinylpyrrolidone), maleic anhydride- alkyl vinyl ether copolymers, pluronic polyols, albumin,
alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid,
oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.
For example, the ingredient is selected from the group consisting of albumin, sucrose, trehalose,
lactitol, gelatin, hydroxypropyl-P- cyclodextrin, methoxypolyethylene glycol and polyethylene
glycol. Another embodiment provides a method for treating a mammal comprising the step of
administering to the mammal an effective amount of the composition disclosed herein.
The invention also provides a pharmaceutical composition comprising a binding protein,
as disclosed herein and a pharmaceutically acceptable carrier. In a further embodiment the
pharmaceutical composition comprises at least one additional therapeutic agent for treating a
disorder. For example, the additional agent is selected from the group consisting of: a therapeutic
agent, an imaging agent, a cytotoxic agent, an angiogenesis inhibitor (including but not limited to
an anti-VEGF antibody or a VEGF-trap), a kinase inhibitor (including but not limited to a KDR
and a TIE-2 inhibitor), a co-stimulation molecule blocker (including but not limited to anti-B7.1,
anti-B7.2, CTLA4-Ig, anti-CD20), an adhesion molecule blocker (including but not limited to an
anti-LFA-1 antibody, an anti-E/L selectin antibody, a small molecule inhibitor), an anti-cytokine
antibody or functional fragment thereof (including but not limited to an anti-IL-18, an anti-TNF,
and an anti-IL-6/cytokine receptor antibody), methotrexate, cyclosporin, rapamycin, FK506, a
detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a
non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local
anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an
anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive,
a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an
antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an
epinephrine or analog, a cytokine, and a cytokine antagonist.
In another aspect, the invention provides a method for treating a human subject suffering
from a disorder in which the target, or targets, that can be bound by the binding protein disclosed
herein is/are detrimental, comprising administering to the human subject a binding protein
disclosed herein such that the activity of the target, or targets in the human subject is inhibited and
one of more symptoms is alleviated or treatment is achieved. For example, the disorder is
arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis,
reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative
colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma,
allergic diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant
rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease,
nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein
purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock,
toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease,
Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart
failure, myocardial infarction, Addison's disease, sporadic polyglandular deficiency type I and
polyglandular deficiency type II, Schmidt's syndrome, adult (acute) respiratory distress syndrome,
alopecia, alopecia areata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic
arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and
salmonella associated arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,
atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus,
pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic
anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal
Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis,
cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired
Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied immunodeficiency
(common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian
failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, postinflammatory
interstitial lung disease, interstitial pneumonitis, connective tissue disease
associated interstitial lung disease, mixed connective tissue disease associated lung disease,
systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial
lung disease, systemic lupus erythematosus associated lung disease,
dermatomyositis/polymyositis associated lung disease, Sjogren's disease associated lung disease,
ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis
associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis,
bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease,
postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune
hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM
antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance with
acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ
transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis,
primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the
kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension
secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic
sclerosis, Sjorgren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia,
idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous
autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism,
primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic
liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis, idiosyncratic liver
disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group B
streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and
Thl Type mediated diseases, acute and chronic pain (different forms of pain), and cancers such as
lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and
hematopoietic malignancies (leukemia and lymphoma), Abetalipoprotemia, Acrocyanosis, acute
and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia
(ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis,
acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcoholinduced
hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft
rejection, alpha-1- antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris,
anterior horn cell degeneration, anti cd3 therapy, antiphospholipid syndrome, anti-receptor
hypersensitivity reactions, aortic and peripheral aneuryisms, aortic dissection, arterial
hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or
paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone
marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiac
arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass
inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar
disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic
myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic
lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate
intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor
pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic
fibrosis, cytokine therapy associated disorders, Dementia pugilistica, demyelinating diseases,
dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus,
diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilated congestive cardiomyopathy,
disorders of the basal ganglia, Down's Syndrome in middle age, drug- induced movement
disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema,
encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus infection,
erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic
lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral
arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, graft rejection
of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular
organisms, hairy cell leukemia, Hallerrorden-Spatz disease, hashimoto's thyroiditis, hay fever,
heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis (A), His bundle
arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders,
hypersensitity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement
disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic
pulmonary fibrosis, antibody mediated cytotoxicity, Asthenia, infantile spinal muscular atrophy,
inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic
neuritis, ischemia- reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile
spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis,
leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphederma,
malaria, malignamt Lymphoma, malignant histiocytosis, malignant melanoma, meningitis,
meningococcemia, metabolic/idiopathic diseases, migraine headache, mitochondrial multi.system
disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple
systems degenerations (Mencel Dejerine- Thomas Shi-Drager and Machado-Joseph), myasthenia
gravis, mycobacterium avium intracellulare, mycobacterium tuberculosis, myelodyplastic
syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma,
neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I
muscular atrophies, neutropenic fever, non- hodgkins lymphoma, occlusion of the abdominal
aorta and its branches, occlusive arterial disorders, okt3 therapy, orchitis/epidydimitis,
orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant
rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,
parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial
disease, peripheral atherlosclerotic disease, peripheral vascular disorders, peritonitis, pernicious
anemia, Pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy,
organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post
perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia,
Progressive supranucleo Palsy, primary pulmonary hypertension, radiation therapy, Raynaud's
phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia,
renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma,
senile chorea, Senile Dementia of Lewy body type, seronegative arthropathies, shock, sickle cell
anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid
tumors, specific arrythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis,
structural lesions of the cerebellum, Subacute sclerosing panencephalitis, Syncope, syphilis of the
cardiovascular system, systemic anaphalaxis, systemic inflammatory response syndrome,
systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis
obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity
reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart
diseases, varicose veins, .vasculitis, venous diseases, venous thrombosis, ventricular fibrillation,
viral and fungal infections, vital encephalitis/aseptic meningitis, vital-associated hemaphagocytic
syndrome, Wernicke- Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or
tissue, acute coronary syndromes, acute idiopathic polyneuritis, acute inflammatory
demyelinating polyradiculoneuropathy, acute ischemia, adult Still's disease, alopecia areata,
anaphylaxis, anti-phospholipid antibody syndrome, aplastic anemia, arteriosclerosis, atopic
eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disorder associated with
streptococcus infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune
lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian
failure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular disease, catastrophic
antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial
pemphigoid, clinically isolated syndrome (cis) with risk for multiple sclerosis, conjunctivitis,
childhood onset psychiatric disorder, chronic obstructive pulmonary disease (COPD),
dacryocystitis, dermatomyositis, diabetic retinopathy, diabetes mellitus, disk herniation, disk
prolaps, drug induced immune hemolytic anemia, endocarditis, endometriosis, endophthalmitis,
episcleritis, erythema multiforme, erythema multiforme major, gestational pemphigoid, Guillain-
Barre syndrome (GBS), hay fever, Hughes syndrome, idiopathic Parkinson's disease, idiopathic
interstitial pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion body myositis,
infectious ocular inflammatory disease, inflammatory demyelinating disease, inflammatory heart
disease, inflammatory kidney disease, IPF/UIP, iritis, keratitis, keratojuntivitis sicca, Kussmaul
disease or Kussmaul-Meier disease, Landry's paralysis, Langerhan's cell histiocytosis, livedo
reticularis, macular degeneration, microscopic polyangiitis, morbus bechterev, motor neuron
disorders, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis,
myelodysplastic syndrome, myocarditis, nerve root disorders, neuropathy, non-A non-B hepatitis,
optic neuritis, osteolysis, ovarian cancer, pauciarticular JRA, peripheral artery occlusive disease
(PAOD), peripheral vascular disease (PVD), peripheral artery, disease (PAD), phlebitis,
polyarteritis nodosa (or periarteritis nodosa), polychondritis, polymyalgia rheumatica, poliosis,
polyarticular JRA, polyendocrine deficiency syndrome, polymyositis, polymyalgia rheumatica
(PMR), post-pump syndrome, primary Parkinsonism, prostate and rectal cancer and
hematopoietic malignancies (leukemia and lymphoma), prostatitis, pure red cell aplasia, primary
adrenal insufficiency, recurrent neuromyelitis optica, restenosis, rheumatic heart disease, SAPHO
(synovitis, acne, pustulosis, hyperostosis, and osteitis), scleroderma, secondary amyloidosis,
shock lung, scleritis, sciatica, secondary adrenal insufficiency, silicone associated connective
tissue disease, sneddon-wilkinson dermatosis, spondilitis ankylosans, Stevens-Johnson syndrome
(SJS), systemic inflammatory response syndrome, temporal arteritis, toxoplasmic retinitis, toxic
epidermal necrolysis, transverse myelitis, TRAPS (tumor necrosis factor receptor, type 1 allergic
reaction, type II diabetes, urticaria, usual interstitial pneumonia (UIP), vasculitis, vernal
conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular
degeneration, wound healing, yersinia and salmonella associated arthropathy.
In an embodiment, diseases that can be treated or diagnosed with the compositions and
methods of the invention include, but are not limited to, primary and metastatic cancers, including
carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,
pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder
and urothelium), female genital tract (including cervix, uterus, and ovaries as well as
choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate,
seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal,
and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those
arising from bone and soft tissues as well as Kaposi's sarcoma), tumors of the brain, nerves, eyes,
and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,
neuroblastomas, Schwannomas, and meningiomas), solid tumors arising from hematopoietic
malignancies such as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin's
lymphomas).
In an embodiment, the antibodies of the invention or antigen-binding portions thereof, are
used to treat cancer or in the prevention or inhibition of metastases from the tumors described
herein either when used alone or in combination with radiotherapy and/or other chemotherapeutic
agents.
In another aspect the invention provides a method of treating a patient suffering from a
disorder comprising the step of administering any one of the binding proteins disclosed herein
before, concurrently, or after the administration of a second agent, as discussed herein. In a
particular embodiment the second agent is selected from the group consisting of budenoside,
epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, 6-
mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine,
balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-ip mAbs,
anti-IL-6 or IL-6 receptor mAbs, growth factors, elastase inhibitors, pyridinyl-imidazole
compounds, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15,
IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8,
CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands, methotrexate,
cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen,
corticosteroids, prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic
agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors,
IL-1ßconverting enzyme inhibitors, TNFa converting enzyme inhibitors, T-cell signalling
inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines,
angiotensin converting enzyme inhibitors, soluble cytokine receptors, soluble p55 TNF receptor,
soluble p75 TNF receptor, sIL-lRI, sIL-lRII, sIL-6R, antiinflammatory cytokines, IL-4, IL-10,
IL-11, IL-13 and TGFß.
In a particular embodiment the pharmaceutical compositions disclosed herein are
administered to the patient by at least one mode selected from parenteral, subcutaneous,
intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac,
intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,
intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal,
sublingual, intranasal, and transdermal.
One aspect of the invention provides at least one anti-idiotypic antibody to at least one
binding protein of the present invention. The anti-idiotypic antibody includes any protein or
peptide containing molecule that comprises at least a portion of an immunoglobulin molecule
such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or
light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a
heavy chain or light chain constant region, a framework region, or any portion thereof, that can be
incorporated into a binding protein of the present invention.
Brief Description of the Drawings
Figure 1A is a schematic representation of Dual Variable Domain (DVD)-Ig constructs and shows
the strategy for generation of a DVD-Ig from two parent antibodies;
Figure IB, is a schematic representation of constructs DVD1-Ig, DVD2-Ig, and two chimeric
mono-specific antibodies from hybridoma clones 2D13.E3 (anti-IL-1α) and 13F5.G5 (anti-
IL-1ß).
Detailed Description of the Invention
This invention pertains to multivalent and/or multispecific binding proteins that bind two
or more antigens. Specifically, the invention relates to dual variable domain immunoglobulins
(DVD-Ig), and pharmaceutical compositions thereof, as well as nucleic acids, recombinant
expression vectors and host cells for making such DVD-Igs. Methods of using the DVD-Igs of
the invention to detect specific antigens, either in vitro or in vivo are also encompassed by the
invention.
Unless otherwise defined herein, scientific and technical terms used in connection with
the present invention shall have the meanings that are commonly understood by those of ordinary
skill in the art. The meaning and scope of the terms should be clear, however, in the event of any
latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic
definition. Further, unless otherwise required by context, singular terms shall include pluralities
and plural terms shall include the singular. In this application, the use of "or" means "and/or"
unless stated otherwise. Furthermore, the use of the term "including," as well as other forms,
such as "includes" and "included," is not limiting. Also, terms such as "element" or "component"
encompass both elements and components comprising one unit and elements and components
that comprise more than one subunit unless specifically stated otherwise.
Generally, nomenclatures used in connection with, and techniques of, cell and tissue
culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well known and commonly used in the
art. The methods and techniques of the present invention are generally performed according to
conventional methods well known in the art and as described in various general and more
specific references that are cited and discussed throughout the present specification unless
otherwise indicated. Enzymatic reactions and purification techniques are performed according to
manufacturer's specifications, as commonly accomplished in the art or as described herein. The
nomenclatures used in connection with, and the laboratory procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well known and commonly used in the art. Standard techniques are
used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
That the present invention may be more readily understood, select terms are defined
below.
The term "polypeptide" as used herein, refers to any polymeric chain of amino acids. The
terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer
to a polymeric chain of amino acids. The term "polypeptide" encompasses native or artificial
proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be
monomelic or polymeric. Use of "polypeptide" herein is intended to encompass polypeptide and
fragments and variants (including fragments of variants) thereof, unless otherwise stated. For an
antigenic polypeptide, a fragment of polypeptide optionally contains at least one contiguous or
nonlinear epitope of polypeptide. The precise boundaries of the at least one epitope fragment can
be confirmed using ordinary skill in the art. The fragment comprises at least about 5 contiguous
amino acids, such as at least about 10 contiguous amino acids, at least about 15 contiguous amino
acids, or at least about 20 contiguous amino acids. A variant of polypeptide is as described
herein.
The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by
virtue of its origin or source of derivation is not associated with naturally associated components
that accompany it in its native state; is substantially free of other proteins from the same species;
is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide
that is chemically synthesized or synthesized in a cellular system different from the cell from
which it naturally originates will be "isolated" from its naturally associated components. A protein
may also be rendered substantially free of naturally associated components by isolation, using
protein purification techniques well known in the art.
The term "recovering" as used herein, refers to the process of rendering a chemical
species such as a polypeptide substantially free of naturally associated components by isolation,
e.g., using protein purification techniques well known in the art.
"Biological activity " as used herein, refers to any one or more inherent biological
properties of a molecule (whether present naturally as found in vivo, or provided or enabled by
recombinant means). Biological properties include but are not limited to binding a receptor,
inducing cell proliferation, inhibiting cell growth, inducing other cytokines, inducing apoptosis,
and enzymatic activity. Biological activity also includes activity of an Ig molecule.
The terms "specific binding" or "specifically binding," as used herein, in reference to the
interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the
interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant
or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific
protein structure rather than to proteins generally. If an antibody is specific for epitope "A," the
presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing
labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.
The term "antibody," as used herein, broadly refers to any immunoglobulin (Ig) molecule
comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any
functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope
binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are
known in the art. Nonlimiting embodiments of which are discussed below.
In a full-length antibody, each heavy chain is comprised of a heavy chain variable region
(abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain
constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is
comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain
constant region. The light chain constant region is comprised of one domain, CL. The VH and
VL regions can be further subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more conserved, termed framework
regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from aminoterminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and
FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),
class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.
The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy
chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a
native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally
comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a
CH4 domain. Replacements of amino acid residues in the Fc portion to alter antibody effector
function are known in the art (US Patent Nos. 5,648,260 and 5,624,821). The Fc portion of an
antibody mediates several important effector functions e.g., cytokine induction, ADCC,
phagocytosis, complement dependent cytotoxicity (CDC) and half-life/ clearance rate of antibody
and antigen-antibody complexes. In some cases these effector functions are desirable for a
therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on
the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate
ADCC and CDC via binding to FcγRs and complement Clq, respectively. Neonatal Fc receptors
(FcRn) are the critical components determining the circulating half-life of antibodies. In still
another embodiment at least one amino acid residue is replaced in the constant region of the
antibody, for example the Fc region of the antibody, such that effector functions of the antibody
are altered. The dimerization of two identical heavy chains of an immunoglobulin is mediated by
the dimerization of CH3 domains and is stabilized by the disulfide bonds within the hinge region
(Huber et al. (1976) Nature 264: 415-20; Thies et al. (1999) J. Mol. Biol. 293: 67-79). Mutation
of cysteine residues within the hinge regions to prevent heavy chain-heavy chain disulfide bonds
will destabilize dimeration of CH3 domains. Residues responsible for CH3 dimerization have
been identified (DalPAcqua (1998) Biochem. 37: 9266-73). Therefore, it is possible to generate a
monovalent half-Ig. Interestingly, these monovalent half Ig molecules have been found in nature
for both IgG and IgA subclasses (Seligman (1978) Ann. Immunol. 129: 855-70; Biewenga et al.
(1983) Clin. Exp. Immunol. 51:395-400). The stoichiometry of FcRn: Ig Fc region has been
determined to be 2:1 (West et al. (2000) Biochem. 39: 9698-708), and half Fc is sufficient for
mediating FcRn binding (Kim et al. (1994) Eur. J. Immunol. 24: 542-548). Mutations to disrupt
the dimerization of CH3 domain may not have greater adverse effect on its FcRn binding as the
residues important for CH3 dimerization are located on the inner interface of CH3 b sheet
structure, whereas the region responsible for FcRn binding is located on the outside interface of
CH2-CH3 domains. However, the half -Ig molecule may have certain advantages in tissue
penetration due to its smaller size in comparison to that of a regular antibody. In one embodiment
at least one amino acid residue is replaced in the constant region of the binding protein of the
invention, for example the Fc region, such that the dimerization of the heavy chains is disrupted,
resulting in half DVD Ig molecules. The anti-inflammatory activity of IgG is completely
dependent on sialylation of the N-linked glycan of the IgG Fc fragment. The precise glycan
requirements for anti-inflammatory activity has been determined, such that an appropriate IgG1
Fc fragment can be created, thereby generating a fully recombinant, sialylated IgGl Fc with
greatly enhanced potency (Anthony, R.M., et al. (2008) Science 320: 373-376).
The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used
herein, refers to one or more fragments of an antibody that retain the ability to bind specifically to
an antigen. It has been shown that the antigen-binding function of an antibody can be performed
by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual
specific, or multi-specific formats; specifically binding to two or more different antigens.
Examples of binding fragments encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody,
(v) a dAb fragment (Ward (1989) Nature 341:544-546: PCT Publication No. WO 90/05144 A1),
which comprises a single variable domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are
coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker
that enables them to be made as a single protein chain in which the VL and VH regions pair to
form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science
242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single
chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of
an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed.
Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a
single polypeptide chain, but using a linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with complementary domains of
another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2: 1121-1123). Such
antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody
Engineering (2001) Springer-Verlag. New York. p. 790 (ISBN 3-540-41354-5). In addition
single chain antibodies also include "linear antibodies" comprising a pair of tandem Fv segments
(VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of
antigen binding regions (Zapata et al. (1995) Protein Eng. 8(10):1057-1062; and US Patent No.
5,641,870).
The term "multivalent binding protein" is used throughout this specification to denote a
binding protein comprising two or more antigen binding sites. In an embodiment, the multivalent
binding protein is engineered to have the three or more antigen binding sites, and is generally not
a naturally occurring antibody. The term "multispecific binding protein" refers to a binding
protein that binds two or more related or unrelated targets. Dual variable domain (DVD) binding
proteins of the invention comprise two or more antigen binding sites and are tetravalent or
multivalent binding proteins. DVDs may be monospecific, i.e., bind one antigen or multispecific,
i.e., capable of binding two or more antigens. DVD binding proteins comprising two heavy chain
DVD polypeptides and two light chain DVD polypeptides are referred to as DVD-Ig. Each half
of a DVD-Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and
two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light
chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site.
The term "bispecific antibody," as used herein, refers to full-length antibodies that are
generated by quadroma technology (see Milstein, C. and Cuello, A.C. (1983) Nature 305(5934):
p. 537-540), by chemical conjugation of two different monoclonal antibodies (see Staerz, U.D. et
al. (1985) Nature 314(6012): 628-631), or by knob-into-hole or similar approaches, which
introduce mutations in the Fc region (see Holliger, P. et al. (1993) Proc. Natl. Acad. Sci USA
90(14): 6444-6448), resulting in multiple different immunoglobulin species of which only one is
the functional bispecific antibody. By molecular function, a bispecific antibody binds one antigen
(or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or
epitope) on its second arm (a different pair of HC/LC). By this definition, a bispecific antibody
has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent
for each antigen it binds to.
The term "dual-specific antibody," as used herein, refers to full-length antibodies that can
bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (see
PCT Publication No. WO 02/02773). Accordingly a dual-specific binding protein has two
identical antigen binding arms, with identical specificity and identical CDR sequences, and is
bivalent for each antigen to which it binds.
A "functional antigen binding site" of a binding protein is one that binds a target antigen.
The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent
antibody from which the antigen binding site is derived, but the ability to bind antigen must be
measurable using any one of a variety of methods known for evaluating antibody binding to an
antigen. Moreover, the antigen binding affinity of each of the antigen binding sites of a
multivalent antibody herein need not be quantitatively the same.
The term "cytokine" is a generic term for proteins released by one cell population, which
act on another cell population as intercellular mediators. Examples of such cytokines are
lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human growth hormone, and
bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;
glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone
(TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin;
placental lactogen; tumor necrosis factor-alpha and - beta; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin;
thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet-growth factor; placental
growth factor, transforming growth factors (TGFs) such as TGF- alpha and TGF-beta; insulin-like
growth factor-1 and -11; erythropoietin (EPO); osteoinductive factors; interferons such as
interferon-alpha, -beta and -gamma colony stimulating factors (CSFs) such as macrophage-CSF
(M-CSF); granulocyte macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins
(ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-
15, IL-18, IL-21, IL-22, IL-23, and IL-33; a tumor necrosis factor such as TNF-alpha or TNFbeta;
and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant cell culture and biologically
active equivalents of the native sequence cytokines.
The term "linker" is used to denote polypeptides comprising two or more amino acid
residues joined by peptide bonds and are used to link one or more antigen binding portions. Such
linker polypeptides are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad.
Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). Exemplary linkers
include, but are not limited to, AKTTPKLEEGEFSEAR (SEQ ID NO: 1);
AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG
(SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ
ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G4S)4 (SEQ ID NO: 9);
SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP
(SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP
(SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17);
AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ
ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22),
GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24);
GPAKELTPLKEAKVS (SEQ ID NO: 25); GHEAAAVMQVQYPAS (SEQ ID NO: 26),
TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO: 27); and
ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28.
An "immunoglobulin constant domain" refers to a heavy or light chain constant domain.
Human IgG heavy chain and light chain constant domain amino acid sequences are known in the
art.
The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained
from a population of substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population are identical except for possible naturally occurring mutations that may
be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a
single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include
different antibodies directed against different determinants (epitopes), each mAb is directed
against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as
requiring production of the antibody by any particular method.
The term "human antibody," as used herein, is intended to include antibodies having
variable and constant regions derived from human germline immunoglobulin sequences. The
human antibodies of the invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific
mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular
CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies
in which CDR sequences derived from the germline of another mammalian species, such as a
mouse, have been grafted onto human framework sequences.
The term "recombinant human antibody," as used herein, is intended to include all human
antibodies that are prepared, expressed, created or isolated by recombinant means, such as
antibodies expressed using a recombinant expression vector transfected into a host cell (described
further in Section IIC, below), antibodies isolated from a recombinant, combinatorial human
antibody library (Hoogenboom H.R. (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W.E.
(2002) Clin. Biochem. 35:425-445; Gavilondo J.V., and Larrick J.W. (2002) BioTechniques
29:128-145; Hoogenboom H., and Chames P. (2000) Immunol. Today 21: 371-378 ), antibodies
isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see,
Taylor, L. D., et al. (1992) Nucl. Acids Res. 20: 6287-6295; Kellermann S-A. and Green L.L.
(2002) Current Opinion in Biotechnol. 13: 593-597; Little M. et al. (2000) Immunol. Today
21:364-370) or antibodies prepared, expressed, created or isolated by any other means that
involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies have variable and constant regions derived from human germline
immunoglobulin sequences. In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig
sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and
VL regions of the recombinant antibodies are sequences that, while derived from and related to
human germline VH and VL sequences, may not naturally exist within the human antibody
germline repertoire in vivo.
An "affinity matured" antibody is an antibody with one or more alterations in one or more
CDRs thereof which result an improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s). Exemplary affinity matured
antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity
matured antibodies are produced by procedures known in the art. Marks et al. (1992)
Bio/Technology 10: 779-783 describes affinity maturation by VH and VL domain shuffling.
Random mutagenesis of CDR and/or framework residues is described by Barbas, et al. (1994)
Proc Nat. Acad. Sci. USA91: 3809-3813; Schieretal. (1995)Gene 169: 147- 155; Yelton etal.,
(1995) J. Immunol. 155: 1994-2004; Jackson et al.(1995) J. Immunol. 154(7): 3310-9; and
Hawkins et al. (1992) J. Mol. Biol. 226: 889-896; and selective mutation at selective mutagenesis
positions, contact or hypermutation positions with an activity enhancing amino acid residue is
described in U.S. Patent No. 6,914,128.
The term "chimeric antibody" refers to antibodies which comprise heavy and light chain
variable region sequences from one species and constant region sequences from another species,
such as antibodies having murine heavy and light chain variable regions linked to human constant
regions.
The term "CDR-grafted antibody" refers to antibodies which comprise heavy and light
chain variable region sequences from one species but in which the sequences of one or more of
the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as
antibodies having murine heavy and light chain variable regions in which one or more of the
murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
The term "humanized antibody" refers to antibodies which comprise heavy and light
chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a
portion of the VH and/or VL sequence has been altered to be more "human-like," i.e., more
similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted
antibody, in which human CDR sequences are introduced into non-human VH and VL sequences
to replace the corresponding nonhuman CDR sequences. Also "humanized antibody"is an
antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to
an antigen of interest and which comprises a framework (FR) region having substantially the
amino acid sequence of a human antibody and a complementary determining region (CDR)
having substantially the amino acid sequence of a non-human antibody. As used herein, the term
"substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least
80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino
acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all
of at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or
substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e.,
donor antibody) and all or substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. In an embodiment, a humanized antibody also comprises at
least a portion of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as
well as at least the variable domain of a heavy chain. The antibody also may include the CH1,
hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized
antibody only contains a humanized light chain. In some embodiments, a humanized antibody
only contains a humanized heavy chain. In specific embodiments, a humanized antibody only
contains a humanized variable domain of a light chain and/or humanized heavy chain.
The terms "Kabat numbering," "Kabat definitions" and "Kabat labeling" are used
interchangeably herein. These terms, which are recognized in the art, refer to a system of
numbering amino acid residues which are more variable (i.e., hypervariable) than other amino
acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding
portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E.A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the
hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions
50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable
region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid
positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
As used herein, the term "CDR" refers to the complementarity determining region within
antibody variable sequences. There are three CDRs in each of the variable regions of the heavy
chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable
regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single
variable region that binds the antigen. The exact boundaries of these CDRs have been defined
differently according to different systems. The system described by Kabat (Kabat et al.,
Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.
(1987) and (1991)) not only provides an unambiguous residue numbering system applicable to
any variable region of an antibody, but also provides precise residue boundaries defining the three
CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia &Lesk
(1987) J. Mol. Biol. 196:901-917 and Chothia et al. (1989) Nature 342:877-883) found that
certain sub- portions within Kabat CDRs adopt nearly identical peptide backbone conformations,
despite having great diversity at the level of amino acid sequence. These sub-portions were
designated as LI, L2 and L3 or HI, H2 and H3 where the "L" and the "H" designate the light
chain and the heavy chain regions, respectively. These regions may be referred to as Chothia
CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs
overlapping with the Kabat CDRs have been described by Padlan (1995) FASEB J. 9: 133-139
and MacCallum (1996) J. Mol. Biol. 262(5): 732-45. Still other CDR boundary definitions may
not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs,
although they may be shortened or lengthened in light of prediction or experimental findings that
particular residues or groups of residues or even entire CDRs do not significantly impact antigen
binding. The methods used herein may utilize CDRs defined according to any of these systems,
although certain embodiments use Kabat or Chothia defined CDRs.
As used herein, the term "framework" or "framework sequence" refers to the remaining
sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence
can be determined by different systems, the meaning of a framework sequence is subject to
correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and
CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and
the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is
positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and
FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework
region, as referred by others, represents the combined FR's within the variable region of a single,
naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four subregions,
and FRs represents two or more of the four sub- regions constituting a framework region.
As used herein, the term "germline antibody gene" or "gene fragment" refers to an
immunoglobulin sequence encoded by non- lymphoid cells that have not undergone the
maturation process that leads to genetic rearrangement and mutation for expression of a particular
immunoglobulin. (See, e.g., Shapiro et al. (2002) Crit. Rev. Immunol. 22(3): 183-200;
Marchalonis et al. (2001) Adv. Exp. Med. Biol. 484: 13-30). One of the advantages provided by
various embodiments of the present invention stems from the recognition that germline antibody
genes are more likely than mature antibody genes to conserve essential amino acid sequence
structures characteristic of individuals in the species, hence less likely to be recognized as from a
foreign source when used therapeutically in that species.
As used herein, the term "neutralizing" refers to counteracting the biological activity of
an antigen when a binding protein specifically binds the antigen. In an embodiment, the
neutralizing binding protein binds the cytokine and reduces its biologically activity by at least
about 20%, 40%, 60%, 80%, 85% or more.
The term "activity" includes activities such as the binding specificity and affinity of a
DVD-Ig for two or more antigens.
The term "epitope" includes any polypeptide determinant that specifically binds to an
immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include
chemically active surface groupings of molecules such as amino acids, sugar side chains,
phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional
structural characteristics, and/or specific charge characteristics. An epitope is a region of an
antigen that is bound by an antibody. An epitope thus consists of the amino acid residues of a
region of an antigen (or fragment thereof) known to bind to the complementary site on the
specific binding partner. An antigenic fragment can contain more than one epitope. In certain
embodiments, an antibody is said to specifically bind an antigen when it recognizes its target
antigen in a complex mixture of proteins and/or macromolecules. Antibodies are said to "bind to
the same epitope" if the antibodies cross-compete (one prevents the binding or modulating effect
of the other). In addition structural definitions of epitopes (overlapping, similar, identical) are
informative, but functional definitions are often more relevant as they encompass structural
(binding) and functional (modulation, competition) parameters.
The term "surface plasmon resonance," as used herein, refers to an optical phenomenon
that allows for the analysis of real-time biospecific interactions by detection of alterations in
protein concentrations within a biosensor matrix, for example using the BIAcore® system
(BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ).
For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et
al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and
Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.
The term "Kon," as used herein, is intended to refer to the on rate constant for association
of a binding protein (e.g., an antibody) to the antigen to form the, e.g., antibody/antigen complex
as is known in the art. The "Kon" also is known by the terms "association rate constant," or "ka,"
as used interchangeably herein. This value indicating the binding rate of an antibody to its target
antigen or the rate of complex formation between an antibody and antigen also is shown by the
equation:
Antibody ("Ab") + Antigen ("Ag") Ab-Ag.
The term "Koff," as used herein, is intended to refer to the off rate constant for
dissociation of a binding protein (e.g., an antibody) from the, e.g., antibody/antigen complex as is
known in the art. The "Koff' also is known by the terms "dissociation rate constant" or "kd" as
used interchangeably herein. This value indicates the dissociation rate of an antibody from its
target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown
by the equation below:
Ab + Ag Ab-Ag.
The terms "equilibrium dissociation constant" or "KD," as used interchangeably herein,
refer to the value obtained in a titration measurement at equilibrium, or by dividing the
dissociation rate constant (koff)by the association rate constant (kon). The association rate
constant, the dissociation rate constant and the equilibrium dissociation constant are used to
represent the binding affinity of an antibody to an antigen. Methods for determining association
and dissociation rate constants are well known in the art. Using fluorescence-based techniques
offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium.
Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction
analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE
Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay)
assay, available from Sapidyne Instruments (Boise, Idaho) can also be used.
"Label" and "detectable label" mean a moiety attached to a specific binding partner, such
as an antibody or an analyte, e.g., to render the reaction between members of a specific binding
pair, such as an antibody and an analyte, detectable, and the specific binding partner, e.g.,
antibody or analyte, so labeled is referred to as "detectably labeled." Thus, the term "labeled
binding protein" as used herein, refers to a protein with a label incorporated that provides for the
identification of the binding protein. In an embodiment, the label is a detectable marker that can
produce a signal that is detectable by visual or instrumental means, e.g., incorporation of a
radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected
by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that
can be detected by optical or colorimetric methods). Examples of labels for polypeptides include,
but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, "Tc,
11'in, 125I, I3,1,177Lu, 166Ho, and 153Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine,
and lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline
phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes
recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, and epitope tags); and magnetic agents, such as
gadolinium chelates. Representative examples of labels commonly employed for immunassays
include moieties that produce light, e.g., acridinium compounds, and moieties that produce
fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety itself
may not be detectably labeled but may become detectable upon reaction with yet another moiety.
Use of "detectably labeled" is intended to encompass the latter type of detectable labeling.
The term "conjugate" refers to a binding protein, such as an antibody, chemically linked
to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term "agent" is used
herein to denote a chemical compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials. In an embodiment, the therapeutic
or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs thereof. When employed in the
context of an immunoassay, the conjugate antibody is a detectably labeled antibody used as the
detection antibody.
The terms "crystal" and "crystallized" as used herein, refer to a binding protein (e.g., an
antibody), or antigen binding portion thereof, that exists in the form of a crystal. Crystals are
one form of the solid state of matter, which is distinct from other forms such as the amorphous
solid state or the liquid crystalline state. Crystals are composed of regular, repeating, threedimensional
arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular
assemblies (e.g., antigen/antibody complexes). These three-dimensional arrays are arranged
according to specific mathematical relationships that are well-understood in the field. The
fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit.
Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined
crystallographic symmetry provides the "unit cell" of the crystal. Repetition of the unit cell by
regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A.
Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-
16, Oxford University Press, New York, New York, (1999)."
The term "polynucleotide" means a polymeric form of two or more nucleotides, either
ribonucleotides or deoxvnucleotides or a modified form of either type of nucleotide. The term
includes single and double stranded forms of DNA.
The term "isolated polynucleotide" shall mean a polynucleotide (e.g., of genomic, cDNA,
or synthetic origin, or some combination thereof) that, by virtue of its origin, the "isolated
polynucleotide" is not associated with all or a portion of a polynucleotide with which the "isolated
polynucleotide" is found in nature; is operably linked to a polynucleotide that it is not linked to in
nature; or does not occur in nature as part of a larger sequence.
The term "vector," is intended to refer to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to
a circular double stranded DNA loop into which additional DNA segments may be ligated.
Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the
viral genome. Certain vectors are capable of autonomous replication in a host cell into which they
are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into
the genome of a host cell upon introduction into the host cell, and thereby are replicated along
with the host genome. Moreover, certain vectors are capable of directing the expression of genes
to which they are operatively linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used
form of vector. However, the invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adenoassociated
viruses), which serve equivalent functions.
The term "operably linked" refers to a juxtaposition wherein the components described
are in a relationship permitting them to function in their intended manner. A control sequence
"operably linked" to a coding sequence is ligated in such a way that expression of the coding
sequence is achieved under conditions compatible with the control sequences. "Operably linked"
sequences include both expression control sequences that are contiguous with the gene of interest
and expression control sequences that act in trans or at a distance to control the gene of interest.
The term "expression control sequence" as used herein refers to polynucleotide sequences which
are necessary to effect the expression and processing of coding sequences to which they are
ligated. Expression control sequences include appropriate transcription initiation, termination,
promoter and enhancer sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance
translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability;
and when desired, sequences that enhance protein secretion. The nature of such control sequences
differs depending upon the host organism; in prokaryotes, such control sequences generally
include a promoter, a ribosomal binding site, and a transcription termination sequence; in
eukaryotes, generally, such control sequences include a promoter and a transcription termination
sequence. The term "control sequences" is intended to include components whose presence is
essential for expression and processing, and can also include additional components whose
presence is advantageous, for example, leader sequences and fusion partner sequences.
"Transformation," refers to any process by which exogenous DNA enters a host cell.
Transformation may occur under natural or artificial conditions using various methods well
known in the art. Transformation may rely on any known method for the insertion of foreign
nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on
the host cell being transformed and may include, but is not limited to, viral infection,
electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably
transformed cells in which the inserted DNA is capable of replication either as an autonomously
replicating plasmid or as part of the host chromosome. They also include cells which transiently
express the inserted DNA or RNA for limited periods of time.
The term "recombinant host cell" (or simply "host cell"), is intended to refer to a cell into
which exogenous DNA has been introduced. In an embodiment, the host cell comprises two or
more (e.g., multiple) nucleic acids encoding antibodies, such as the host cells described in US
Patent No. 7,262,028, for example. Such terms are intended to refer not only to the particular
subject cell, but, also to the progeny of such a cell. Because certain modifications may occur in
succeeding generations due to either mutation or environmental influences, such progeny may
not, in fact, be identical to the parent cell, but are still included within the scope of the term "host
cell" as used herein. In an embodiment, host cells include prokaryotic and eukaryotic cells
selected from any of the Kingdoms of life. In another embodiment, eukaryotic cells include
protist, fungal, plant and animal cells. In another embodiment, host cells include but are not
limited to the prokaryotic cell line E.coli; mammalian cell lines CHO, HEK 293, COS, NSO, SP2
and PER.C6; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and
tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to manufacturer's specifications or as
commonly accomplished in the art or as described herein. The foregoing techniques and
procedures may be generally performed according to conventional methods well known in the art
and as described in various general and more specific references that are cited and discussed
throughout the present specification. See e.g., Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
"Transgenic organism," as known in the art, refers to an organism having cells that
contain a transgene, wherein the transgene introduced into the organism (or an ancestor of the
organism) expresses a polypeptide not naturally expressed in the organism. A "transgene" is a
DNA construct, which is stably and operably integrated into the genome of a cell from which a
transgenic organism develops, directing the expression of an encoded gene product in one or more
cell types or tissues of the transgenic organism.
The term "regulate"and "modulate" are used interchangeably, and, as used herein, refers
to a change or an alteration in the activity of a molecule of interest (e.g., the biological activity of
a cytokine). Modulation may be an increase or a decrease in the magnitude of a certain activity or
function of the molecule of interest. Exemplary activities and functions of a molecule include, but
are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal
transduction.
Correspondingly, the term "modulator" is a compound capable of changing or altering an
activity or function of a molecule of interest (e.g., the biological activity of a cytokine). For
example, a modulator may cause an increase or decrease in the magnitude of a certain activity or
function of a molecule compared to the magnitude of the activity or function observed in the
absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases
the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but
are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic
molecules. Peptibodies are described, e.g., in WOO 1/83525.
The term "agonist," refers to a modulator that, when contacted with a molecule of
interest, causes an increase in the magnitude of a certain activity or function of the molecule
compared to the magnitude of the activity or function observed in the absence of the agonist.
Particular agonists of interest may include, but are not limited to, polypeptides, nucleic acids,
carbohydrates, and any other molecules that bind to the antigen.
The term "antagonist" or "inhibitor," refers to a modulator that, when contacted with a
molecule of interest, causes a decrease in the magnitude of a certain activity or function of the
molecule compared to the magnitude of the activity or function observed in the absence of the
antagonist. Particular antagonists of interest include those that block or modulate the biological or
immunological activity of of the antigen. Antagonists and inhibitors of antigens may include, but
are not limited to, proteins, nucleic acids, carbohydrates, and any other molecules, which bind to
the antigen.
As used herein, the term "effective amount" refers to the amount of a therapy which is
sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more
symptoms thereof, inhibit or prevent the advancement of a disorder, cause regression of a
disorder, inhibit or prevent the recurrence, development, onset or progression of one or more
symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or
therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).
"Patient" and "subject" may be used interchangeably herein to refer to an animal, such as
a mammal, including a primate (for example, a human, a monkey, and a chimpanzee), a nonprimate
(for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a
guinea pig, a cat, a dog, a rat, a mouse, and a whale), a bird (e.g., a duck or a goose), and a shark.
Preferably, the patient or subject is a human, such as a human being treated or assessed for a
disease, disorder or condition, a human at risk for a disease, disorder or condition, a human
having a disease, disorder or condition, and/or human being treated for a disease, disorder or
condition.
The term "sample," as used herein, is used in its broadest sense. A "biological sample,"
as used herein, includes, but is not limited to, any quantity of a substance from a living thing or
formerly living thing. Such living things include, but are not limited to, humans, mice, rats,
monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood,
(e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells,
leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.
"Component," "components," and "at least one component," refer generally to a capture
antibody, a detection or conjugate antibody, a control, a calibrator, a series of calibrators, a
sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a
detection reagent, a pretreatment reagent/solution, a substrate (e.g., as a solution), a stop solution,
and the like that can be included in a kit for assay of a test sample, such as a patient urine, serum
or plasma sample, in accordance with the methods described herein and other methods known in
the art. Thus, in the context of the present disclosure, "at least one component," "component,"
and "components" can include a polypeptide or other analyte as above, such as a composition
comprising an analyte such as polypeptide, which is optionally immobilized on a solid support,
such as by binding to an anti-analyte (e.g., anti-polypeptide) antibody. Some components can be
in solution or lyophilized for reconstitution for use in an assay.
"Control" refers to a composition known to not contain analyte ("negative control") or to
contain analyte ("positive control"). A positive control can comprise a known concentration of
analyte. "Control," "positive control," and "calibrator" may be used interchangeably herein to
refer to a composition comprising a known concentration of analyte. A "positive control" can be
used to establish assay performance characteristics and is a useful indicator of the integrity of
reagents (e.g., analytes).
"Predetermined cutoff" and "predetermined level" refer generally to an assay cutoff value
that is used to assess diagnostic/prognostic/therapeutic efficacy results by comparing the assay
results against the predetermined cutoff/level, where the predetermined cutoff/level already has
been linked or associated with various clinical parameters (e.g., severity of disease,
progression/nonprogression/improvement, etc.). While the present disclosure may provide
exemplary predetermined levels, it is well-known that cutoff values may vary depending on the
nature of the immunoassay (e.g., antibodies employed, etc.). It further is well within the ordinary
skill of one in the art to adapt the disclosure herein for other immunoassays to obtain
immunoassay-specific cutoff values for those other immunoassays based on this disclosure.
Whereas the precise value of the predetermined cutoff/level may vary between assays,
correlations as described herein (if any) should be generally applicable.
"Pretreatment reagent," e.g., lysis, precipitation and/or solubilization reagent, as used in a
diagnostic assay as described herein is one that lyses any cells and/or solubilizes any analyte that
is/are present in a test sample. Pretreatment is not necessary for all samples, as described further
herein. Among other things, solubilizing the analyte (e.g., polypeptide of interest) may entail
release of the analyte from any endogenous binding proteins present in the sample. A
pretreatment reagent may be homogeneous (not requiring a separation step) or heterogeneous
(requiring a separation step). With use of a heterogeneous pretreatment reagent there is removal
of any precipitated analyte binding proteins from the test sample prior to proceeding to the next
step of the assay.
"Quality control reagents" in the context of immunoassays and kits described herein,
include, but are not limited to, calibrators, controls, and sensitivity panels. A "calibrator" or
"standard" typically is used (e.g., one or more, such as a plurality) in order to establish calibration
(standard) curves for interpolation of the concentration of an analyte, such as an antibody or an
analyte. Alternatively, a single calibrator, which is near a predetermined positive/negative cutoff,
can be used. Multiple calibrators (i.e., more than one calibrator or a varying amount of
calibrator(s)) can be used in conjunction so as to comprise a "sensitivity panel."
"Risk" refers to the possibility or probability of a particular event occurring either
presently or at some point in the future. "Risk stratification" refers to an array of known clinical
risk factors that allows physicians to classify patients into a low, moderate, high or highest risk of
developing a particular disease, disorder or condition.
"Specific" and "specificity" in the context of an interaction between members of a
specific binding pair (e.g., an antigen (or fragment thereof) and an antibody (or antigenically
reactive fragment thereof)) refer to the selective reactivity of the interaction. The phrase
"specifically binds to" and analogous phrases refer to the ability of antibodies (or antigenically
reactive fragments thereof) to bind specifically to analyte (or a fragment thereof) and not bind
specifically to other entities.
"Specific binding partner" is a member of a specific binding pair. A specific binding pair
comprises two different molecules, which specifically bind to each other through chemical or
physical means. Therefore, in addition to antigen and antibody specific binding pairs of common
immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin),
carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules,
cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific
binding pairs can include members that are analogs of the original specific binding members, for
example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen
fragments, and antibodies, including monoclonal and polyclonal antibodies as well as complexes,
fragments, and variants (including fragments of variants) thereof, whether isolated or
recombinantly produced.
"Variant" as used herein means a polypeptide that differs from a given polypeptide (e.g.,
IL-18, BNP, NGAL or HIV polypeptide or anti-polypeptide antibody) in amino acid sequence by
the addition (e.g., insertion), deletion, or conservative substitution of amino acids, but that retains
the biological activity of the given polypeptide (e.g., a variant IL-18 can compete with anti-IL-18
antibody for binding to IL-18). A conservative substitution of an amino acid, i.e., replacing an
amino acid with a different amino acid of similar properties (e.g., hydrophilicity and degree and
distribution of charged regions) is recognized in the art as typically involving a minor change.
These minor changes can be identified, in part, by considering the hydropathic index of amino
acids, as understood in the art (see, e.g., Kyte et al. (1982) J. Mol. Biol. 157: 105-132). The
hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge.
It is known in the art that amino acids of similar hydropathic indexes can be substituted and still
retain protein function. In one aspect, amino acids having hydropathic indexes of ± 2 are
substituted. The hydrophilicity of amino acids also can be used to reveal substitutions that would
result in proteins retaining biological function. A consideration of the hydrophilicity of amino
acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of
that peptide, a useful measure that has been reported to correlate well with antigenicity and
immunogenicity (see, e.g., U.S. Patent No. 4,554,101. Substitution of amino acids having similar
hydrophilicity values can result in peptides retaining biological activity, for example
immunogenicity, as is understood in the art. In one aspect, substitutions are performed with
amino acids having hydrophilicity values within ± 2 of each other. Both the hydrophobicity index
and the hydrophilicity value of amino acids are influenced by the particular side chain of that
amino acid. Consistent with that observation, amino acid substitutions that are compatible with
biological function are understood to depend on the relative similarity of the amino acids, and
particularly the side chains of those amino acids, as revealed by the hydrophobicity,
hydrophilicity, charge, size, and other properties. "Variant" also can be used to describe a
polypeptide or fragment thereof that has been differentially processed, such as by proteolysis,
phosphorylation, or other post-translational modification, yet retains its biological activity or
antigen reactivity, e.g., the ability to bind to IL-18. Use of "variant" herein is intended to
encompass fragments of a variant unless otherwise contradicted by context.
I. Generation of DVD binding protein
The invention pertains to Dual Variable Domain binding proteins that bind one or more
targets and methods of making the same. In an embodiment, the binding protein comprises a
polypeptide chain, wherein said polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein
VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1
represents an amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1. The binding
protein of the invention can be generated using various techniques. The invention provides
expression vectors, host cell and methods of generating the binding protein.
A. Generation of parent monoclonal antibodies
The variable domains of the DVD binding protein can be obtained from parent antibodies,
including polyclonal and mAbs that bind antigens of interest. These antibodies may be naturally
occurring or may be generated by recombinant technology.
MAbs can be prepared using a wide variety of techniques known in the art including the
use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For
example, mAbs can be produced using hybridoma techniques including those known in the art
and taught, for example, in Harlow et al. (1988) Antibodies: A Laboratory Manual, (Cold Spring
Harbor Laboratory Press, 2nd ed.); Hammerling, et al. (1981) in: Monoclonal Antibodies and TCell
Hybridomas 563-681 (Elsevier, N.Y.). The term "monoclonal antibody" as used herein is not
limited to antibodies produced through hybridoma technology. The term "monoclonal antibody"
refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or
phage clone, and not the method by which it is produced. Hybridomas are selected, cloned and
further screened for desirable characteristics, including robust hybridoma growth, high antibody
production and desirable antibody characteristics, as discussed in Example 1 below. Hybridomas
may be cultured and expanded in vivo in syngeneic animals, in animals that lack an immune
system, e.g., nude mice, or in cell culture in vitro. Methods of selecting, cloning and expanding
hybridomas are well known to those of ordinary skill in the art. In a particular embodiment, the
hybridomas are mouse hybridomas. In another embodiment, the hybridomas are produced in a
non-human, non-mouse species such as rats, sheep, pigs, goats, cattle or horses. In another
embodiment, the hybridomas are human hybridomas, in which a human non-secretory myeloma is
fused with a human cell expressing an antibody that binds a specific antigen.
Recombinant mAbs are also generated from single, isolated lymphocytes using a
procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described
in U.S. Patent No. 5,627,052; PCT Publication No. WO 92/02551; and Babcock, J.S. et al. (1996)
Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method, single cells secreting antibodies of
interest, e.g., lymphocytes derived from an immunized animal, are identified, and, heavy- and
light-chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR.
These variable regions can then be expressed, in the context of appropriate immunoglobulin
constant regions (e.g., human constant regions), in mammalian host cells, such as COS or CHO
cells. The host cells transfected with the amplified immunoglobulin sequences, derived from in
vivo selected lymphocytes, can then undergo further analysis and selection in vitro, for example
by panning the transfected cells to isolate cells expressing antibodies to the antigen of interest.
The amplified immunoglobulin sequences further can be manipulated in vitro, such as by in vitro
affinity maturation methods such as those described in PCT Publication Nos. WO 97/29131 and
WO 00/56772.
Monoclonal antibodies are also produced by immunizing a non-human animal
comprising some, or all, of the human immunoglobulin locus with an antigen of interest. In an
embodiment, the non-human animal is a XENOMOUSE transgenic mouse, an engineered
mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient
in mouse antibody production. See, e.g., Green et al. (1994) Nature Genet. 7: 13-21 and U.S.
Patent Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598; and
6,130,364. See also PCT Publication Nos. WO 91/10741; WO 94/02602; WO 96/34096; WO
96/33735; WO 98/16654; WO 98/24893; WO 98/50433; WO 99/45031; WO 99/53049; WO 00/
09560; and WO 00/037504. The XENOMOUSE transgenic mouse produces an adult-like
human repertoire of fully human antibodies, and generates antigen-specific human monoclonal
antibodies. The XENOMOUSE transgenic mouse contains approximately 80% of the human
antibody repertoire through introduction of megabase sized, germline configuration YAC
fragments of the human heavy chain loci and x light chain loci. See Mendez et al. (1997) Nature
Genet. 15: 146-156; Green and Jakobovits (1998) J. Exp. Med. 188: 483-495.
In vitro methods also can be used to make the parent antibodies, wherein an antibody
library is screened to identify an antibody having the desired binding specificity. Methods for
such screening of recombinant antibody libraries are well known in the art and include methods
described in, for example, Ladner et al., U.S. Patent No. 5,223,409; PCT Publication Nos. WO
92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO
92/09690 and WO 97/29131; Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay et al. (1992)
Hum. Antibod. Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; McCafferty et
al. (1990) Nature 348: 552-554; Griffiths et al. (1993) EMBO J. 12: 725-734; Hawkins et al.
(1992) J. Mol. Biol. 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al.
(1992) Proc. Natl. Acad. Sci. USA 89: 3576-3580; Garrad et al. (1991) Bio/Technology 9: 1373-
1377; Hoogenboom et al. (1991) Nucl. Acid Res. 19: 4133-4137; and Barbas et al. (1991) Proc.
Natl. Acad. Sci. USA 88: 7978-7982, and U.S. Patent Publication No. 2003/0186374.
Parent antibodies of the present invention can also be generated using various phage
display methods known in the art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the polynucleotide sequences encoding
them. In a particular, such phage can be utilized to display antigen-binding domains expressed
from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an
antigen binding domain that binds the antigen of interest can be selected or identified with
antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage
used in these methods are typically filamentous phage including fd and M13 binding domains
expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly
fused to either the phage gene III or gene VIII protein. Examples of phage display methods that
can be used to make the antibodies of the present invention include those disclosed in Brinkman
et al. (1995) J. Immunol. Methods 182: 41-50; Ames et al. (1995) J. Immunol. Methods 184: 177-
186; Kettleborough et al. (1994) Eur. J. Immunol. 24: 952-958; Persic et al. (1997) Gene 187: 9-
18; Burton et al. (1994) Advances in Immunol. 57: 191-280; PCT Application No.
PCT/GB91/01134; PCT Publication Nos. WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; and WO 95/20401; and U.S. Patent Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.
As described in the herein references, after phage selection, the antibody coding regions
from the phage can be isolated and used to generate whole antibodies including human antibodies
or any other desired antigen binding fragment, and expressed in any desired host, including
mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
For example, techniques to produce recombinantly Fab, Fab' and F(ab')2 fragments can also be
employed using methods known in the art such as those disclosed in PCT Publication No. WO
92/22324; Mullinax et al. (1992) BioTechniques 12(6): 864-869; Sawai et al. (1995) AJRI 34: 26-
34; and Better et al. (1988) Science 240: 1041-1043. Examples of techniques, which can be used
to produce single-chain Fvs and antibodies, include those described in U.S. Patent Nos. 4,946,778
and 5,258,498; Huston et al. (1991), Methods Enzymol. 203:46-88; Shu et al. (1993) Proc. Natl.
Acad. Sci. USA 90: 7995-7999; and Skerra et al. (1988) Science 240: 1038-1040.
Alternative to screening of recombinant antibody libraries by phage display, other
methodologies known in the art for screening large combinatorial libraries can be applied to the
identification of parent antibodies. One type of alternative expression system is one in which the
recombinant antibody library is expressed as RNA-protein fusions, as described in PCT
Publication No. WO 98/31700 and in Roberts, R.W. and Szostak, J.W. (1997) Proc. Natl. Acad.
Sci. USA 94:12297-12302. In this system, a covalent fusion is created between an mRNA and the
peptide or protein that it encodes by in vitro translation of synthetic mRN As that carry puromycin,
a peptidyl acceptor antibiotic, at their 3' end. Thus, a specific mRNA can be enriched from a
complex mixture of mRN As {e.g., a combinatorial library) based on the properties of the encoded
peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion
thereof, to the dual specificity antigen. Nucleic acid sequences encoding antibodies, or portions
thereof, recovered from screening of such libraries can be expressed by recombinant means as
described herein (e.g., in mammalian host cells) and, moreover, can be subjected to further
affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which
mutations have been introduced into the originally selected sequence(s), or by other methods for
affinity maturation in vitro of recombinant antibodies, as described herein.
In another approach the parent antibodies can also be generated using yeast display
methods known in the art. In yeast display methods, genetic methods are used to tether antibody
domains to the yeast cell wall and display them on the surface of yeast. In particular, such yeast
can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial
antibody library (e.g., human or murine). Examples of yeast display methods that can be used to
make the parent antibodies include those disclosed in U.S. Patent No. 6,699,658.
The antibodies described herein can be further modified to generate CDR grafted and
humanized parent antibodies. CDR-grafted parent antibodies comprise heavy and light chain
variable region sequences from a human antibody wherein one or more of the CDR regions of VH
and/or VL are replaced with CDR sequences of murine antibodies that bind an antigen of interest.
A framework sequence from any human antibody may serve as the template for CDR grafting.
However, straight chain replacement onto such a framework often leads to some loss of binding
affinity to the antigen. The more homologous a human antibody is to the original murine
antibody, the less likely the possibility that combining the murine CDRs with the human
framework will introduce distortions in the CDRs that could reduce affinity. Therefore, in an
embodiment, the human variable framework that is chosen to replace the murine variable
framework apart from the CDRs have at least a 65% sequence identity with the murine antibody
variable region framework. In an embodiment, the human and murine variable regions apart from
the CDRs have at least 70% sequence identify. In a particular embodiment, that the human and
murine variable regions apart from the CDRs have at least 75% sequence identity. In another
embodiment, the human and murine variable regions apart from the CDRs have at least 80%
sequence identity. Methods for producing such antibodies are known in the art (see EP 239,400;
PCT Publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089),
veneering or resurfacing (EP 592,106; EP 519,596; Padlan (1991) Mol. Immunol. 28(4/5): 489-
498; Studnicka et al. (1994) Prot. Engineer. 7(6): 805-814; and Roguska et al. (1994) Proc. Acad.
Sci. USA 91: 969-973), chain shuffling (U.S. Patent No. 5,565,352), and anti-idiotypic antibodies.
Humanized antibodies are antibody molecules from non-human species that bind the
desired antigen and have one or more CDRs from the non-human species and framework regions
from a human immunoglobulin molecule. Known human Ig sequences are disclosed, e.g.,
www.ncbi.nlm.nih.gov/entrez- /query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/;
www.abcam.com/; www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/.about.pedro/research_tools.html; www.mgen.uniheidelberg.
de/SD/IT/IT.html; www.whfreeman.com/immunology/CH-05/kuby05.htm;
www.library.thinkquest.org/12429/Immune/Antibody.html;
www.hhmi.org/grants/lectures/1996/vlab/; www.path.cam.ac.uk/.about.mrc7/m- ikeimages.html;
www.antibodyresource.com/; mcb.harvard.edu/BioLinks/Immunology.
html.www.immunologylink.com/; pafhbox.wustl.edu/.about.hcenter/index.- html;
www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html-;
www.nal.usda.gov/awic/pubs/antibody/; www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;
www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin- ks.html;
www.biotech.ufl.edu/.about.fccl/protocol.html; www.isac-net.org/sites_geo.html; aximtl.imt.unimarburg.
de/.about.rek/AEP- Starthtml; baserv.uci.kun.nl/.about.jraats/linksl.html;
www.recab.uni-hd.de/immuno.bme.nwu.edu/; www.mrc-cpe.cam.ac.uk/imt-doc/public/
INTRO.html; www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;
www.biochem.ucl.ac.uk/.about.martin/abs/index.html; antibody.bath.ac.uk/;
abgen.cvm.tamu.edu/lab/wwwabgen.html; www.unizh.ch/.about.honegger/AHOseminar/
SlideO 1 .html; www.cryst.bbk.ac.uk/.about.ubcg07s/;
www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; www.path.cam.ac.uk/.about.mrc7/humanisation/
TAHHP.html; www.ibt.unam.mx/vir/structure/stat_aim.html;
www.biosci.missouri.edu/smithgp/index.html; www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Webpages/
Pept/spottech.html; www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html.; and
Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983). Such
imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding,
affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as
known in the art.
Framework residues in the human framework regions may be substituted with the
corresponding residue from the CDR donor antibody to alter, e.g., improve, antigen binding.
These framework substitutions are identified by methods well known in the art, e.g., by modeling
of the interactions of the CDR and framework residues to identify framework residues important
for antigen binding and sequence comparison to identify unusual framework residues at particular
positions. (See, e.g., U.S. Patent No. 5,585,089; Riechmann et al. (1988) Nature 332:323). Threedimensional
immunoglobulin models are commonly available and are familiar to those skilled in
the art. Computer programs are available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin sequences. Inspection of these
displays permits analysis of the likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from
the consensus and import sequences so that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most
substantially involved in influencing antigen binding. Antibodies can be humanized using a
variety of techniques known in the art, such as but not limited to those described in Jones et al.
(1986) Nature 321: 522; Verhoeyen et al. (1988) Science 239: 1534; Sims et al. (1993) J.
Immunol. 151: 2296; Chothia and Lesk (1987) J. Mol. Biol. 196: 901; Carter et al. (1992) Proc.
Natl. Acad. Sci. USA 89: 4285; Presta et al. (1993) J. Immunol. 151: 2623; Padlan (1991) Mol.
Immunol. 28(4/5): 489-498; Studnicka et al. (1994) Prot. Engineer. 7(6): 805-814; Roguska et al.,
(1994) Proc. Natl. Acad. Sci. USA 91: 969-973; PCT Publication No. WO 91/09967:
US98/16280; US96/18978; US91/09630; US91/05939; US94/01234; GB89/01334; GB91/01134;
GB92/01755; WO90/14443; WO90/14424; and WO90/14430; European Patent Publication Nos.
EP 229246; EP 592,106; EP 519,596; and EP 239,400; and U.S. Patent Nos. 5,565,332;
5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886;
5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.
B. Criteria for selecting parent monoclonal antibodies
An embodiment of the invention pertains to selecting parent antibodies with at least one
or more properties desired in the DVD-Ig molecule. In an embodiment, the desired property is
selected from one or more antibody parameters. In another embodiment, the antibody parameters
are selected from the group consisting of antigen specificity, affinity to antigen, potency,
biological function, epitope recognition, stability, solubility, production efficiency,
immunogenicity, pharmacokinetics, bioavailability, tissue cross reactivity, and orthologous
antigen binding.
Bl. Affinity to Antigen
The desired affinity of a therapeutic mAb may depend upon the nature of the antigen, and
the desired therapeutic end-point. In an embodiment, monoclonal antibodies have higher
affinities (Kd = 0.01 - 0.50 pM) when blocking a cytokine-cytokine receptor interaction as such
interactions are usually high affinity interactions (e.g., nM range) could be therapeutically
effective e.g.,in clearing circulating potentially pathogenic proteins e.g.,monoclonal antibodies
that bind to, sequester, and clear circulating species of A-p amyloid. In other instances, reducing
the affinity of an existing high affinity mAb by site-directed mutagenesis or using a mAb with
lower affinity for its target could be used to avoid potential side-effects e.g.,a high affinity mAb
may sequester/neutralize all of its intended target, thereby completely depleting/eliminating the
function(s) of the targeted protein. In this scenario, a low affinity mAb may sequester/neutralize a
fraction of the target that may be responsible for the disease symptoms (the pathological or overproduced
levels), thus allowing a fraction of the target to continue to perform its normal
physiological function(s). Therefore, it may be possible to reduce the Kd to adjust dose and/or
reduce side-effects. The affinity of the parental mAb might play a role in appropriately targeting
cell surface molecules to achieve desired therapeutic out-come. For example, if a target is
expressed on cancer cells with high density and on normal cells with low density, a lower affinity
mAb will bind a greater number of targets on tumor cells than normal cells, resulting in tumor cell
elimination via ADCC or CDC, and therefore might have therapeutically desirable effects. Thus
selecting a mAb with desired affinity may be relevant for both soluble and surface targets.
Signaling through a receptor upon interaction with its ligand may depend upon the
affinity of the receptor-ligand interaction. Similarly, it is conceivable that the affinity of a mAb
for a surface receptor could determine the nature of intracellular signaling and whether the mAb
may deliver an agonist or an antagonist signal. The affinity-based nature of mAb-mediated
signaling may have an impact of its side-effect profile. Therefore, the desired affinity and desired
functions of therapeutic monoclonal antibodies need to be determined carefully by in vitro and in
vivo experimentation.
The desired Kd of a binding protein (e.g., an antibody) may be determined experimentally
depending on the desired therapeutic outcome. In an embodiment parent antibodies with affinity
(Kd) for a particular antigen equal to, or better than, the desired affinity of the DVD-Ig for the
same antigen are selected. The parent antibodies for a given DVD-Ig molecule can be the same
antibody or different antibodies. The antigen binding affinity and kinetics are assessed by
Biacore or another similar technique. In one embodiment, each parent antibody has a dissociation
constant (Kd) to its antigen selected from the group consisting of: at most about 10-7 M; at most
about 10-8 M; at most about 10"9 M; at most about 10-10 M; at most about 10-11 M; at most about
10-12 M; and at most 10-13 M. The first parent antibody from which VD1 is obtained and the
second parent antibody from which VD2 is obtained may have similar or different affinity (KD)
for the respective antigen. Each parent antibody has an on rate constant (Kon) to the antigen
selected from the group consisting of: at least about 102M-1s-1; at least about 103M-1s-1; at least
about 104M-1s-1; at least about 105M-1s-1; and at least about 106M-1s-1, as measured by surface
plasmon resonance. The first parent antibody from which VD1 is obtained and the second parent
antibody from which VD2 is obtained may have similar or different on rate constant (Kon) for the
respective antigen. In one embodiment, each parent antibody has an off rate constant (Koff) to
the antigen selected from the group consisting of: at most about 10-3s-1; at most about 10-4-s-1; at
most about 10-s-1; and at most about 10-6s-1, as measured by surface plasmon resonance. The first
parent antibody from which VD1 is obtained and the second parent antibody from which VD2 is
obtained may have similar or different off rate constants (Koff) for the respective antigen.
B2. Potency
The desired affinity/potency of parental monoclonal antibodies will depend on the desired
therapeutic outcome. For example, for receptor-ligand (R-L) interactions the affinity (kd) is equal
to or better than the R-L kd (pM range). For simple clearance of a pathologic circulating protein,
the kd could be in low nM range e.g.,clearance of various species of circulating A-P peptide. In
addition, the kd will also depend on whether the target expresses multiple copies of the same
epitope e.g., a mAb targeting conformational epitope in A0 oligomers.
Where VDI and VD2 bind the same antigen, but distint epitopes, the DVD-Ig will contain
four binding sites for the same antigen, thus increasing avidity and thereby the apparent kd of the
DVD-Ig. In an embodiment, parent antibodies with equal or lower kd than that desired in the
DVD-Ig are chosen. The affinity considerations of a parental mAb may also depend upon
whether the DVD-Ig contains four or more identical antigen binding sites (i.e., a DVD-Ig from a
single mAb). In this case, the apparent kd would be greater than the mAb due to avidity. Such
DVD-Igs can be employed for cross-linking surface receptor, increase neutralization potency,
enhance clearance of pathological proteins, etc.
In an embodiment parent antibodies with neutralization potency for specific antigen equal
to or better than the desired neutralization potential of the DVD-Ig for the same antigen are
selected. The neutralization potency can be assessed by a target-dependent bioassay where cells
of appropriate type produce a measurable signal (i.e., proliferation or cytokine production) in
response to target stimulation, and target neutralization by the mAb can reduce the signal in a
dose-dependent manner.
B3. Biological functions
Monoclonal antibodies can perform potentially several functions. Some of these functions
are listed in Table 1. These functions can be assessed by both in vitro assays (e.g., cell-based and
biochemical assays) and in vivo animal models.
Table 1: Some Potential Applications For Therapeutic Antibodies
MAbs with distinct functions described in the examples herein in Table 1 can be selected
to achieve desired therapeutic outcomes. Two or more selected parent monoclonal antibodies can
then be used in DVD-Ig format to achieve two distinct functions in a single DVD-Ig molecule.
For example, a DVD-Ig can be generated by selecting a parent mAb that neutralizes function of a
specific cytokine, and selecting a parent mAb that enhances clearance of a pathological protein.
Similarly, two parent monoclonal antibodies that recognize two different cell surface receptors
can be selected, e.g., one mAb with an agonist function on one receptor and the other mAb with
an antagonist function on a different receptor. These two selected monoclonal antibodies each
with a distinct function, can be used to construct a single DVD-Ig molecule that will possess the
two distinct functions (agonist and antagonist) of the selected monoclonal antibodies in a single
molecule. Similarly, two antagonistic monoclonal antibodies to cell surface receptors each
blocking binding of respective receptor ligands (e.g., EGF and IGF), can be used in a DVD-Ig
format. Conversely, an antagonistic anti-receptor mAb (e.g., anti-EGFR) and a neutralizing antisoluble
mediator (e.g., anti-IGFl/2) mAb can be selected to make a DVD-Ig.
B4. Epitope Recognition:
Different regions of proteins may perform different functions. For example specific
regions of a cytokine interact with the cytokine receptor to bring about receptor activation
whereas other regions of the protein may be required for stabilizing the cytokine. In this instance
one may select a mAb that binds specifically to the receptor interacting region(s) on the cytokine
and thereby blocks cytokine-receptor interaction. In some cases, for example, certain chemokine
receptors that bind multiple ligands, a mAb that binds to the epitope (region on chemokine
receptor) that interacts with only one ligand can be selected. In other instances, monoclonal
antibodies can bind to epitopes on a target that are not directly responsible for physiological
functions of the protein, but binding of a mAb to these regions could either interfere with
physiological functions (steric hindrance) or alter the conformation of the protein such that the
protein cannot function (mAb to receptors with multiple ligand which alter the receptor
conformation such that none of the ligand can bind). Anti-cytokine monoclonal antibodies that do
not block binding of the cytokine to its receptor, but block signal transduction have also been
identified (e.g., 125-2H, an anti-IL-18 mAb).
Examples of epitopes and mAb functions include, but are not limited to, blocking
Receptor-Ligand (R-L) interaction (neutralizing mAb that binds R-interacting site); steric
hindrance resulting in diminished or no R-binding. An Ab can bind the target at a site other than
a receptor binding site, but still interfere with receptor binding and functions of the target by
inducing conformational change and eliminating function (e.g., Xolair), e.g., binding to R but
blocking signaling (125-2H).
In an embodiment, the parental mAb needs to target the appropriate epitope for maximum
efficacy. Such epitope should be conserved in the DVD-Ig. The binding epitope of a mAb can be
determined by several approaches, including co-crystallography, limited proteolysis of mAbantigen
complex plus mass spectrometric peptide mapping (Legros V. et al. (2000) Protein Sci.
9:1002-10), phage displayed peptide libraries (O'Connor, K.H. et al. (2005) J. Immunol. Methods.
299:21-35), as well as mutagenesis (Wu C. et al. (2003) J. Immunol. 170:5571-7).
B5. Physicochemical and pharmaceutical properties:
Therapeutic treatment with antibodies often requires administration of high doses, often
several mg/kg (due to a low potency on a mass basis as a consequence of a typically large
molecular weight). In order to accommodate patient compliance and to address adequately
chronic disease therapies and outpatient treatment, subcutaneous (s.c.) or intramuscular (i.m.)
administration of therapeutic mAbs is desirable. For example, the maximum desirable volume for
s.c. administration is~1.0mL, and therefore, concentrations of >100 mg/mL are desirable to limit
the number of injections per dose. In an embodiment, the therapeutic antibody is administered in
one dose. The development of such formulations is constrained, however, by protein-protein
interactions (e.g., aggregation, which potentially increases immunogenicity risks) and by
limitations during processing and delivery (e.g., viscosity). Consequently, the large quantities
required for clinical efficacy and the associated development constraints limit full exploitation of
the potential of antibody formulation and s.c. administration in high-dose regimens. It is apparent
that the physicochemical and pharmaceutical properties of a protein molecule and the protein
solution are of utmost importance, e.g., stability, solubility and viscosity features.
BS.l. Stability:
A "stable" antibody formulation is one in which the antibody therein essentially retains its
physical stability and/or chemical stability and/or biological activity upon storage. Stability can be
measured at a selected temperature for a selected time period. In an embodiment,, the antibody in
the formulation is stable at room temperature (about 30°C) or at 40°C for at least 1 month and/or
stable at about 2-8°C. for at least 1 year, such as, for at least 2 years. Furthermore, in an
embodiment, the formulation is stable following freezing (to, e.g., -70°C) and thawing of the
formulation, hereinafter referred to as a "freeze/thaw cycle." In another example, a "stable"
formulation may be one wherein less than about 10% and less than about 5% of the protein is
present as an aggregate in the formulation.
A DVD-Ig stable that is in vitro at various temperatures for an extended time period is
desirable. One can achieve this by rapid screening of parental mAbs that are stable in vitro at
elevated temperature, e.g., at 40°C for 2-4 weeks, and then assess stability. During storage at 2-
8DC, the protein reveals stability for at least 12 months, e.g., at least 24 months. Stability (% of
monomeric, intact molecule) can be assessed using various techniques such as cation exchange
chromatography, size exclusion chromatography, SDS-PAGE, as well as bioactivity testing. For a
more comprehensive list of analytical techniques that may be employed to analyze covalent and
conformational modifications see Jones, A. J. S. (1993) Analytical methods for the assessment of
protein formulations and delivery systems. In: Cleland, J. L.; Langer, R., editors. Formulation and
delivery of peptides and proteins, la edition, Washington, ACS, pg. 22-45; and Pearlman, R.;
Nguyen, T. H.(1990) Analysis of protein drugs. In: Lee, V. H., editor. Peptide and protein drug
delivery, 1st edition, New York, Marcel Dekker, Inc., pg. 247-301.
Heterogeneity and aggregate formation: stability of the antibody may be such that the
formulation may reveal less than about 10%, such as less than about 5%, such as less than about
2%, or, within the range of 0.5% to 1.5% or less in the GMP antibody material that is present as
aggregate. Size exclusion chromatography is a method that is sensitive, reproducible, and very
robust in the detection of protein aggregates.
In addition to low aggregate levels, the antibody must, in an embodiment, be chemically
stable. Chemical stability may be determined by ion exchange chromatography (e.g., cation or
anion exchange chromatography), hydrophobic interaction chromatography, or other methods
such as isoelectric focusing or capillary electrophoresis. For instance, chemical stability of the
antibody may be such that after storage of at least 12 months at 2-8°C the peak representing
unmodified antibody in a cation exchange chromatography may increase not more than 20%, such
as not more than 10%, or not more than 5% as compared to the antibody solution prior to storage
testing.
In an embodiment, the parent antibodies display structural integrity; correct disulfide
bond formation, and correct folding. Chemical instability due to changes in secondary or tertiary
structure of an antibody may impact antibody activity. For instance, stability, as indicated by
activity of the antibody may be such that after storage of at least 12 months at 2-8°C, the activity
of the antibody may decrease not more than 50%, such as not more than 30%, not more than 10%,
or not more than 5% or 1% as compared to the antibody solution prior to storage testing. Suitable
antigen-binding assays can be employed to determine antibody activity.
B5.2. Solubility:
The "solubility" of a mAb correlates with the production of correctly folded, monomelic
IgG. The solubility of the IgG may therefore be assessed by HPLC. For example, soluble
(monomeric) IgG will give rise to a single peak on the HPLC chromatograph, whereas insoluble
(e.g., multimeric and aggregated) will give rise to a plurality of peaks. A person skilled in the art
will therefore be able to detect an increase or decrease in solubility of an IgG using routine HPLC
techniques. For a more comprehensive list of analytical techniques that may be employed to
analyze solubility (see Jones, A. G. (1993) Dep. Chem. Biochem. Eng., Univ. Coll. London,
London, UK. Editor(s): Shamlou, P. Ayazi. Process. Solid-Liq. Suspensions, 93-117. Publisher:
Butterworth-Heinemann, Oxford, UK and Peariman et al. (1990) Adv. in Parenteral Sci. 4 (Pept.
Protein Drug Delivery): 247-301). Solubility of a therapeutic mAb is critical for formulating to
high concentration often required for adequate dosing. As outlined herein, solubilities of
>100 mg/mL may be required to accommodate efficient antibody dosing. For instance, antibody
solubility may be not less than about 5 mg/mL in early research phase, not less than about 25
mg/mL in advanced process science stages, or not less than about 100 mg/mL, or not less than
about 150 mg/mL. It is obvious to a person skilled in the art that the intrinsic properties of a
protein molecule are important the physico-chemical properties of the protein solution, e.g.,
stability, solubility, viscosity. However, a person skilled in the art will appreciate that a broad
variety of excipients exist that may be used as additives to beneficially impact the characteristics
of the final protein formulation. These excipients may include: (i) liquid solvents, cosolvents (e.g.,
alcohols such as ethanol); (ii) buffering agents (e.g., phosphate, acetate, citrate, and amino acid
buffers); (iii) sugars or sugar alcohols (e.g., sucrose, trehalose, fructose, raffmose, mannitol,
sorbitol, and dextrans); (iv) surfactants (e.g., polysorbate 20,40, 60, 80, and poloxamers); (v)
isotonicity modifiers (e.g., salts such as NaCl, sugars, and sugar alcohols); and (vi) others (e.g.,
preservatives, chelating agents, antioxidants, chelating substances (e.g., EDTA), biodegradable
polymers, and carrier molecules (e.g., HSA and PEGs)).
Viscosity is a parameter of high importance with regard to antibody manufacture and
antibody processing (e.g.,diafiltration/ultrafiltration), fill-finish processes (pumping aspects,
filtration aspects) and delivery aspects (syringeability, sophisticated device delivery). Low
viscosities enable the liquid solution of the antibody having a higher concentration. This enables
the same dose may be administered in smaller volumes. Small injection volumes inhere the
advantage of lower pain on injection sensations, and the solutions not necessarily have to be
isotonic to reduce pain on injection in the patient. The viscosity of the antibody solution may be
such that at shear rates of 100 (1/s) antibody solution viscosity is below 200 mPa s, below 125
mPa s, below 70 mPa s, and below 25 mPa s, or even below 10 mPa s.
B S3. Production efficiency
The generation of a DVD-Ig that is efficiently expressed in mammalian cells, such as
Chinese hamster ovary cells (CHO), will in an embodiment require two parental monoclonal
antibodies, which are themselves expressed efficiently in mammalian cells. The production yield
from a stable mammalian line (i.e., CHO) should be above about 0.5g/L, above about lg/L, or in
the range of about 2 to about 5 g/L or more (Kipriyanov, S.M. and Little, M. (1999) Mol.
Biotechnol. 12:173-201; Carroll, S. and Al-Rubeai, M. (2004) Expert Opin. Biol. Ther. 4: 1821-
9).
Production of antibodies and Ig fusion proteins in mammalian cells is influenced by
several factors. Engineering of the expression vector via incorporation of strong promoters,
enhancers and selection markers can maximize transcription of the gene of interest from an
integrated vector copy. The identification of vector integration sites that are permissive for high
levels of gene transcription can augment protein expression from a vector (Wurm et al. (2004)
Nature Biotechnol. 22(11): 1393-1398). Furthermore, levels of production are affected by the
ratio of antibody heavy and light chains and various steps in the process of protein assembly and
secretion (Jiang et al. (2006) Biotechnol. Prog. 22(1): 313-8).
B 6. Immunogenicity
Administration of a therapeutic mAb may result in certain incidence of an immune
response (i.e., the formation of endogenous antibodies directed against the therapeutic mAb).
Potential elements that might induce immunogenicity should be analyzed during selection of the
parental monoclonal antibodies, and steps to reduce such risk can be taken to optimize the
parental monoclonal antibodies prior to DVD-Ig construction. Mouse-derived antibodies have
been found to be highly immunogenic in patients. The generation of chimeric antibodies
comprised of mouse variable and human constant regions presents a logical next step to reduce
the immunogenicity of therapeutic antibodies (Morrison and Schlom, 1990). Alternatively,
immunogenicity can be reduced by transferring murine CDR sequences into a human antibody
framework (reshaping/CDR grafting/humanization), as described for a therapeutic antibody by
Riechmann et al. (1988) Nature 332: 323-327. Another method is referred to as "resurfacing" or
"veneering," starting with the rodent variable light and heavy domains, only surface-accessible
framework amino acids are altered to human ones, while the CDR and buried amino acids remain
from the parental rodent antibody (Roguska et al. (1996) Prot. Engineer 9: 895-904). In another
type of humanization, instead of grafting the entire CDRs, one technique grafts only the
"specificity-determining regions" (SDRs), defined as the subset of CDR residues that are involved
in binding of the antibody to its target (Kashmiri et al. (2005) Methods 36(1): 25-34). This
necessitates identification of the SDRs either through analysis of available three-dimensional
structures of antibody-target complexes or mutational analysis of the antibody CDR residues to
determine which interact with the target. Alternatively, fully human antibodies may have reduced
immunogenicity compared to murine, chimeric or humanized antibodies.
Another approach to reduce the immunogenicity of therapeutic antibodies is the
elimination of certain specific sequences that are predicted to be immunogenic. In one approach,
after a first generation biologic has been tested in humans and found to be unacceptably
immunogenic, the B-cell epitopes can be mapped and then altered to avoid immune detection.
Another approach uses methods to predict and remove potential T-cell epitopes. Computational
methods have been developed to scan and to identify the peptide sequences of biologic
therapeutics with the potential to bind to MHC proteins (Desmet et al. (2005) Proteins 58: 53-69).
Alternatively a human dendritic cell-based method can be used to identify CD4+ T-cell epitopes in
potential protein allergens (Stickler et al. (2000) J. Immunother. 23: 654-60; S.L. Morrison and J.
Schlom, (1990) Important Adv. Oncol. 3-18; Riechmann et al. (1988) Nature 332: 323-327;
Roguska et al. (1996) Protein Engineer. 9: 895-904; Kashmiri et al. (2005) Methods 36(1): 25-34;
Desmet et al. (2005) Proteins 58: 53-69; and Stickler et al. (2000) J. Immunotherapy 23: 654-60.)
B 7. In vivo efficacy
To generate a DVD-Ig molecule with desired in vivo efficacy, it is important to generate
and select mAbs with similarly desired in vivo efficacy when given in combination. However, in
some instances the DVD-Ig may exhibit in vivo efficacy that cannot be achieved with the
combination of two separate mAbs. For instance, a DVD-Ig may bring two targets in close
proximity leading to an activity that cannot be achieved with the combination of two separate
mAbs. Additional desirable biological functions are described herein in section B 3. Parent
antibodies with characteristics desirable in the DVD-Ig molecule may be selected based on factors
such as pharmacokinetic t lA; tissue distribution; soluble versus cell surface targets; and target
concentration- soluble/density -surface.
B 8. In vivo tissue distribution
To generate a DVD-Ig molecule with desired in vivo tissue distribution, in an
embodiment parent mAbs with similar desired in vivo tissue distribution profile must be selected.
In this regard, the parent mAbs can be the same antibody or different antibodies. Alternatively,
based on the mechanism of the dual-specific targeting strategy, it may at other times not be
required to select parent mAbs with the similarly desired in vivo tissue distribution when given in
combination, (e.g., in the case of a DVD-Ig in which one binding component targets the DVD-Ig
to a specific site thereby bringing the second binding component to the same target site). For
example, one binding specificity of a DVD-Ig could target pancreas (islet cells) and the other
specificity could bring GLP1 to the pancreas to induce insulin.
B 9. Isotype:
To generate a DVD-Ig molecule with desired properties including, but not limited to,
isotype, effector functions, and the circulating half-life, in an embodiment parent mAbs with
appropriate Fc-effector functions depending on the therapeutic utility and the desired therapeutic
end-point are selected. The parent mAbs can be the same antibody or different antibodies. There
are five main heavy-chain classes or isotypes, some of which have several sub-types, and these
determine the effector functions of an antibody molecule. These effector functions reside in the
hinge region, CH2 and CH3 domains of the antibody molecule. However, residues in other parts
of an antibody molecule may have effects on effector functions as well. The hinge region Fceffector
functions include: (i) antibody-dependent cellular cytotoxicity, (ii) complement (Clq)
binding, activation and complement-dependent cytotoxicity (CDC), (iii) phagocytosis/clearance
of antigen-antibody complexes, and (iv) cytokine release in some instances. These Fc-effector
functions of an antibody molecule are mediated through the interaction of the Fc-region with a set
of class-specific cell surface receptors. Antibodies of the IgGl isotype are most active while IgG2
and IgG4 having minimal or no effector functions. The effector functions of the IgG antibodies
are mediated through interactions with three structurally homologous cellular Fc receptor types
(and sub-types) (FcgRl, FcgRII and FcgRIII). These effector functions of an IgGl can be
eliminated by mutating specific amino acid residues in the lower hinge region (e.g., L234A,
L235A) that are required for FcgR and Clq binding. Amino acid residues in the Fc region, in
particular the CH2-CH3 domains, also determine the circulating half-life of the antibody
molecule. This Fc function is mediated through the binding of the Fc-region to the neonatal Fc
receptor (FcRn), which is responsible for recycling of antibody molecules from the acidic
lysosomes back to the general circulation.
Whether a mAb should have an active or an inactive isotype will depend on the desired
therapeutic end-point for an antibody. Some examples of usage of isotypes and desired
therapeutic outcome are listed below:
a) If the desired end-point is functional neutralization of a soluble cytokine, then an inactive
isotype may be used;
b) If the desired out-come is clearance of a pathological protein, an active isotype may be
used;
c) If the desired out-come is clearance of protein aggregates, an active isotype may be used;
d) If the desired outcome is to antagonize a surface receptor, an inactive isotype is used
(Tysabri, IgG4; OKT3, mutated IgGl);
e) If the desired outcome is to eliminate target cells, an active isotype is used (Herceptin,
IgGl (and with enhanced effector functions); and
f) If the desired outcome is to clear proteins from circulation without entering the CNS, an
IgM isotype may be used (e.g., clearing circulating Ab peptide species).
The Fc effector functions of a parental mAb can be determined by various in vitro methods well
known in the art.
As discussed, the selection of isotype, and thereby the effector functions will depend upon
the desired therapeutic end-point. In cases where simple neutralization of a circulating target is
desired, for example, blocking receptor-ligand interactions, the effector functions may not be
required. In such instances isotypes or mutations in the Fc-region of an antibody that eliminate
effector functions are desirable. In other instances, where elimination of target cells is the
therapeutic end-point, for example, elimination of tumor cells, isotypes or mutations or defucosylation
in the Fc-region that enhance effector functions are desirable (Presta, G.L. (2006)
Adv. Drug Deliv. Rev. 58:640-656 and Satoh, M. et al. (2006) Expert Opin. Biol. Ther. 6: 1161-
1173). Similarly, depending up on the therapeutic utility, the circulating half-life of an antibody
molecule can be reduced/prolonged by modulating antibody-FcRn interactions by introducing
specific mutations in the Fc region (Dall'Acqua, W.F. et al. (2006) J. Biol. Chem. 281: 23514-
23524; Petkova, S.B. (2006) et al., Intemat. Immunol. 18:1759-1769; Vaccaro, C. et al. (2007)
Proc. Natl. Acad. Sci. USA 103: 18709-18714).
The published information on the various residues that influence the different effector
functions of a normal therapeutic mAb may need to be confirmed for DVD-Ig. It may be possible
that in a DVD-Ig format additional (different) Fc-region residues, other than those identified for
the modulation of monoclonal antibody effector functions, may be important.
Overall, the decision as to which Fc-effector functions (isotype) will be critical in the
final DVD-Ig format will depend upon the disease indication, therapeutic target, and desired
therapeutic end-point and safety considerations. Listed below are exemplary appropriate heavy
chain and light chain constant regions including, but not limited to:
o IgG 1 - allotype: G1 mz
o IgG 1 mutant - A234, A235
o IgG2 - allotype: G2m(n-)
o Kappa - Km3
o Lambda
Fc Receptor and Clq Studies: The possibility of unwanted antibody-dependent cellmediated
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by antibody
complexing to any overexpressed target on cell membranes can be abrogated by the (for example,
L234A, L235A) hinge-region mutations. These substituted amino acids, present in the IgGl
hinge region of mAb, are expected to result in diminished binding of mAb to human Fc receptors
(but not FcRn), as FcgR binding is thought to occur within overlapping sites on the IgGl hinge
region. This feature of mAb may lead to an improved safety profile over antibodies containing a
wild-type IgG. Binding of mAb to human Fc receptors can be determined by flow cytometry
experiments using cell lines (e.g.,THP-l, K562) and an engineered CHO cell line that expresses
FcgRIIb (or other FcgRs). Compared to IgGl control monoclonal antibodies, mAb show reduced
binding to FcgRI and FcgRIIa, whereas binding to FcgRIIb is unaffected. The binding and
activation of Clq by antigen/IgG immune complexes triggers the classical complement cascade
with consequent inflammatory and/or immunoregulatory responses. The Clq binding site on
IgGs has been localized to residues within the IgG hinge region. Clq binding to increasing
concentrations of mAb was assessed by Clq ELISA. The results demonstrate that mAb is unable
to bind to Clq, as expected when compared to the binding of a wildtype control IgGl. Overall,
the L234A, L235A hinge region mutation abolishes binding of mAb to FcgRI, FcgRIIa and Clq
but does not impact the interaction of mAb with FcgRIIb. These data suggest that in vivo, mAb
with mutant Fc will interact normally with the inhibitory FcgRIIb but will likely fail to interact
with the activating FcgRI and FcgRIIa receptors or Clq.
Human FcRn binding: The neonatal receptor (FcRn) is responsible for transport of IgG
across the placenta and to control the catabolic half-life of the IgG molecules. It might be
desirable to increase the terminal half-life of an antibody to improve efficacy, to reduce the dose
or frequency of administration, or to improve localization to the target. Alternatively, it might be
advantageous to do the converse that is, to decrease the terminal half-life of an antibody to reduce
whole body exposure or to improve the target-to-non-target binding ratios. Tailoring the
interaction between IgG and its salvage receptor, FcRn, offers a way to increase or decrease the
terminal half-life of IgG. Proteins in the circulation, including IgG, are taken up in the fluid phase
through micropinocytosis by certain cells, such as those of the vascular endothelia. IgG can bind
FcRn in endosomes under slightly acidic conditions (pH 6.0-6.5) and can recycle to the cell
surface, where it is released under almost neutral conditions (pH 7.0-7.4). Mapping of the Fcregion-
binding site on FcRn80, 16, 17 showed that two histidine residues that are conserved
across species, His310 and His435, are responsible for the pH dependence of this interaction.
Using phage-display technology, a mouse Fc-region mutation that increases binding to FcRn and
extends the half-life of mouse IgG was identified (see Victor, G. et al. (1997) Nature Biotechnol.
15(7): 637-640). Fc-region mutations that increase the binding affinity of human IgG for FcRn at
pH 6.0, but not at pH 7.4, have also been identified (see Dall'Acqua, William F., et al. (2002) J.
Immunol. 169(9): 5171 -80). Moreover, in one case, a similar pH-dependent increase in binding
(up to 27-fold) was also observed for rhesus FcRn, and this resulted in a twofold increase in
serum half-life in rhesus monkeys compared with the parent IgG (see Hinton, P.R. et al. (2004) J.
Biol. Chem. 279(8), 6213-6216). These findings indicate that it is feasible to extend the plasma
half-life of antibody therapeutics by tailoring the interaction of the Fc region with FcRn.
Conversely, Fc-region mutations that attenuate interaction with FcRn can reduce antibody halflife.
B.10 Pharmacokinetics (PK):
To generate a DVD-Ig molecule with desired pharmacokinetic profile, in an embodiment
parent mAbs with the similarly desired pharmacokinetic profile are selected. One consideration is
that immunogenic response to monoclonal antibodies (i.e., HAHA, human anti-human antibody
response; HACA, human anti-chimeric antibody response) further complicates the
pharmacokinetics of these therapeutic agents. In an embodiment, monoclonal antibodies with
minimal or no immunogenicity are used for constructing DVD-Ig molecules such that the
resulting DVD-Igs will also have minimal or no immunogenicity. Some of the factors that
determine the PK of a mAb include, but are not limited to, intrinsic properties of the mAb (VH
amino acid sequence); immunogenicity; FcRn binding and Fc functions.
The PK profile of selected parental monoclonal antibodies can be easily determined in
rodents as the PK profile in rodents correlates well with (or closely predicts) the PK profile of
monoclonal antibodies in cynomolgus monkey and humans. The PK profile is determined as
described in Example section I.2.2.3.A.
After the parental monoclonal antibodies with desired PK characteristics (and other desired
functional properties as discussed herein) are selected, the DVD-Ig is constructed. As the DVD-Ig
molecules contain two antigen-binding domains from two parental monoclonal antibodies, the PK
properties of the DVD-Ig are assessed as well. Therefore, while determining the PK properties of
the DVD-Ig, PK assays may be employed that determine the PK profile based on functionality of
both antigen-binding domains derived from the two parent monoclonal antibodies. The PK profile
of a DVD-Ig can be determined as described in Example 1.2.2.3.A. Additional factors that may
impact the PK profile of DVD-Ig include the antigen-binding domain (CDR) orientation; linker
size; and Fc / FcRn interactions. PK characteristics of parent antibodies can be evaluated by
assessing the following parameters: absorption, distribution, metabolism and excretion.
Absorption: To date, administration of therapeutic monoclonal antibodies is via
parenteral routes (e.g., intravenous [IV], subcutaneous [SC], or intramuscular [IM]). Absorption
of a mAb into the systemic circulation following either SC or IM administration from the
interstitial space is primarily through the lymphatic pathway. Saturable, presystemic, proteolytic
degradation may result in variable absolute bioavailability following extravascular administration.
Usually, increases in absolute bioavailability with increasing doses of monoclonal antibodies may
be observed due to saturated proteolytic capacity at higher doses. The absorption process for a
mAb is usually quite slow as the lymph fluid drains slowly into the vascular system, and the
duration of absorption may occur over hours to several days.The absolute bioavailability of
monoclonal antibodies following SC administration generally ranges from 50% to 100%. In the
case of a transport-mediating structure at the blood-brain barrier targeted by the DVD-Ig
construct, circulation times in plasma may be reduced due to enhanced trans-cellular transport at
the blood brain barrier (BBB) into the CNS compartment, where the DVD-Ig is liberated to
enable interaction via its second antigen recognition site.
Distribution: Following IV administration, monoclonal antibodies usually follow a
biphasic serum (or plasma) concentration-time profile, beginning with a rapid distribution phase,
followed by a slow elimination phase. In general, a biexponential pharmacokinetic model best
describes this kind of pharmacokinetic profile. The volume of distribution in the central
compartment (Vc) for a mAb is usually equal to or slightly larger than the plasma volume (2-3
liters). A distinct biphasic pattern in serum (plasma) concentration versus time profile may not be
apparent with other parenteral routes of administration, such as IM or SC, because the distribution
phase of the serum (plasma) concentration-time curve is masked by the long absorption portion.
Many factors, including physicochemical properties, site-specific and target-oriented receptor
mediated uptake, binding capacity of tissue, and mAb dose can influence biodistribution of a
mAb. Some of these factors can contribute to nonlinearity in biodistribution for a mAb.
Metabolism and Excretion: Due to the molecular size, intact monoclonal antibodies are
not excreted into the urine via kidney. They are primarily inactivated by metabolism (e.g.,
catabolism). For IgG-based therapeutic monoclonal antibodies, half-lives typically ranges from
hours or 1-2 days to over 20 days. The elimination of a mAb can be affected by many factors,
including, but not limited to, affinity for the FcRn receptor, immunogenicity of the mAb, the
degree of glycosylation of the mAb, the susceptibility for the mAb to proteolysis, and receptormediated
elimination.
B.ll Tissue cross-reactivity pattern on human and tox species:
Identical staining pattern suggests that potential human toxicity can be evaluated in tox
species. Tox species are those animal in which unrelated toxicity is studied.
The individual antibodies are selected to meet two criteria: (1) tissue staining appropriate
for the known expression of the antibody target; and (2) similar staining pattern between human
and tox species tissues from the same organ.
Criterion 1: Immunizations and/or antibody selections typically employ recombinant or
synthesized antigens (proteins, carbohydrates or other molecules). Binding to the natural
counterpart and counterscreen against unrelated antigens are often part of the screening funnel for
therapeutic antibodies. However, screening against a multitude of antigens is often unpractical.
Therefore tissue cross-reactivity studies with human tissues from all major organs serve to rule
out unwanted binding of the antibody to any unrelated antigens.
Criterion 2: Comparative tissue cross reactivity studies with human and tox species
tissues (cynomolgus monkey, dog, possibly rodents and others, the same 36 or 37 tissues are
being tested as in the human study) help to validate the selection of a tox species. In the typical
tissue cross-reactivity studies on frozen tissue sections therapeutic antibodies may demonstrate
the expected binding to the known antigen and/or to a lesser degree binding to tissues based either
on low level interactions (unspecific binding, low level binding to similar antigens, low level
charge based interactions, etc.). In any case the most relevant toxicology animal species is the one
with the highest degree of coincidence of binding to human and animal tissue.
Tissue cross reactivity studies follow the appropriate regulatory guidelines including EC
CPMP Guideline HI/5271/94 "Production and quality control of mAbs" and the 1997 U.S.
FDA/CBER "Points to Consider in the Manufacture and Testing of Monoclonal Antibody
Products for Human Use". Cryosections (5 μm) of human tissues obtained at autopsy or biopsy
were fixed and dried on object glass. The peroxidase staining of tissue sections was performed,
using the avidin-biotin system. FDA's Guidance "Points to Consider in the Manufacture and
Testing of Monoclonal Antibody Products for Human Use".
Tissue cross reactivity studies are often done in two stages, with the first stage including
cryosections of 32 tissues (typically: Adrenal Gland, Gastrointestinal Tract, Prostate, Bladder,
Heart, Skeletal Muscle, Blood Cells, Kidney, Skin, Bone Marrow, Liver, Spinal Cord, Breast,
Lung, Spleen, Cerebellum, Lymph Node, Testes, Cerebral Cortex, Ovary, Thymus, Colon,
Pancreas, Thyroid, Endothelium, Parathyroid, Ureter, Eye, Pituitary, Uterus, Fallopian Tube and
Placenta) from one human donor. In the second phase a full cross reactivity study is performed
with up to 38 tissues (including adrenal, blood, blood vessel, bone marrow, cerebellum, cerebrum,
cervix, esophagus, eye, heart, kidney, large intestine, liver, lung, lymph node, breast mammary
gland, ovary, oviduct, pancreas, parathyroid, peripheral nerve, pituitary, placenta, prostate,
salivary gland, skin, small intestine, spinal cord, spleen, stomach, striated muscle, testis, thymus,
thyroid, tonsil, ureter, urinary bladder, and uterus) from 3 unrelated adults. Studies are done
typically at minimally two dose levels.
The therapeutic antibody (i.e., test article) and isotype matched control antibody may be
biotinylated for avidin-biotin complex (ABC) detection; other detection methods may include
tertiary antibody detection for a FITC (or otherwise) labeled test article, or precomplexing with a
labeled anti-human IgG for an unlabeled test article.
Briefly, cryosections (about 5 μm) of human tissues obtained at autopsy or biopsy are
fixed and dried on object glass. The peroxidase staining of tissue sections is performed, using the
avidin-biotin system. First (in case of a precomplexing detection system), the test article is
incubated with the secondary biotinylated anti-human IgG and developed into immune complex.
The immune complex at the final concentrations of 2 and 10 ug/mL of test article is added onto
tissue sections on object glass and then the tissue sections are reacted for 30 minutes with a
avidin-biotin-peroxidase kit. Subsequently, DAB (3,3'-diaminobenzidine), a substrate for the
peroxidase reaction, is applied for 4 minutes for tissue staining. Antigen-Sepharose beads are
used as positive control tissue sections.
Any specific staining is judged to be either an expected (e.g.,consistent with antigen
expression) or unexpected reactivity based upon known expression of the target antigen in
question. Any staining judged specific is scored for intensity and frequency. Antigen or serum
competion or blocking studies can assist further in determining whether observed staining is
specific or nonspecific.
If two selected antibodies are found to meet the selction criteria - appropriate tissue
staining, and matching staining between human and toxicology animal specific tissue - they can
be selected for DVD-Ig generation.
The tissue cross-reactivity study has to be repeated with the final DVD-Ig construct, but
while these studies follow the same protocol as outline herein, they are more complex to evaluate
because any binding can come from any of the two parent antibodies, and any unexplained
binding needs to be confirmed with complex antigen competition studies.
It is readily apparent that the complex undertaking of tissue crossreactivity studies with a
multispecific molecule like a DVD-Ig is greatly simplified if the two parental antibodies are
selected for (1) lack of unexpected tissue cross reactivity findings and (2) for appropriate
similarity of tissue cross reactivity findings between the corresponding human and toxicology
animal species tissues.
B.12 Specificity and selectivity:
To generate a DVD-Ig molecule with desired specificity and selectivity, one needs to
generate and select parent mAbs with the similarly desired specificity and selectivity profile. In
this regard, parent mAbs can be the same antibody or different antibodies.
Binding studies for specificity and selectivity with a DVD-Ig can be complex due to the
four or more binding sites, two each for each antigen. Briefly, binding studies using an enzyme
linked immunosorbent assay (ELISA), BIAcore, KinExA, or other interaction studies with a
DVD-Ig need to monitor the binding of one, two or more antigens to the DVD-Ig molecule. While
BIAcore technology can resolve the sequential, independent binding of multiple antigens, more
traditional methods, including ELISA, or more modern techniques, such as KinExA, cannot.
Therefore, careful characterization of each parent antibody is critical. After each individual
antibody has been characterized for specificity, confirmation of specificity retention of the
individual binding sites in the DVD-Ig molecule is greatly simplified.
It is readily apparent that the complex undertaking of determining the specificity of a
DVD-Ig is greatly simplified if the two parental antibodies are selected for specificity prior to
being combined into a DVD-Ig.
Antigen-antibody interaction studies can take many forms, including many classical
protein protein interaction studies, ELISA, mass spectrometry, chemical cross-linking, SEC with
light scattering, equilibrium dialysis, gel permeation, ultrafiltration, gel chromatography, largezone
analytical SEC, micropreparative ultracentrigugation (sedimentation equilibrium),
spectroscopic methods, titration microcalorimetry, sedimentation equilibrium (in analytical
ultracentrifuge), sedimentation velocity (in analytical centrifuge), and surface plasmon resonance
(including BIAcore). Relevant references include "Current Protocols in Protein Science,"
Coligan, J.E. et al. (eds.) Volume 3, chapters 19 and 20, published by John Wiley & Sons Inc.,
and "Current Protocols in Immunology," Coligan, J.E. et al. (eds.) published by John Wiley &
Sons Inc., and relevant references included therein.
Cytokine Release in Whole Blood: The interaction of mAb with human blood cells can
be investigated by a cytokine release (Wing, M. G. (1995)-Therapeut. Immunol. 2(4): 183-190;
"Current Protocols in Pharmacology," Enna, S.J. et al. (eds.) published by John Wiley & Sons
Inc; Madhusudan, S. (2004) Clin. Cancer Res. 10(19): 6528-6534; Cox, J. (2006) Methods 38(4):
274-282; Choi, 1.(2001) Eur. J. Immunol. 31(1): 94-106). Briefly, various concentrations of
mAb are incubated with human whole blood for 24 hours. The concentration tested should cover a
wide range including final concentrations mimicking typical blood levels in patients (including
but not limited to 100 ng/ml - 100 g/ml). Following the incubation, supernatants and cell
lysates were analyzed for the presence of IL-1R , TNF- , IL-lb, IL-6andIL-8. Cytokine
concentration profiles generated for mAb were compared to profiles produced by a negative
human IgG control and a positive LPS or PHA control. The cytokine profile displayed by mAb
from both cell supernatants and cell lysates was comparable to control human IgG. In an
embodiment, the monoclonal antibody does not interact with human blood cells to spontaneously
release inflammatory cytokines.
Cytokine release studies for a DVD-Ig are complex due to the four or more binding sites,
two each for each antigen. Briefly, cytokine release studies as described herein measure the effect
of the whole DVD-Ig molecule on whole blood or other cell systems, but can resolve which
portion of the molecule causes cytokine release. Once cytokine release has been detected, the
purity of the DVD-Ig preparation has to be ascertained, because some co-purifying cellular
components can cause cytokine release on their own. If purity is not the issue, fragmentation of
DVD-Ig (including but not limited to removal of Fc portion, separation of binding sites etc.),
binding site mutagenesis or other methods may need to be employed to deconvolute any
observations. It is readily apparent that this complex undertaking is greatly simplified if the two
parental antibodies are selected for lack of cytokine release prior to being combined into a DVDIg-
B.13 Cross reactivity to other species for toxicological studies:
In an embodiment, the individual antibodies are selected with sufficient cross-reactivity to
appropriate tox species, for example, cynomolgus monkey. Parental antibodies need to bind to
orthologous species target (i.e., cynomolgus monkey) and elicit appropriate response (modulation,
neutralization, activation). In an embodiment, the cross-reactivity (affinity/potency) to
orthologous species target should be within 10-fold of the human target. In practice, the parental
antibodies are evaluated for multiple species, including mouse, rat, dog, monkey (and other nonhuman
primates), as well as disease model species (i.e., sheep for asthma model). The acceptable
cross-reactivity to tox species from the perental monoclonal antibodies allows future toxicology
studies of DVD-Ig-Ig in the same species. For that reason, the two parental monoclonal
antibodies should have acceptable cross-reactivity for a common tox species, thereby allowing
toxicology studies of DVD-Ig in the same species.
Parent mAbs may be selected from various mAbs that bind specific targets and are well
known in the art. The parent antibodies can be the same antibody or different antibodies. These
include, but are not limited to anti-TNF antibody (U.S. Patent No. 6,258,562), anti-IL-12 and/or
anti-IL-12p40 antibody (U.S. Patent No. 6,914,128); anti-IL-18 antibody (U.S. Patent Publication
No. 2005/0147610), anti-C5, anti-CBL, anti-CD147, anti-gpl20, anti-VLA-4, anti-CDl la, anti-
CD18, anti-VEGF, anti-CD40L, anti CD-40 (e.g., see PCT Publication No. WO 2007/124299)
anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2, anti-HGF, anticMet,
anti DLL-4, anti-NPRl, anti-PLGF, anti-ErbB3, anti-E-selectin, anti-Fact VII, anti-
Her2/neu, anti-F gp, anti-CDl 1/18, anti-CD14, anti-ICAM-3, anti-RON, anti-SOST, anti CD-19,
anti-CD80 (e.g., see PCT Publication No. WO 2003/039486, anti-CD4, anti-CD3, anti-CD23,
anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti-HLA DR, anti-CD22 (e.g., see U.S. Patent
No. 5,789,554), anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alpha beta, anti-CD2, anti-Hep
B, anti-CA 125, anti-EpCAM, anti-gpl20, anti-CMV, anti-gpllbllla, anti-IgE, anti-CD25, anti-
CD33, anti-HLA, anti-IGFl,2, anti IGFR, anti-VNRintegrin, anti-IL-1 alpha, anti-IL-lbeta, anti-
IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5 receptor, anti-
IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17, and anti-IL-23 (see Presta,
L.G. (2005) J. Allergy Clin. Immunol. 116: 731 -6 and
www.path.cam.ac.uk/~mrc7/humanisation/antibodies.html).
Parent mAbs may also be selected from various therapeutic antibodies approved for use,
in clinical trials, or in development for clinical use. Such therapeutic antibodies include, but are
not limited to, rituximab (Rituxan®, IDEC/Genentech/Roche) (see, for example, U. S. Patent
No. 5,736,137), a chimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma;
HuMax-CD20, an anti-CD20 currently being developed by Genmab, an anti-CD20 antibody
described in U.S. Patent No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20
(Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT Application No.
PCT/US2003/040426), trastuzumab (Herceptin®, Genentech) (see, for example, U.S. Patent No.
5,677,171), a humanized anti- Her2/neu antibody approved to treat breast cancer, pertuzumab
(rhuMab-2C4, Omnitarg®), currently being developed by Genentech; an anti-Her2 antibody
(U.S. Patent No. 4,753,894; cetuximab (Erbitux®, Imclone) (U.S. Patent No. 4,943,533; PCT
Publication No. WO 96/40210), a chimeric anti-EGFR antibody in clinical trials for a variety of
cancers; ABX-EGF (U.S. Patent No. 6,235,883), currently being developed by Abgenix-
Immunex-Amgen; HuMax- EGFr (U.S. Patent No. 7,247,301), currently being developed by
Genmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Patent No.
5,558,864; Murthy, et al. (1987) Arch. Biochem. Biophys. 252(2): 549-60; Rodeck, et al. (1987)
J. Cell. Biochem. 35(4): 315-20; Kettleborough, et al. (1991) Protein Eng. 4(7): 773-83); ICR62
(Institute of Cancer Research) (PCT Publication No. WO 95/20045; Modjtahedi, et al. (1993) J.
Cell. Biophys. 22(1-3): 129-46; Modjtahedi, et al. (1993) Br. J. Cancer 67(2): 247-53;
Modjtahedi, et al. (1996) Br. J. Cancer^ 73(2): 228-35; Modjtahedi, et al. (2003) Int. J. Cancer^
105(2): 273-80); TheraCIM hR3 (YM Biosciences, Canada and Centra de Immunologia
Molecular, Cuba (U.S. Patent No. 5,891,996; U.S. Patent No. 6,506,883; Mateo, et al. (1997)
Immunotechnol. 3(1): 71-81); mAb-806 (Ludwig Institue for Cancer Research, Memorial Sloan-
Kettering) (Jungbluth, et al. (2003) Proc. Natl. Acad. Sci. USA. 100(2): 639-44); KSB-102 (KS
Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT Publication No. WO 01/62931A2);
and SC100 (Scancell) (PCT Publication No. WO 01/88138); alemtuzumab (Campath®,
Millenium), a humanized mAb currently approved for treatment of B-cell chronic lymphocytic
leukemia; muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho
Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), an anti-CD20 antibody
developed by IDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67
protein) antibody developed by Celltech/Wyeth, alefacept (Amevive®), an anti-LFA-3 Fc fusion
developed by Biogen), abciximab (ReoPro®), developed by Centocor/Lilly, basiliximab
(Simulect®), developed by Novartis, palivizumab (Synagis®), developed by Medimmune,
infliximab (Remicade®), an anti-TNFalpha antibody developed by Centocor, adalimumab
(Humira®), an anti-TNFalpha antibody developed by Abbott, Humicade®, an anti-TNFalpha
antibody developed by Celltech, golimumab (CNTO-148), a fully human TNF antibody
developed by Centocor, etanercept (Enbrel®), an p75 TNF receptor Fc fusion developed by
Immunex/Amgen, lenercept, an p55TNF receptor Fc fusion previously developed by Roche,
ABX-CBL, an anti-CD147 antibody being developed by Abgenix, ABX-IL8, an anti-IL8
antibody being developed by Abgenix, ABX-MA1, an anti-MUC 18 antibody being developed by
Abgenix, Pemtumomab (R1549, 90Y-muHMFGl), an anti-MUC 1 in development by Antisoma,
Therex (R1550), an anti-MUC 1 antibody being developed by Antisoma, AngioMab (AS 1405),
being developed by Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS 1407)
being developed by Antisoma, Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) and
alpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, an anti-VLA-1 integrin
antibody being developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR)
antibody being developed by Biogen, CAT-152, an anti-TGF-p2 antibody being developed by
Cambridge Antibody Technology, ABT 874 (J695), an anti- IL-12 p40 antibody being developed
by Abbott, CAT-192, an anti-TGFp"l antibody being developed by Cambridge Antibody
Technology and Genzyme, CAT-213, an anti-Eotaxinl antibody being developed by Cambridge
Antibody Technology, LymphoStat-B® an anti-Blys antibody being developed by Cambridge
Antibody Technology and Human Genome Sciences Inc., TRAIL-RlmAb, an anti-TRAIL-Rl
antibody being developed by Cambridge Antibody Technology and Human Genome Sciences,
Inc., Avastin® bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed by
Genentech, an anti-HER receptor family antibody being developed by Genentech, Anti-Tissue
Factor (ATF), an anti-Tissue Factor antibody being developed by Genentech, Xolair®
(Omalizumab), an anti-IgE antibody being developed by Genentech, Raptiva® (Efalizumab), an
anti- CD1 la antibody being developed by Genentech and Xoma, MLN-02 Antibody (formerly
LDP-02), being developed by Genentech and Millenium Pharmaceuticals, HuMax CD4, an anti-
CD4 antibody being developed by Genmab, HuMax-IL15, an anti-ILl 5 antibody being
developed by Genmab and Amgen, HuMax-Inflam, being developed by Genmab and Medarex,
HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmab and Medarex and
Oxford GcoSciences, HuMax-Lymphoma, being developed by Genmab and Amgen, HuMax-
TAC, being developed by Genmab, IDEC-131, and anti-CD40L antibody being developed by
IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti- CD4 antibody being developed by
IDEC Pharmaceuticals, IDEC-114, an anti- CD80 antibody being developed by IDEC
Pharmaceuticals, IDEC-152, an anti- CD23 being developed by IDEC Pharmaceuticals, antimacrophage
migration factor (MIF) antibodies being developed by IDEC Pharmaceuticals,
BEC2, an anti-idiotypic antibody being developed by Imclone, IMC-1C11, an anti-KDR
antibody being developed by Imclone, DC 101, an anti-flk-1 antibody being developed by
Imclone, anti-VE cadherin antibodies being developed by Imclone, CEA-Cide® (labetuzumab),
an anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics,
LymphoCide® (Epratuzumab), an anti-CD22 antibody being developed by Immunomedics,
AFP-Cide, being developed by Immunomedics, MyelomaCide, being developed by
Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide, being developed by
Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by Medarex, MDX-060,
an anti-CD30 antibody being developed by Medarex, MDX-070 being developed by Medarex,
MDX-018 being developed by Medarex, Osidem® (IDM-1), and anti-Her2 antibody being
developed by Medarex and Immuno-Designed Molecules, HuMax®-CD4, an anti-CD4 antibody
being developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody being developed
by Medarex and Genmab, CNTO 148, an anti-TNFa antibody being developed by Medarex and
Centocor/J&J, CNTO 1275, an anti-cytokine antibody being developed by Centocor/J&J,
MORI01 and MORI 02, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies
being developed by MorphoSys, MOR201, an anti-fibroblast growth factor receptor 3 (FGFR-3)
antibody being developed by MorphoSys, Nuvion® (visilizumab), an anti-CD3 antibody being
developed by Protein Design Labs, HuZAF®, an anti-gamma interferon antibody being
developed by Protein Design Labs, Anti-a 501 Integrin, being developed by Protein Design
Labs, anti-IL-12, being developed by Protein Design Labs, ING-1, an anti-Ep-CAM antibody
being developed by Xoma, Xolair® (Omalizumab) a humanized anti-IgE antibody developed by
Genentech and Novartis, and MLN01, an anti-Beta2 integrin antibody being developed by
Xoma. In another embodiment, the therapeutics include KRN330 (Kirin); huA33 antibody (A33,
Ludwig Institute for Cancer Research); CNTO 95 (alpha V integrins, Centocor); MEDI-522
(alpha V|33 integrin, Medimmune); volociximab (alpha Vß1 integrin, Biogen/PDL); Human
mAb 216 (B cell glycosolated epitope, NCI); BiTE MT103 (bispecific CD 19 x CD3,
Medimmune); 4G7xH22 (Bispecific BcellxFcgammaRl, Medarex/Merck KGa); rM28
(Bispecific CD28 x MAPG, EP Patent No. EP1444268); MDX447 (EMD 82633) (Bispecific
CD64 x EGFR, Medarex); Catumaxomab (removab) (Bispecific EpCAM x anti-CD3,
Trion/Fres); Ertumaxomab (bispecific HER2/CD3, Fresenius Biotech); oregovomab (OvaRex)
(CA-125, ViRexx); Rencarex® (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2,
Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers
Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, Medimmune);
Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech);
veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab
(IDEC 152) (CD23, Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDL
Biopharma); HeFi-1, CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex);
SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics);
Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40,
Seattle Genentics); Campathlh (Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70,
Medarex); hLLl (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen);
MT293 (TRC093/D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab
(MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (Ticilimumab, CP-675,2)
(CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4 TRAIL-R1 agonist, Human Genome
Science /Glaxo Smith Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008
(DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5 TRAIL-R2 agonist, HGS); Cetuximab
(Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio);
Panitumumab (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab);
CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201) (Epcam,
Merck); edrecolomab (Panorex, 17-1 A) (Epcam, Glaxo/Centocor); MORAb-003 (folate receptor
a, Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech);
CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin) (HER2, Celldex);
Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL
Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-lR R1507 (IGF1-R, Roche); CP 751871
(IGF1-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen); Mik-beta-1 (IL2Rb
(CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); Anti-KIR (1-7F9) (Killer cell Iglike
Receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research);
hCBE-11 (LTfiR, Biogen); HuHMFGl (MUC1, Antisoma/NCI); RAV12 (N-linked carbohydrate
epitope, Raven); CAL (parathyroid hormone-related protein (PTH-rP), University of California);
CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1, Medarex/Ono); MAb CT-011 (PD1,
Curetech); IMC-3G3 (PDGFRa, Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591
(PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation);
GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNFa, Centocor);
A27.15 (transferrin receptor, Salk Institute, INSERN WO 2005/111082); E2.3 (transferrin
receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF,
Tsukuba Research Lab-, PCT Publication No. WO/2000/034337, University of Texas); IMC-
18F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone).
B. Construction of DVD molecules:
The dual variable domain immunoglobulin (DVD-Ig) molecule is designed such that two
different light chain variable domains (VL) from the two parent monoclonal antibodies, which can
be the same or different, are linked in tandem directly or via a short linker by recombinant DNA
techniques, followed by the light chain constant domain, and optionally, an Fc region. Similarly,
the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem,
followed by the constant domain CH1 and Fc region (Figure 1 A).
The variable domains can be obtained using recombinant DNA techniques from a parent
antibody generated by any one of the methods described herein. In an embodiment, the variable
domain is a murine heavy or light chain variable domain. In another embodiment, the variable
domain is a CDR grafted or a humanized variable heavy or light chain domain. In an
embodiment, the variable domain is a human heavy or light chain variable domain.
In one embodiment the first and second variable domains are linked directly to each other
using recombinant DNA techniques. In another embodiment the variable domains are linked via
a linker sequence. In an embodiment, two variable domains are linked. Three or more variable
domains may also be linked directly or via a linker sequence. The variable domains may bind the
same antigen or may bind different antigens. DVD-Ig molecules of the invention may include
one immunoglobulin variable domain and one non- immunoglobulin variable domain, such as
ligand binding domain of a receptor, or an active domain of an enzyme. DVD-Ig molecules may
also comprise two or more non-Ig domains.
The linker sequence may be a single amino acid or a polypeptide sequence. In an
embodiment, the linker sequences are selected from the group consisting of
AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2);
AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5);
RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO:
8); RADAAAA(G4S)4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP
(SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13);
TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID
NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID
NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21);
ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23);
GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); and
GHEAAAVMQVQYPAS (SEQ ID NO: 26). The choice of linker sequences is based on crystal
structure analysis of several Fab molecules. There is a natural flexible linkage between the
variable domain and the CH1/CL constant domain in Fab or antibody molecular structure. This
natural linkage comprises approximately 10-12 amino acid residues, contributed by 4-6 residues
from C-terminus of V domain and 4-6 residues from the N-terminus of CL/CH1 domain. DVD
Igs of the invention were generated using N-terminal 5-6 amino acid residues, or 11-12 amino
acid residues, of CL or CH1 as linker in light chain and heavy chain of DVD-Ig, respectively.
The N-terminal residues of the CL or CH1 domain, particularly the first 5-6 amino acid residues,
adopt a loop conformation without strong secondary structure, and, therefore, can act as a flexible
linker between the two variable domains. The N-terminal residues of the CL or CH1 domain are
a natural extension of the variable domains, as they are part of the Ig sequences, and, therefore,
minimize to a large extent any immunogenicity potentially arising from the linkers and junctions.
Other linker sequences may include any sequence of any length of the CL/CH1 domain
but not all residues of the CL/CH1 domain (for example, the first 5-12 amino acid residues of the
CL/CH1 domains) the light chain linkers can be from CK or CX; and the heavy chain linkers can
be derived from CH1 of any isotypes, including Cyl, Cy2, Cy3, Cy4, Cod, Ca2, CS, Ce, and Cu.
Linker sequences may also be derived from other proteins such as Ig-like proteins, (e.g.TCR,
FcR, KIR); G/S based sequences (e.g., G4S repeats) (SEQ ID NO:29); hinge region-derived
sequences; and other natural sequences from other proteins.
In an embodiment a constant domain is linked to the two linked variable domains using
recombinant DNA techniques. In an embodiment, sequence comprising linked heavy chain
variable domains is linked to a heavy chain constant domain and sequence comprising linked light
chain variable domains is linked to a light chain constant domain. In an embodiment, the constant
domains are human heavy chain constant domain and human light chain constant domain
respectively. In an embodiment, the DVD heavy chain is further linked to an Fc region. The Fc
region may be a native sequence Fc region, or a variant Fc region. In another embodiment, the Fc
region is a human Fc region. In another embodiment the Fc region includes Fc region from IgGl,
IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.
In another embodiment two heavy chain DVD polypeptides and two light chain DVD
polypeptides are combined to form a DVD-Ig molecule. Table 2 lists amino acid sequences of
VH and VL regions of exemplary antibodies for targets useful for treating disease, e.g., for
treating cancer. In an embodiment, the invention provides a DVD comprising at least two of the
VH and/or VL regions listed in Table 2, in any orientation.
Table 2: List of Amino Acid Sequences of VH and VL regions of Antibodies for Generating
DVD-Igs
Detailed description of specific DVD-Ig molecules capable of binding specific targets,
and methods of making the same, is provided in the Examples section below.
C. Production of DVD proteins
Binding proteins of the present invention may be produced by any of a number of
techniques known in the art. For example, expression from host cells, wherein expression
vector(s) encoding the DVD heavy and DVD light chains is (are) transfected into a host cell by
standard techniques. The various forms of the term "transfection" are intended to encompass a
wide variety of techniques commonly used for the introduction of exogenous DNA into a
prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAEdextran
transfection and the like. Although it is possible to express the DVD proteins of the
invention in either prokaryotic or eukaryotic host cells, DVD proteins are expressed in eukaryotic
cells, for example, mammalian host cells, because such eukaryotic cells (and in particular
mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded
and immunologically active DVD protein.
Exemplary mammalian host cells for expressing the recombinant antibodies of the
invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in
Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR
selectable marker, e.g., as described in Kaufman, R.J. and Sharp, P.A. (1982) Mol. Biol. 159:601-
621), NSO myeloma cells, COS cells, SP2 and PER.C6 cells. When recombinant expression
vectors encoding DVD proteins are introduced into mammalian host cells, the DVD proteins are
produced by culturing the host cells for a period of time sufficient to allow for expression of the
DVD proteins in the host cells or secretion of the DVD proteins into the culture medium in which
the host cells are grown. DVD proteins can be recovered from the culture medium using standard
protein purification methods.
In an exemplary system for recombinant expression of DVD proteins of the invention, a
recombinant expression vector encoding both the DVD heavy chain and the DVD light chain is
introduced into dhfr- CHO cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the DVD heavy and light chain genes are each operatively linked
to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of
the genes. The recombinant expression vector also carries a DHFR gene, which allows for
selection of CHO cells that have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are cultured to allow for expression
of the DVD heavy and light chains and intact DVD protein is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the recombinant expression vector,
transfect the host cells, select for transformants, culture the host cells and recover the DVD
protein from the culture medium. Still further the invention provides a method of synthesizing a
DVD protein of the invention by culturing a host cell of the invention in a suitable culture
medium until a DVD protein of the invention is synthesized. The method can further comprise
isolating the DVD protein from the culture medium.
An important feature of DVD-Ig is that it can be produced and purified in a similar way
as a conventional antibody. The production of DVD-Ig results in a homogeneous, single major
product with desired dual-specific activity, without any sequence modification of the constant
region or chemical modifications of any kind. Other previously described methods to generate
"bi-specific," "multi-specific," and "multi-specific multivalent" full length binding proteins do
not lead to a single primary product but instead lead to the intracellular or secreted production of a
mixture of assembled inactive, mono-specific, multi-specific, multivalent, full length binding
proteins, and multivalent full length binding proteins with combination of different binding sites.
As an example, based on the design described by Miller and Presta (PCT Publication No.
WO2001/077342(Al), there are 16 possible combinations of heavy and light chains.
Consequently, only 6.25% of protein is likely to be in the desired active form, and not as a single
major product or single primary product compared to the other 15 possible combinations.
Separation of the desired, fully active forms of the protein from inactive and partially active forms
of the protein using standard chromatography techniques, typically used in large scale
manufacturing, is yet to be demonstrated.
Surprisingly, the design of the "dual-specific multivalent full length binding proteins" of
the present invention leads to a dual variable domain light chain and a dual variable domain heavy
chain which assemble primarily to the desired "dual-specific multivalent full length binding
proteins".
At least 50%, at least 75% and at least 90% of the assembled, and expressed dual variable
domain immunoglobulin molecules are the desired dual-specific tetravalent protein. This aspect of
the invention particularly enhances the commercial utility of the invention. Therefore, the present
invention includes a method to express a dual variable domain light chain and a dual variable
domain heavy chain in a single cell leading to a single primary product of a "dual-specific
tetravalent full length binding protein".
The present invention provides methods of expressing a dual variable domain light chain
and a dual variable domain heavy chain in a single cell leading to a "primary product" of a "dualspecific
tetravalent full length binding protein," where the "primary product" is more than 50% of
all assembled protein, comprising a dual variable domain light chain and a dual variable domain
heavy chain.
The present invention provides methods of expressing a dual variable domain light chain
and a dual variable domain heavy chain in a single cell leading to a single "primary product" of a
"dual-specific tetravalent full length binding protein," where the "primary product" is more than
75% of all assembled protein, comprising a dual variable domain light chain and a dual variable
domain heavy chain.
The present invention provides methods of expressing a dual variable domain light chain
and a dual variable domain heavy chain in a single cell leading to a single "primary product" of a
"dual-specific tetravalent full length binding protein," where the "primary product" is more than
90% of all assembled protein, comprising a dual variable domain light chain and a dual variable
domain heavy chain.
II. Derivatized DVD binding proteins:
One embodiment provides a labeled binding protein wherein the binding protein of the
invention is derivatized or linked to another functional molecule (e.g., another peptide or protein).
For example, a labeled binding protein of the invention can be derived by functionally linking a
binding protein of the invention (by chemical coupling, genetic fusion, noncovalent association or
otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific
antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a
protein or peptide that can mediate association of the binding protein with another molecule (such
as a streptavidin core region or a polyhistidine tag).
Useful detectable agents with which a binding protein of the invention may be derivatized
include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein,
fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride,
phycoerythrin and the like. A binding protein may also be derivatized with detectable enzymes,
such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When a
binding protein is derivatized with a detectable enzyme, it is detected by adding additional
reagents that the enzyme uses to produce a detectable reaction product. For example, when the
detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and
diaminobenzidine leads to a colored reaction product, which is detectable. A binding protein may
also be derivatized with biotin, and detected through indirect measurement of avidin or
streptavidin binding.
Another embodiment of the invention provides a crystallized binding protein and
formulations and compositions comprising such crystals. In one embodiment the crystallized
binding protein has a greater half-life in vivo than the soluble counterpart of the binding protein.
In another embodiment the binding protein retains biological activity after crystallization.
Crystallized binding protein of the invention may be produced according to methods
known in the art and as disclosed in PCT Publication No. WO 02/072636.
Another embodiment of the invention provides a glycosylated binding protein wherein the
antibody or antigen-binding portion thereof comprises one or more carbohydrate residues.
Nascent in vivo protein production may undergo further processing, known as post-translational
modification. In particular, sugar (glycosyl) residues may be added enzymatically, a process
known as glycosylation. The resulting proteins bearing covalently linked oligosaccharide side
chains are known as glycosylated proteins or glycoproteins. Antibodies are glycoproteins with
one or more carbohydrate residues in the Fc domain, as well as the variable domain.
Carbohydrate residues in the Fc domain have an important effect on the effector function of the
Fc domain, with minimal effect on antigen binding or half-life of the antibody (Jefferis, R. (2005)
Biotechnol. Prog. 21:11-16). In contrast, glycosylation of the variable domain may have an
effect on the antigen binding activity of the antibody. Glycosylation in the variable domain may
have a negative effect on antibody binding affinity, likely due to steric hindrance (Co, M.S., et al.
(1993) Mol. Immunol. 30:1361- 1367), or result in increased affinity for the antigen (Wallick,
S.C., et al. (1988) Exp. Med. 168:1099-1109; Wright, A., et al. (1991) EMBO J. 10:2717-2723).
One aspect of the present invention is directed to generating glycosylation site mutants in
which the O- or N-linked glycosylation site of the binding protein has been mutated. One skilled
in the art can generate such mutants using standard well-known technologies. Glycosylation site
mutants that retain the biological activity but have increased or decreased binding activity are
another object of the present invention.
In still another embodiment, the glycosylation of the antibody or antigen-binding portion
of the invention is modified. For example, an aglycoslated antibody can be made (i.e., the
antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity
of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for
example, altering one or more sites of glycosylation within the antibody sequence. For example,
one or more amino acid substitutions can be made that result in elimination of one or more
variable region glycosylation sites to thereby eliminate glycosylation at that site. Such
aglycosylation may increase the affinity of the antibody for antigen. Such an approach is
described in further detail in PCT Publication WO 2003/016466A2, and U.S. Patent Nos.
5,714,350 and 6,350,861.
Additionally or alternatively, a modified binding protein of the invention can be made
that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced
amounts of fucosyl residues (see Kanda, Y. et al. (2007) J. Biotech. 130(3): 300-310.) or an
antibody having increased bisecting GlcNAc structures. Such altered glycosylation patterns have
been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications
can be accomplished by, for example, expressing the antibody in a host cell with altered
glycosylation machinery. Cells with altered glycosylation machinery have been described in the
art and can be used as host cells in which to express recombinant antibodies of the invention to
thereby produce an antibody with altered glycosylation. See, for example, Shields, R. L. et al.
(2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as,
European Patent No. EP 1,176,195 and PCT Publication Nos. WO 03/035835 and WO 99/54342
80.
Protein glycosylation depends on the amino acid sequence of the protein of interest, as
well as the host cell in which the protein is expressed. Different organisms may produce different
glycosylation enzymes (e.g., glycosyltransferases and glycosidases), and have different substrates
(nucleotide sugars) available. Due to such factors, protein glycosylation pattern, and composition
of glycosyl residues, may differ depending on the host system in which the particular protein is
expressed. Glycosyl residues useful in the invention may include, but are not limited to, glucose,
galactose, mannose, fucose, n-acetylglucosamine and sialic acid. In an embodiment, the
glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is
human.
It is known to those skilled in the art that differing protein glycosylation may result in
differing protein characteristics. For instance, the efficacy of a therapeutic protein produced in a
microorganism host, such as yeast, and glycosylated utilizing the yeast endogenous pathway may
be reduced compared to that of the same protein expressed in a mammalian cell, such as a CHO
cell line. Such glycoproteins may also be immunogenic in humans and show reduced half-life in
vivo after administration. Specific receptors in humans and other animals may recognize specific
glycosyl residues and promote the rapid clearance of the protein from the bloodstream. Other
adverse effects may include changes in protein folding, solubility, susceptibility to proteases,
trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors,
antigenicity, or allergenicity. Accordingly, a practitioner may choose a therapeutic protein with a
specific composition and pattern of glycosylation, for example glycosylation composition and
pattern identical, or at least similar, to that produced in human cells or in the species-specific cells
of the intended subject animal.
Expressing glycosylated proteins different from that of a host cell may be achieved by
genetically modifying the host cell to express heterologous glycosylation enzymes. Using
techniques known in the art a practitioner may generate antibodies or antigen-binding portions
thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically
modified to express non-naturally occurring glycosylation enzymes such that glycosylated
proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to
that of animal cells, especially human cells (U.S Patent Nos. 7,449,308 and 7,029,872 and PCT
Publication No/ WO2005/100584).
In addition to the binding proteins, the present invention is also directed to anti-idiotypic
(anti-Id) antibodies specific for such binding proteins of the invention. An anti-Id antibody is an
antibody, which recognizes unique determinants generally associated with the antigen-binding
region of another antibody. The anti-Id can be prepared by immunizing an animal with the
binding protein or a CDR containing region thereof. The immunized animal will recognize, and
respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id
antibody. It is readily apparent that it may be easier to generate anti-idiotypic antibodies to the
two or more parent antibodies incorporated into a DVD-Ig molecule; and confirm binding studies
by methods well recognized in the art (e.g., BIAcore, ELISA) to verify that anti-idiotypic
antibodies specific for the idiotype of each parent antibody also recognize the idiotype (e.g.,
antigen binding site) in the context of the DVD-Ig. The anti-idiotypic antibodies specific for each
of the two or more antigen binding sites of a DVD-Ig provide ideal reagents to measure DVD-Ig
concentrations of a human DVD-Ig in patrient serum; DVD-Ig concentration assays can be
established using a "sandwich assay ELISA format" with an antibody to a first antigen binding
region coated on the solid phase (e.g., BIAcore chip, ELISA plate etc.), rinsing with rinsing
buffer, incubating with the serum sample, rinsing again and ultimately incubating with another
anti-idiotypic antibody to the another antigen binding site, itself labeled with an enzyme for
quantitation of the binding reaction. In an embodiment, for a DVD-Ig with more than two
different binding sites, anti-idiotypic antibodies to the two outermost binding sites (most distal
and proximal from the constant region) will not only help in determining the DVD-Ig
concentration in human serum but also document the integrity of the molecule in vivo. Each anti-
Id antibody may also be used as an "immunogen" to induce an immune response in yet another
animal, producing a so-called anti-anti-Id antibody.
Further, it will be appreciated by one skilled in the art that a protein of interest may be
expressed using a library of host cells genetically engineered to express various glycosylation
enzymes, such that member host cells of the library produce the protein of interest with variant
glycosylation patterns. A practitioner may then select and isolate the protein of interest with
particular novel glycosylation patterns. In an embodiment, the protein having a particularly
selected novel glycosylation pattern exhibits improved or altered biological properties.
III. Uses of DVD-Ig
Given their ability to bind to two or more antigens the binding proteins of the invention
can be used to detect the antigens (e.g., in a biological sample, such as serum or plasma), using a
conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), a
radioimmunoassay (RIA) or tissue immunohistochemistry. The DVD-Ig is directly or indirectly
labeled with a detectable substance to facilitate detection of the bound or unbound antibody.
Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, P-galactosidase, and acetylcholinesterase; examples
of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; an example of
a luminescent material includes luminol; and examples of suitable radioactive material include 3H,
14C 35S, 90Y, 99Tc, 111In, 1251,13,1,177Lu, 166Ho, and 153Sm.
In an embodiment, the binding proteins of the invention neutralize the activity of the
antigens both in vitro and in vivo. Accordingly, such DVD-Igs can be used to inhibit antigen
activity, e.g., in a cell culture containing the antigens, in human subjects or in other mammalian
subjects having the antigens with which a binding protein of the invention cross-reacts. In
another embodiment, the invention provides a method for reducing antigen activity in a subject
suffering from a disease or disorder in which the antigen activity is detrimental. A binding
protein of the invention can be administered to a human subject for therapeutic purposes.
As used herein, the term "a disorder in which antigen activity is detrimental" is intended
to include diseases and other disorders in which the presence of the antigen in a subject suffering
from the disorder has been shown to be or is suspected of being either responsible for the
pathophysiology of the disorder or a factor that contributes to a worsening of the disorder.
Accordingly, a disorder in which antigen activity is detrimental is a disorder in which reduction of
antigen activity is expected to alleviate the symptoms and/or progression of the disorder. Such
disorders may be evidenced, for example, by an increase in the concentration of the antigen in a
biological fluid of a subject suffering from the disorder {e.g., an increase in the concentration of
antigen in serum, plasma, synovial fluid, etc. of the subject). Non-limiting examples of disorders
that can be treated with the binding proteins of the invention include those disorders discussed
below and in the section pertaining to pharmaceutical compositions of the antibodies of the
invention.
The DVD-Igs of the invention may bind one antigen or multiple antigens. Such antigens
include, but are not limited to, the targets listed in the following databases. These target databases
include those listings:
Therapeutic targets (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp);
Cytokines and cytokine receptors (http://www.cytokinewebfacts.com/,
http://www.copewithcytokines.de/cope.cgi, and
http://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine.medic.kumamotou.
ac.jp/CFC/indexR.html);
Chemokines (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html);
Chemokine receptors and GPCRs (http://csp.medic.kumamoto-u.ac.jp/CSP/Receptor.html, and
http://www.gpcr.org/7tm/);
Olfactory Receptors (http://senselab.med.yale.edu/senselab/ORDB/default.asp);
Receptors (http ://www.iuphar-db.org/iuphar-rd/list/index.htm);
Cancer targets (http://cged.hgc.jp/cgi-bin/input.cgi);
Secreted proteins as potential antibody targets (http://spd.cbi.pku.edu.cn/);
Protein kinases (http://spd.cbi.pku.edu.cn/), and
Human CD markers (http://content.labvelocity.eom/tools/6/1226/CD_table_final_locked.pdf) and
(Zola H, (2005) Blood 106: 3123-6).
DVD-Igs are useful as therapeutic agents to simultaneously block two different targets to
enhance efficacy/safety and/or increase patient coverage. Such targets may include soluble
targets (e.g., TNF) and cell surface receptor targets (e.g., VEGFR and EGFR). It can also be used
to induce redirected cytotoxicity between tumor cells and T cells (e.g., Her2 and CD3) for cancer
therapy, or between autoreactive cell and effector cells for autoimmune disease or transplantation,
or between any target cell and effector cell to eliminate disease-causing cells in any given disease.
In addition, DVD-Ig can be used to trigger receptor clustering and activation when it is
designed to target two different epitopes on the same receptor. This may have benefit in making
agonistic and antagonistic anti-GPCR therapeutics. In this case, DVD-Ig can be used to target
two different epitopes (including epitopes on both the loop regions and the extracellular domain)
on one cell for clustering/signaling (two cell surface molecules) or signaling (on one molecule).
Similarly, a DVD-Ig molecule can be designed to triger CTLA-4 ligation, and a negative signal
by targeting two different epitopes (or 2 copies of the same epitope) of CTLA-4 extracellular
domain, leading to down regulation of the immune response. CTLA-4 is a clinically validated
target for therapeutic treatment of a number of immunological disorders. CTLA-4/B7 interactions
negatively regulate T cell activation by attenuating cell cycle progression, IL-2 production, and
proliferation of T cells following activation, and CTLA-4 (CD152) engagement can downregulate
T cell activation and promote the induction of immune tolerance. However, the strategy
of attenuating T cell activation by agonistic antibody engagement of CTLA-4 has been
unsuccessful since CTLA-4 activation requires ligation. The molecular interaction of CTLA-4/B7
is in "skewed zipper" arrays, as demonstrated by crystal structural analysis (Stamper (2001)
Nature 410: 608). However none of the currently available CTLA-4 binding reagents have
ligation properties, including anti-CTLA-4 mAbs. There have been several attempts to address
this issue. In one case, a cell member-bound single chain antibody was generated, and
significantly inhibited allogeneic rejection in mice (Hwang (2002) J. Immunol. 169:633). In a
separate case, artificial APC surface-linked single-chain antibody to CTLA-4 was generated and
demonstrated to attenuate T cell responses (Griffin (2000) J. Immunol. 164:4433). In both cases,
CTLA-4 ligation was achieved by closely localized member-bound antibodies in artificial
systems. While these experiments provide proof-of-concept for immune down-regulation by
triggering CTLA-4 negative signaling, the reagents used in these reports are not suitable for
therapeutic use. To this end, CTLA-4 ligation may be achieved by using a DVD-Ig molecule,
which target two different epitopes (or 2 copies of the same epitope) of CTLA-4 extracellular
domain. The rationale is that the distance spanning two binding sites of an IgG, approximately
150-170A, is too large for active ligation of CTLA-4 (30-50 A between 2 CTLA-4 homodimer).
However the distance between the two binding sites on DVD-Ig (one arm) is much shorter, also in
the range of 30-50 A, allowing proper ligation of CTLA-4.
Similarly, DVD-Ig can target two different members of a cell surface receptor complex
(e.g.,IL-12R alpha and beta). Furthermore, DVD-Ig can target CR1 and a soluble
protein/pathogen to drive rapid clearance of the target soluble protein/pathogen.
Additionally, DVD-Igs of the invention can be employed for tissue-specific delivery
(target a tissue marker and a disease mediator for enhanced local PK thus higher efficacy and/or
lower toxicity), including intracellular delivery (targeting an internalizing receptor and a
intracellular molecule), and delivery to the inside of the brain (targeting transferrin receptor and a
CNS disease mediator for crossing the blood-brain barrier). DVD-Ig can also serve as a carrier
protein to deliver an antigen to a specific location via binding to a non-neutralizing epitope of that
antigen and also to increase the half-life of the antigen. Furthermore, DVD-Ig can be designed to
either be physically linked to medical devices implanted into patients or target these medical
devices (see Burke, S. E. et al. (2006) Adv. Drug Deliv. Rev. 58(3): 437-446; Hildebrand, H. F. et
al. (2006) Surface and Coatings Technol. 200(22-23): 6318-6324; Wu, P. et al. (2006)
Biomaterials 27(11): 2450-2467; Marques, A. P. et al. (2005) Biodegrad. Syst. Tissue Eng..and
Regen. Med. 377-397). Briefly, directing appropriate types of cell to the site of medical implant
may promote healing and restoring normal tissue function. Alternatively, inhibition of mediators
(including but not limited to cytokines), released upon device implantation by a DVD coupled to
or target to a device is also provided. For example, stents have been used for years in
interventional cardiology to clear blocked arteries and to improve the flow of blood to the heart
muscle. However, traditional bare metal stents have been known to cause restenosis (re-narrowing
of the artery in a treated area) in some patients and can lead to blood clots. Recently, an anti-
CD34 antibody coated stent has been described which reduced restenosis and prevents blood clots
from occurring by capturing endothelial progenitor cells (EPC) circulating throughout the blood.
Endothelial cells are cells that line blood vessels, allowing blood to flow smoothly. The EPCs
adhere to the hard surface of the stent forming a smooth layer that not only promotes healing but
prevents restenosis and blood clots, complications previously associated with the use of stents
(Aoji et al. (2005) J. Am. Coll. Cardiol. 45(10):1574-9). In addition to improving outcomes for
patients requiring stents, there are also implications for patients requiring cardiovascular bypass
surgery. For example, a prosthetic vascular conduit (artificial artery) coated with anti-EPC
antibodies would eliminate the need to use arteries from patients legs or arms for bypass surgery
grafts. This would reduce surgery and anesthesia times, which in turn will reduce coronary
surgery deaths. DVD-Ig are designed in such a way that it binds to a cell surface marker (such as
CD34) as well as a protein (or an epitope of any kind, including but not limited to proteins, lipids
and polysaccharides) that has been coated on the implanted device to facilitate the cell
recruitment. Such approaches can also be applied to other medical implants in general.
Alternatively, DVD-Igs can be coated on medical devices and upon implantation and releasing all
DVDs from the device (or any other need which may require additional fresh DVD-Ig, including
aging and denaturation of the already loaded DVD-Ig) the device could be reloaded by systemic
administration of fresh DVD-Ig to the patient, where the DVD-Ig is designed to binds to a target
of interest (a cytokine, a cell surface marker (such as CD34) etc.) with one set of binding sites and
to a target coated on the device (including a protein, an epitope of any kind, including but not
limited to lipids, polysaccharides and polymers ) with the other. This technology has the
advantage of extending the usefulness of coated implants.
A. Use of DVD-Igs in various diseases
DVD-Ig molecules of the invention are also useful as therapeutic molecules to treat
various diseases. Such DVD molecules may bind one or more targets involved in a specific
disease. Examples of such targets in various diseases are described below.
1. Human Autoimmune and Inflammatory Response
Many proteins have been implicated in general autoimmune and inflammatory responses,
including C5, CCL1 (1-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15 (MlP-ld), CCL16 (HCC-
4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2),
CCL23 (MPIF-1), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MlP-1a), CCL4
(MlP-lb), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10),
CXCL11 (I-TAC / IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5
(ENA-78 / UX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3,
CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10,
IL13, IL17C, ILIA, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9,
LTA, LTB, MIF, SCYE1 (endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5,
IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A,
CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAKI, IRAK2,
MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6,
ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80,
CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1,
P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2,
CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19,
CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,
CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6,
CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1,
AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orfl0 (IL27w), CER1, CSF1, CSF2, CSF3,
DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, ILIA, IL1B, IL1R1, IL1R2, IL2,
IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA,
IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2,
IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20,
KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1,
TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF,
TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21,
TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). In one aspect,
DVD-Igs that bind one or more of the targets listed herein are provided.
DVD Igs capable of binding the following pairs of targets to treat inflammatory disease
are contemplated: TNF and IL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST (seq. 1);
TNF and DKK; TNF and alphaVbeta3; TNF and NGF; TNF and IL-23pl9; TNF and IL-6; TNF
and SOST (seq. 2); TNF and IL-6R; TNF and CD-20; IgE and IL-13 (seq. 1); IL-13 (seq. 1) and
IL23pl9; IgE and IL-4; IgE and IL-9 (seq. 1); IgE and IL-9 (seq. 2); IgE and IL-13 (seq. 2); IL-13
(seq. 1) and IL-9 (seq. 1); IL-13 (seq. 1) and IL-4; IL-13 (seq. 1) and IL-9 (seq. 2); IL-13 (seq. 2)
and IL-9 (seq. 1); IL-13 (seq. 2) and IL-4; IL-13 (seq. 2) and IL-23pl9; IL-13 (seq. 2) and IL-9
(seq. 2); IL-6R and VEGF; IL-6R and IL-17A; IL-6R and RANKL; IL-17A and IL-lbeta (seq. 1);
IL-lbeta (seq. 1) and RANKL; IL-lbeta (seq. 1) and VEGF; RANKL and CD-20; IL-lalpha and
IL-lbeta (seq. 1); IL-lalpha and IL-lbeta (seq. 2); NKG2D and CD-20; NKG2D and CD-19 (seq.
1); NKG2D and EGFR (seq. 1); NKG2D and EGFR (seq. 2); NKG2D and HER-2 (seq. 1); and
NKG2D and IGF 1R (seq. 1); and NKG2D and EGFR (seq. 3).
2. Asthma
Allergic asthma is characterized by the presence of eosinophilia, goblet cell metaplasia,
epithelial cell alterations, airway hyperreactivity (AHR), and Th2 and Thl cytokine expression, as
well as elevated serum IgE levels. It is now widely accepted that airway inflammation is the key
factor underlying the pathogenesis of asthma, involving a complex interplay of inflammatory cells
such as T cells, B cells, eosinophils, mast cells and macrophages, and of their secreted mediators
including cytokines and chemokines. Corticosteroids are the most important anti-inflammatory
treatment for asthma today, however their mechanism of action is non-specific and safety
concerns exist, especially in the juvenile patient population. The development of more specific
and targeted therapies is therefore warranted. There is increasing evidence that IL-13 in mice
mimics many of the features of asthma, including AHR, mucus hypersecretion and airway
fibrosis, independently of eosinophilic inflammation (Finotto et al. (2005) Int. Immunol. 17(8):
993-1007; Padilla et al. (2005) J. Immunol. 174(12): 8097-8105).
IL-13 has been implicated as having a pivotal role in causing pathological responses
associated with asthma. The development of anti-IL-13 mAb therapy to reduce the effects of IL-
13 in the lung is an exciting new approach that offers considerable promise as a novel treatment
for asthma. However other mediators of differential immunological pathways are also involved in
asthma pathogenesis, and blocking these mediators, in addition to IL-13, may offer additional
therapeutic benefit. Such target pairs include, but are not limited to, IL-13 and a proinflammatory
cytokine, such as tumor necrosis factor-a (TNF-a). TNF-a may amplify the
inflammatory response in asthma and may be linked to disease severity (McDonnell, et al. (2001)
Progr. Respir. Res. 31: 247-250). This suggests that blocking both IL-13 and TNF-a may have
beneficial effects, particularly in severe airway disease. In another embodiment the DVD-Ig of
the invention binds the targets IL-13 and TNF and is used for treating asthma.
Animal models such as OVA-induced asthma mouse model, where both inflammation
and AHR can be assessed, are known in the art and may be used to determine the ability of
various DVD-Ig molecules to treat asthma. Animal models for studying asthma are disclosed in
Coffman, etal. (2005) J. Exp. Med. 201(12): 1875-1879; Lloyd et al. (2001) Adv. Immunol. 77:
263-295; Boyce et al. (2005) J. Exp. Med. 201(12): 1869-1873; and Snibson et al. (2005) J. Brit.
Soc. Allerg. Clin. Immunol. 35(2): 146-52. In addition to routine safety assessments of these
target pairs, specific tests for the degree of immunosuppression may be warranted and helpful in
selecting the best target pairs (see Luster et al. (1994) Toxicology 92(1-3): 229-43; Descotes, et
al. (1992) Devel. Biol. Stand. 77: 99-102; Hart et al. (2001) J. Allerg. Clin. Immunol. 108(2):
250-257).
Based on the rationale disclosed herein and using the same evaluation model for efficacy
and safety other pairs of targets that DVD-Ig molecules can bind and be useful to treat asthma
may be determined. In an embodiment, such targets include, but are not limited to, IL-13 and ILlbeta,
since IL-1 beta is also implicated in inflammatory response in asthma; IL-13 and cytokines
and chemokines that are involved in inflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13
and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-0;
IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13 and
ADAM8. The present invention also provides DVD-Igs capable of binding one or more targets
involved in asthma selected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3
(GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histamine receptors, ILIA, IL1B, IL2, IL3,
IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18,
IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1,
IL13RA2, IL18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17,
CCL18, CCL19, CCL20, CCL22, CCL24.CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2,
CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1,
JAK3, STAT6, TBX21, TGFB1, TNF, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R,
TB4R2, LTBR, and Chitinase.
3. Rheumatoid arthritis
Rheumatoid arthritis (RA), a systemic disease, is characterized by a chronic inflammatory
reaction in the synovium of joints and is associated with degeneration of cartilage and erosion of
juxta-articular bone. Many pro-inflammatory cytokines including TNF, chemokines, and growth
factors are expressed in diseased joints. Systemic administration of anti-TNF antibody or sTNFR
fusion protein to mouse models of RA was shown to be anti-inflammatory and joint protective.
Clinical investigations in which the activcity of TNF in RA patients was blocked with
intravenously administered infliximab (Harriman, G. et al. (1999) Ann. Rheum. Dis. 58 (Suppl 1):
161-4), a chimeric anti-TNF mAb, has provided evidence that TNF regulates IL-6, IL-8, MCP-1,
and VEGF production, recruitment of immune and inflammatory cells into joints, angiogenesis,
and reduction of blood levels of matrix metalloproteinases-1 and -3. A better understanding of
the inflammatory pathway in rheumatoid arthritis has led to identification of other therapeutic
targets involved in rheumatoid arthritis. Promising treatments such as interleukin-6 antagonists
(IL-6 receptor antibody MRA, developed by Chugai, Roche (see Nishimoto, N. et al. (2004)
Arthrit. Rheum. 50(6): 1761-1769), CTLA4Ig (abatacept, Genovese, M. et al. (2005) N. Engl. J.
Med. 353: 1114-23.), and anti-B cell therapy (rituximab; Okamoto, H. and Kamatani, N. (2004)
N. Engl. J. Med. 351: 1909), have already been tested in randomized controlled trials over the
past year. Other cytokines have been identified and have been shown to be of benefit in animal
models, including interleukin-15 (therapeutic antibody HuMax-IL_15, AMG 714 (see Baslund, B.
et al. (2005) Arthrit. Rheum. 52(9): 2686-2692)), interleukin-17, and interleukin-18, and clinical
trials of these agents are currently under way. Dual-specific antibody therapy, combining anti-
TNF and another mediator, has great potential in enhancing clinical efficacy and/or patient
coverage. For example, blocking both TNF and VEGF can potentially eradicate inflammation
and angiogenesis, both of which are involved in pathophysiology of RA. Blocking other pairs of
targets involved in RA including, but not limited to, TNF and IL-18; TNF and IL-12; TNF and
IL-23; TNF and IL-lbeta; TNF and MIF; TNF and IL-17; TNF and IL-15, TNF and SOST with
specific DVD Igs is also contemplated. In addition to routine safety assessments of these target
pairs, specific tests for the degree of immunosuppression may be warranted and helpful in
selecting the best target pairs (see Luster et al. (1994) Toxicol. 92(1-3): 229-43; Descotes et al.
(1992) Devel. Biol. Stand. 77: 99-102; Hart et al. (2001) J. Allerg. Clin. Immunol. 108(2): 250-
257). Whether a DVD Ig molecule will be useful for the treatment of rheumatoid arthritis can be
assessed using pre-clinical animal RA models such as the collagen-induced arthritis mouse model.
Other useful models are also well known in the art (see Brand, D.D. (2005) Comp. Med. 55(2):
114-22). Based on the cross-reactivity of the parental antibodies for human and mouse othologues
(e.g., reactivity for human and mouse TNF, human and mouse IL-15, etc.) validation studies in
the mouse CIA model may be conducted with "matched surrogate antibody" derived DVD-Ig
molecules; briefly, a DVD-Ig based on two (or more) mouse target specific antibodies may be
matched to the extent possible to the characteristics of the parental human or humanized
antibodies used for human DVD-Ig construction (similar affinity, similar neutralization potency,
similar half-life etc.).
4. SLE
The immunopathogenic hallmark of SLE is the polyclonal B cell activation, which leads
to hyperglobulinemia, autoantibody production and immune complex formation. The fundamental
abnormality appears to be the failure of T cells to suppress the forbidden B cell clones due to
generalized T cell dysregulation. In addition, B and T-cell interaction is facilitated by several
cytokines such as IL-10 as well as co-stimulatory molecules such as CD40 and CD40L, B7 and
CD28 and CTLA-4, which initiate the second signal. These interactions together with impaired
phagocytic clearance of immune complexes and apoptotic material, perpetuate the immune
response with resultant tissue injury. The following targets may be involved in SLE and can
potentially be used for a DVD-Ig approach for therapeutic intervention: B cell targeted therapies:
CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6,
TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1,
RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK,
GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA,
KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8,
TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3,
MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA, andNT5E.; co-stimulatory signals:
CTLA4 or B7.1/B7.2; inhibition of B cell survival: BlyS or BAFF; Complement inactivation: C5;
Cytokine modulation: the key principle is that the net biologic response in any tissue is the result
of a balance between local levels of proinflammatory or anti-inflammatory cytokines (see
Sfikakis, P.P. et al. (2005) Curr. Opin. Rheumatol. 17:550-7). SLE is considered to be a Th-2
driven disease with documented elevations in serum IL-4, IL-6, IL-10. DVD-Igs yhat bind one or
more targets selected from the group consisting of IL-4, IL-6, IL-10, IFN-ct, and TNF-a are also
contemplated. Combination of targets discussed herein will enhance therapeutic efficacy for SLE
which can be tested in a number of lupus preclinical models (see Peng, S.L. (2004) Methods Mol.
Med. 102:227-72). Based on the cross-reactivity of the parental antibodies for human and mouse
othologues (e.g.,reactivity for human and mouse CD20, human and mouse Interferon alpha etc.)
validation studies in a mouse lupus model may be conducted with "matched surrogate antibody"
derived DVD-Ig molecules. Briefly, a DVD-Ig based two (or more) mouse target specific
antibodies may be matched to the extent possible to the characteristics of the parental human or
humanized antibodies used for human DVD-Ig construction (similar affinity, similar
neutralization potency, similar half-life etc.).
5. Multiple sclerosis
Multiple sclerosis (MS) is a complex human autoimmune-type disease with a
predominantly unknown etiology. Immunologic destruction of myelin basic protein (MBP)
throughout the nervous system is the major pathology of multiple sclerosis. MS is a disease of
complex pathologies, which involves infiltration by CD4+ and CD8+ T cells and of response
within the central nervous system. Expression in the CNS of cytokines, reactive nitrogen species
and costimulator molecules have all been described in MS. Of major consideration are
immunological mechanisms that contribute to the development of autoimmunity. In particular,
antigen expression, cytokine and leukocyte interactions, and regulatory T-cells, which help
balance/modulate other T-cells such as Th1 and Th2 cells, are important areas for therapeutic
target identification.
IL-12 is a proinflammatory cytokine that is produced by APC and promotes
differentiation of Thl effector cells. IL-12 is produced in the developing lesions of patients with
MS as well as in EAE-affected animals. Previously it was shown that interference in IL-12
pathways effectively prevents EAE in rodents, and that in vivo neutralization of IL-12p40 using a
anti-IL-12 mAb has beneficial effects in the myelin-induced EAE model in common marmosets.
TWEAK is a member of the TNF family, constitutively expressed in the central nervous
system (CNS), with pro-inflammatory, proliferative or apoptotic effects depending upon cell
types. Its receptor, Fnl4, is expressed in CNS by endothelial cells, reactive astrocytes and
neurons. TWEAK and Fnl4 mRNA expression increased in spinal cord during experimental
autoimmune encephalomyelitis (EAE). Anti-TWEAK antibody treatment in myelin
oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice resulted in a reduction of
disease severity and leukocyte infiltration when mice were treated after the priming phase.
One aspect of the invention pertains to DVD Ig molecules capable of binding one or
more, for example two, targets selected from the group consisting of IL-12, TWEAK, IL-23,
CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF,
FGF, C5, CD52, and CCR2. An embodiment includes a dual-specific anti-IL-12A"WEAK DVD
Ig as a therapeutic agent beneficial for the treatment of MS.
Several animal models for assessing the usefulness of the DVD molecules to treat MS are
known in the art (see Steinman. L. et al. (2005) Trends Immunol. 26(11): 565-71; Lublin, F.D. et
al. (1985) Springer Semin. Immunopathol. 8(3): 197-208; Genain, C.P. et al. (1997) J. Mol. Med.
75(3): 187-97; Tuohy, V.K. et al. (1999) J. Exp. Med. 189(7): 1033-42; Owens, T. et al. (1995)
Neurol. Clin.l3(1): 51-73; and Hart, B.A. et al. (2005) J. Immunol. 175(7): 4761-8. Based on the
cross-reactivity of the parental antibodies for human and animal species othologues
(e.g.,reactivity for human and mouse IL-12, human and mouse TWEAK etc.), validation studies
in the mouse EAE model may be conducted with "matched surrogate antibody" derived DVD-Ig
molecules. Briefly, a DVD-Ig based on two (or more) mouse target specific antibodies may be
matched to the extent possible to the characteristics of the parental human or humanized
antibodies used for human DVD-Ig construction (similar affinity, similar neutralization potency,
similar half-life etc.). The same concept applies to animal models in other non-rodent species,
where a "matched surrogate antibody" derived DVD-Ig would be selected for the anticipated
pharmacology and possibly safety studies. In addition to routine safety assessments of these
target pairs specific tests for the degree of immunosuppression may be warranted and helpful in
selecting the best target pairs (see Luster et al. (1994) Toxicol. 92(1-3): 229-43; Descotes et al.
(1992) Devel. Biol. Stand. 77: 99-102; Jones, R. (2000) IDrugs 3(4): 442-6).
6. Sepsis
The pathophysiology of sepsis is initiated by the outer membrane components of both
gram-negative organisms (lipopolysaccharide [LPS], lipid A, endotoxin) and gram-positive
organisms (lipoteichoic acid, peptidoglycan). These outer membrane components are able to bind
to the CD 14 receptor on the surface of monocytes. By virtue of the recently described toll-like
receptors, a signal is then transmitted to the cell, leading to the eventual production of the
proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1).
Overwhelming inflammatory and immune responses are essential features of septic shock and
play a central part in the pathogenesis of tissue damage, multiple organ failure, and death induced
by sepsis. Cytokines, especially tumor necrosis factor (TNF) and interleukin (IL-1), have been
shown to be critical mediators of septic shock. These cytokines have a direct toxic effect on
tissues; they also activate phospholipase A2. These and other effects lead to increased
concentrations of platelet-activating factor, promotion of nitric oxide synthase activity, promotion
of tissue infiltration by neutrophils, and promotion of neutrophil activity.
The treatment of sepsis and septic shock remains a clinical conundrum, and recent
prospective trials with biological response modifiers (i.e., anti-TNF and anti-MIF) aimed at the
inflammatory response have shown only modest clinical benefit. Recently, interest has shifted
toward therapies aimed at reversing the accompanying periods of immune suppression. Studies in
experimental animals and critically ill patients have demonstrated that increased apoptosis of
lymphoid organs and some parenchymal tissues contribute to this immune suppression, anergy,
and organ system dysfunction. During sepsis syndromes, lymphocyte apoptosis can be triggered
by the absence of IL-2 or by the release of glucocorticoids, granzymes, or the so-called 'death'
cytokines: tumor necrosis factor alpha or Fas ligand. Apoptosis proceeds via auto-activation of
cytosolic and/or mitochondrial caspases, which can be influenced by the pro- and anti-apoptotic
members of the Bcl-2 family. In experimental animals, not only can treatment with inhibitors of
apoptosis prevent lymphoid cell apoptosis; it may also improve outcome. Although clinical trials
with anti-apoptotic agents remain distant due in large part to technical difficulties associated with
their administration and tissue targeting, inhibition of lymphocyte apoptosis represents an
attractive therapeutic target for the septic patient. Likewise, a dual-specific agent targeting both
inflammatory mediator and an apoptotic mediator, may have added benefit. One aspect of the
invention pertains to DVD Igs capable of binding one or more targets involved in sepsis, in an
embodiment two targets, selected from the group consisting of TNF, IL-1, MIF, IL-6, IL-8, IL-18,
IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1,
CASP4, IL-10, IL-1B, NFKB1, PROC, TNFRSF1A, CSF3, CGR3, IL1RN, MIF, NFKB1,
PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine, IRAKI, NFKB2, SERPINA1, SERPINE1,
and TREM1. The efficacy of such DVD Igs for sepsis can be assessed in preclinical animal
models known in the art (see Buras, J.A., et al. (2005) Nat. Rev. Drug Discov. 4(10):854-65 and
Calandra T, et al. (2000) Nat Med. 6(2): 164-70).
7. Neurological disorders
7.1. Neurodegenerative Diseases
Neurodegenerative diseases are either chronic in which case they are usually agedependent
or acute (e.g., stroke, traumatic brain injury, spinal cord injury, etc.). They are
characterized by progressive loss of neuronal functions (neuronal cell death, demyelination), loss
of mobility and loss of memory. Emerging knowledge of the mechanisms underlying chronic
neurodegenerative diseases (e.g., Alzheimer's disease disease) show a complex etiology and a
variety of factors have been recognized to contribute to their development and progression
e.g.,age, glycemic status, amyloid production and multimerization, accumulation of advanced
glycation-end products (AGE) which bind to their receptor RAGE (receptor for AGE), increased
brain oxidative stress, decreased cerebral blood flow, neuroinflammation including release of
inflammatory cytokines and chemokines, neuronal dysfunction and microglial activation. Thus
these chronic neurodegenerative diseases represent a complex interaction between multiple cell
types and mediators. Treatment strategies for such diseases are limited and mostly constitute
either blocking inflammatory processes with non-specific anti-inflammatory agents (e.g.,
corticosteroids, COX inhibitors) or agents to prevent neuron loss and/or synaptic functions.
These treatments fail to stop disease progression. Recent studies suggest that more targeted
therapies such as antibodies to soluble A-b peptide (including the A-b oligomeric forms) can not
only help stop disease progression but may help maintain memory as well. These preliminary
observations suggest that specific therapies targeting more than one disease mediator (e.g.,A-b
and a pro-inflammatory cytokine, such as TNF) may provide even better therapeutic efficacy for
chronic neurodegenerative diseases than observed with targeting a single disease mechanism (e.g.,
soluble A-ßalone) Several animal models for assessing the usefulness of the DVD molecules to
treat MS are known in the art (see Steinman. L. et al. (2005) Trends Immunol. 26(11): 565-71;
Lublin, F.D. et al. (1985) Springer Semin. Immunopathol. 8(3): 197-208; Genain, C.P. et al.
(1997) J. Mol. Med. 75(3): 187-97; Tuohy, V.K. et al. (1999) J. Exp. Med. 189(7): 1033-42;
Owens, T. et al. (1995) Neurol. Clin. 13(1): 51-73; and Hart, B.A. et al. (2005) J. Immunol.
175(7): 4761-8. Based on the cross-reactivity of the parental antibodies for human and animal
species othologues (e.g., reactivity for human and mouse IL-12, human and mouse TWEAK etc.),
validation studies in the mouse EAE model may be conducted with "matched surrogate antibody"
derived DVD-Ig molecules. Briefly, a DVD-Ig based on two (or more) mouse target specific
antibodies may be matched to the extent possible to the characteristics of the parental human or
humanized antibodies used for human DVD-Ig construction (similar affinity, similar
neutralization potency, similar half-life etc.). The same concept applies to animal models in other
non-rodent species, where a "matched surrogate antibody" derived DVD-Ig would be selected for
the anticipated pharmacology and possibly safety studies. In addition to routine safety
assessments of these target pairs specific tests for the degree of immunosuppression may be
warranted and helpful in selecting the best target pairs (see Luster et al. (1994) Toxicol. 92(1-3):
229-43; Descotes et al. (1992) Devel. Biol. Stand. 77: 99-102; Jones, R. (2000) IDrugs 3(4): 442-
6).
The DVD-Ig molecules of the invention can bind one or more targets involved in chronic
neurodegenerative diseases such as Alzheimers. Such targets include, but are not limited to, any
mediator, soluble or cell surface, implicated in AD pathogenesis e.g AGE (SI00 A, amphoterin),
pro-inflammatory cytokines (e.g., IL-1), chemokines (e.g., MCP 1), molecules that inhibit nerve
regeneration (e.g., Nogo, RGM A), molecules that enhance neurite growth (neurotrophins) and
molecules that can mediate transport at the blood brain barrier (e.g., transferrin receptor, insulin
receptor or RAGE). The efficacy of DVD-Ig molecules can be validated in pre-clinical animal
models such as the transgenic mice that over-express amyloid precursor protein or RAGE and
develop Alzheimer's disease-like symptoms. In addition, DVD-Ig molecules can be constructed
and tested for efficacy in the animal models and the best therapeutic DVD-Ig can be selected for
testing in human patients. DVD-Ig molecules can also be employed for treatment of other
neurodegenerative diseases such as Parkinson's disease. Alpha-Synuclein is involved in
Parkinson's pathology. A DVD-Ig capable of targeting alpha-synuclein and inflammatory
mediators such as TNF, IL-1, MCP-1 can prove effective therapy for Parkinson's disease and are
contemplated in the invention.
7.2 Neuronal Regeneration and Spinal Cord Injury
Despite an increase in knowledge of the pathologic mechanisms, spinal cord injury (SCI)
is still a devastating condition and represents a medical indication characterized by a high medical
need. Most spinal cord injuries are contusion or compression injuries and the primary injury is
usually followed by secondary injury mechanisms (inflammatory mediators e.g., cytokines and
chemokines) that worsen the initial injury and result in significant enlargement of the lesion area,
sometimes more than 10-fold. These primary and secondary mechanisms in SCI are very similar
to those in brain injury caused by other means e.g., stroke. No satisfying treatment exists and
high dose bolus injection of methylprednisolone (MP) is the only used therapy within a narrow
time window of 8 h post injury. This treatment, however, is only intended to prevent secondary
injury without causing any significant functional recovery. It is heavily critisized for the lack of
unequivocal efficacy and severe adverse effects, like immunosuppression with subsequent
infections and severe histopathological muscle alterations. No other drugs, biologies or small
molecules, stimulating the endogenous regenerative potential are approved, but promising
treatment principles and drug candidates have shown efficacy in animal models of SCI in recent
years. To a large extent the lack of functional recovery in human SCI is caused by factors
inhibiting neurite growth, at lesion sites, in scar tissue, in myelin as well as on injury-associated
cells. Such factors are the myelin-associated proteins NogoA, OMgp and MAG, RGM A, the
scar-associated CSPG (Chondroitin Sulfate Proteoglycans) and inhibitory factors on reactive
astrocytes (some semaphorins and ephrins). However, at the lesion site not only growth
inhibitory molecules are found but also neurite growth stimulating factors like neurotrophins,
laminin, LI and others. This ensemble of neurite growth inhibitory and growth promoting
molecules may explain that blocking single factors, like NogoA or RGM A, resulted in significant
functional recovery in rodent SCI models, because a reduction of the inhibitory influences could
shift the balance from growth inhibition to growth promotion. However, recoveries observed
with blocking a single neurite outgrowth inhibitory molecule were not complete. To achieve
faster and more pronounced recoveries either blocking two neurite outgrowth inhibitory
molecules, e.g., Nogo and RGM A, or blocking an neurite outgrowth inhibitory molecule and
enhancing functions of a neurite outgrowth enhancing molecule e.g Nogo and neurotrophins, or
blocking a neurite outgrowth inhibitory moleclule e.g.,Nogo and a pro-inflammatory molecule
e.g.,TNF, may be desirable (see McGee, A.W. et al. (2003) Trends Neurosci. 26: 193;
Domeniconi, M. et al. (2005) J. Neurol. Sci. 233: 43; Makwanal, M. et al. (2005) FEBS J. 272:
2628; Dickson, B.J. (2002) Science 298: 1959; Yu, F. and Teng, H. et al. (2005) J. Neurosci. Res.
79: 273; Karnezis, T. et al. (2004) Nature Neurosci. 7: 736; Xu, G. et al. (2004) J. Neurochem. 91:
1018).
In one aspect, DVD-Igs capable of binding target pairs such as NgR and RGM A; NogoA
and RGM A; MAG and RGM A; OMGp and RGM A; RGM A and RGM B; CSPGs and RGM A;
aggrecan, midkine, neurocan, versican, phosphacan, Te38 and TNF-a; A8 globulomer-specific
antibodies combined with antibodies promoting dendrite & axon sprouting are provided.
Dendrite pathology is a very early sign of AD and it is known that NOGO A restricts dendrite
growth. One can combine one such type of Ab with any of the SCI-candidate (myelin-proteins)
Abs. Other DVD-Ig targets may include any combination of NgR-p75, NgR-Troy, NgR-Nogo66
(Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG and Omgp. Additionally, targets may also
include any mediator, soluble or cell surface, implicated in inhibition of neurite e.g., Nogo,
Ompg, MAG, RGM A, semaphorins, ephrins, soluble A-b, pro-inflammatory cytokines (e.g., IL-
1), chemokines (e.g.,MIP la), molecules that inhibit nerve regeneration. The efficacy of antinogo
/ anti-RGM A or similar DVD-Ig molecules can be validated in pre-clinical animal models
of spinal cord injury. In addition, these DVD-Ig molecules can be constructed and tested for
efficacy in the animal models and the best therapeutic DVD-Ig can be selected for testing in
human patients. In addition, DVD-Ig molecules can be constructed that target two distinct ligand
binding sites on a single receptor e.g., Nogo receptor, which binds the three ligand Nogo, Ompg,
and MAG and RAGE that binds A-b and SI00 A. Furthermore, neurite outgrowth inihibitors e.g.,
nogo and nogo receptor, also play a role in preventing nerve regeneration in immunological
diseases like multiple sclerosis. Inhibition of nogo-nogo receptor interaction has been shown to
enhance recovery in animal models of multiple sclerosis. Therefore, DVD-Ig molecules that can
block the function of one immune mediator, e.g., a cytokine, like IL-12, and a neurite outgrowth
inhibitor molecule eg nogo or RGM may offer faster and greater efficacy than blocking either an
immune or an neurite outgrowth inhibitor molecule alone.
In general, antibodies do not cross the blood brain barrier (BBB) in an efficient and
relevant manner. However, in certain neurologic diseases, e.g., stroke, traumatic brain injury,
multiple sclerosis, etc., the BBB may be compromised and allows for increased penetration of
DVD-Igs and antibodies into the brain. In other neurological conditions, where BBB leakage is
not occuring, one may employ the targeting of endogenous transport systems, including carriermediated
transporters such as glucose and amino acid carriers and receptor-mediated transcytosismediating
cell structures/receptors at the vascular endothelium of the BBB, thus enabling transBBB
transport of the DVD-Ig. Structures at the BBB enabling such transport include but are not
limited to the insulin receptor, transferrin receptor, LRP and RAGE. In addition, strategies enable
the use of DVD-Igs also as shuttles to transport potential drugs into the CNS including low
molecular weight drugs, nanoparticles and nucleic acids (Coloma, M.J. et al. (2000) Pharm Res.
17(3):266-74; Boado, R.J. et al. (2007) Bioconjug. Chem. 18(2):447-55).
8. Oncological disorders
Monoclonal antibody therapy has emerged as an important therapeutic modality for
cancer (von Mehren, M, et al. (2003) Annu. Rev. Med. 54:343-69). Antibodies may exert
antitumor effects by inducing apoptosis, redirecting cytotoxicity, interfering with ligand-receptor
interactions, or preventing the expression of proteins that are critical to the neoplastic phenotype.
In addition, antibodies can target components of the tumor microenvironment, perturbing vital
structures such as the formation of tumor-associated vasculature. Antibodies can also target
receptors whose ligands are growth factors, such as the epidermal growth factor receptor. The
antibody thus inhibits natural ligands that stimulate cell growth from binding to targeted tumor
cells. Alternatively, antibodies may induce an anti-idiotype network, complement-mediated
cytotoxicity, or antibody-dependent cellular cytotoxicity (ADCC). The use of dual-specific
antibody that targets two separate tumor mediators will likely give additional benefit compared to
a mono-specific therapy. DVD Igs capable of binding the following pairs of targets to treat
oncological disease are also contemplated: IGF1 and IGF2; IGF 1/2 and HER-2; VEGFR and
EGFR; CD20 and CD3; CD138 and CD20; CD38 and CD20; CD38 and CD138; CD40 and
CD20; CD138 and CD40; CD38 and CD40; CD-20 and CD-19; CD-20 and EGFR; CD-20 and
CD-80; CD-20 and CD-22; CD-3 and HER-2; CD-3 and CD-19; EGFR and HER-2; EGFR and
CD-3; EGFR and IGF1.2; EGFR and IGF1R; EGFR and RON; EGFR and HGF; EGFR and c-
MET; HER-2 and IGF 1,2; HER-2 and IGF1R; RON and HGF; VEGF and EGFR; VEGF and
HER-2; VEGF and CD-20; VEGF and IGF 1,2; VEGF and DLL4; VEGF and HGF; VEGF and
RON; VEGF and NRP1; CD20 and CD3; VEGF and PLGF; DLL4 and PLGF; ErbB3 and EGFR;
HGF and ErbB3, HER-2 and ErbB3; c-Met and ErbB3; HER-2 and PLGF; HER-2 and HER-2;
NKG2D and CD-20; NKG2D and CD-19 (seq. 1); NKG2D and EGFR (seq. 1); NKG2D and
HER-2 (seq. 1); NKG2D and IGF1R (seq. 1); and NKG2D and EGFR (seq. 3).
In another embodiment, a DVD of the invention is capable of binding VEGF and
phosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and ROB04; VEGF and BSG2;
VEGF and CDCP1; VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and
BSG2; HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR and
CDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20; CD20
and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52; CD20 and
CD4; HGF and c-MET; HGF and NRP1; HGF and phosphatidylserine; ErbB3 and IGF1R; ErbB3
and IGF 1,2; c-Met and Her-2; c-Met andNRPl; c-Met and IGF1R; IGF 1,2 and PDGFR; IGF 1,2
and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 and
VEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF; PDGFRa
and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2; PDGFRb and c-
Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP1; RON and MSP; RON and
CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR; VEGFR2 and PLGF;
VEGFR2 and NRPl; VEGFR2 and RON; VEGFR2 and DLL4; VEGFR2 and EGFR; VEGFR2
and ROB04; VEGFR2 and CD55; LPA and SIP; EPHB2 and RON; CTLA4 and VEGF; CD3
and EPCAM; CD40 and IL6; CD40 and IGF; CD40 and CD56; CD40 and CD70; CD40 and
VEGFR1; CD40 and DR5; CD40 and DR4; CD40 and APRIL; CD40 and BCMA; CD40 and
RANKL; CD28 and MAPG; CD80 and CD40; CD80 and CD30; CD80 and CD33; CD80 and
CD74; CD80 and CD2; CD80 and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52;
CD80 and VEGF; CD80 and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80;
CD22 and CD40; CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22
and DR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 and
CD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30 and CD19;
CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52; CD30 and CD4; CD138
and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 and VEGFR2; CD33 and CD44; CD33
and DR4; CD33 and DR5; DR4 and CD137; DR4 and IGF1.2; DR4 and IGF1R; DR4 and DR5;
DR5 and CD40; DR5 and CD137; DR5 and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and
IGFR, DR5 and HER-2, and EGFR and DLL4. Other target combinations include one or more
members of the EGF/erb-2/erb-3 family. Other targets (one or more) involved in oncological
diseases that DVD Igs may bind include, but are not limited to those selected from the group
consisting of: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, ILIA, IL1B, IL2, IL24,
INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16,
FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6,
FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, ILIA, IL1B, IL2, INHA, TGFA, TGFB1,
TGFB2, TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2,
CD 164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1,
IGFBP6, ILIA, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5,
CDK6, CDK7, CDK9, E2F1, EGFR, ENOl, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2,
INSL4, MYC,NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23,
FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3, INSL4,
PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2,
NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1,
NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3,
KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8,
FGF1, FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22,
FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2, IGFBP3,
IGFBP6, IL12A, ILIA, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10, KLK12, KLK13,
KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1,
PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19,
CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8,
CDH9, ROB02, CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2,
AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP,
DES, DNCL1, ELAC2, EN02, EN03, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1,
GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PARTI, PATE, PCA3,
PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1,
TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1,
KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAI1,
COL4A3, IL8, LAMA5, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2,
SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9,
IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4,
FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5,
COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1,
CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kipl), CDKN2A
(pl6INK4a), COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1,
ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST
(glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67),
NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin),
SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6
(FasL), TOP2A (topoisomerase Iia), TP53, AZGP1 (zinc-a-glycoprotem), BPAG1 (plectin),
CDKN1A (p21Wapl/Cipl), CLDN7 (claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1,
FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4
integrin), KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type II keratin),
MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2
(p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2
(mammaglobin 1), SPRR1B (Sprl), THBS1, THBS2, THBS4, and TNFAIP2 (B94), RON, c-Met,
CD64, DLL4, PLGF, CTLA4, phophatidylserine, ROB04, CD80, CD22, CD40, CD23, CD28,
CD80, CD55, CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5,
RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM,
PGE2, NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha, PDGFR beta, ROR1,
PSMA, PSCA, SCD1, and CD59.
IV. Pharmaceutical Compositions
The invention also provides pharmaceutical compositions comprising a binding protein,
of the invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions
comprising binding proteins of the invention are for use in, but not limited to, diagnosing,
detecting, or monitoring a disorder, in preventing (e.g., inhibiting or delaying the onset of a
disease, disorder or other condition), treating, managing, or ameliorating of a disorder or one or
more symptoms thereof, and/or in research. In a specific embodiment, a composition comprises
one or more binding proteins of the invention. In another embodiment, the pharmaceutical
composition comprises one or more binding proteins of the invention and one or more
prophylactic or therapeutic agents other than binding proteins of the invention for treating a
disorder. In an embodiment, the prophylactic or therapeutic agents are useful for or have been or
currently are being used in the prevention, treatment, management, or amelioration of a disorder
or one or more symptoms thereof. In accordance with these embodiments, the composition may
further comprise a carrier, diluent or excipient.
The binding proteins of the invention can be incorporated into pharmaceutical
compositions suitable for administration to a subject. Typically, the pharmaceutical composition
comprises a binding protein of the invention and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like
that are physiologically compatible. Examples of pharmaceutically acceptable carriers include
one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as
well as combinations thereof. In some embodiments, isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride, are included in the composition.
Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances
such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody or antibody portion.
Various delivery systems are known and can be used to administer one or more antibodies
of the invention or the combination of one or more antibodies of the invention and a prophylactic
agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or
one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the antibody or antibody fragment, receptor- mediated
endocytosis (see, e. g., Wu and Wu (1987) J. Biol. Chem. 262:4429-4432), construction of a
nucleic acid as part of a retroviral or other vector, etc. Methods of administering a prophylactic or
therapeutic agent of the invention include, but are not limited to, parenteral administration (e.g.,
intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural
administration, intratumoral administration, and mucosal adminsitration (e.g., intranasal and oral
routes). In addition, pulmonary administration can be employed, e.g., by use of an inhaler or
nebulizer, and a formulation with an aerosolizing agent. See, e.g., U.S. Patent Nos. 6,019,968;
5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT
Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO
99/66903. In one embodiment, a binding protein of the invention, combination therapy, or a
composition of the invention is administered using Alkermes AIR® pulmonary drug delivery
technology (Alkermes, Inc., Cambridge, Mass.). In a specific embodiment, prophylactic or
therapeutic agents of the invention are administered intramuscularly, intravenously,
intratumorally, orally, intranasally, pulmonary, or subcutaneously. The prophylactic or therapeutic
agents may be administered by any convenient route, for example by infusion or bolus injection,
by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.) and may be administered together with other biologically active agents.
Administration can be systemic or local.
In an embodiment, specific binding of antibody-coupled carbon nanotubes (CNTs) to
tumor cells in vitro, followed by their highly specific ablation with near-infrared (MR) light can
be used to target tumor cells. For example, biotinylated polar lipids can be used to prepare stable,
biocompatible, noncytotoxic CNT dispersions that are then attached to one or two different
neutralite avidin-derivatized DVD-Igs directed against one or more tumor antigens (e.g., CD22)
(Chakravarty, P. et al. (2008) Proc. Natl. Acad. Sci. USA 105:8697-8702.
In a specific embodiment, it may be desirable to administer the prophylactic or
therapeutic agents of the invention locally to the area in need of treatment; this may be achieved
by, for example, and not by way of limitation, local infusion, by injection, or by means of an
implant, said implant being of a porous or non-porous material, including membranes and
matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen
matrices. In one embodiment, an effective amount of one or more antibodies of the invention
antagonists is administered locally to the affected area to a subject to prevent, treat, manage,
and/or ameliorate a disorder or a symptom thereof. In another embodiment, an effective amount
of one or more antibodies of the invention is administered locally to the affected area in
combination with an effective amount of one or more therapies (e. g., one or more prophylactic or
therapeutic agents) other than a binding protein of the invention of a subject to prevent, treat,
manage, and/or ameliorate a disorder or one or more symptoms thereof.
In another embodiment, the prophylactic or therapeutic agent can be delivered in a
controlled release or sustained release system. In one embodiment, a pump may be used to
achieve controlled or sustained release (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed.
Eng. 14: 20; Buchwald et al. (1980) Surgery 88: 507; Saudek et al. (1989) N. Engl. J. Med. 321:
574). In another embodiment, polymeric materials can be used to achieve controlled or sustained
release of the therapies of the present disclosure (see e.g., Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York
(1984); Ranger and Peppas (1983) J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also
Levy et al. (1985) Science 228: 190; During et al. (1989) Ann. Neurol. 25: 351; Howard et al.
(1989) J.Neurosurg. 71: 105); U.S. Patent Nos. 5,679,377; 5, 916,597; 5,912,015; 5,989,463; and
5,128,326; and PCT Publication Nos. WO 99/15154; WO 99/20253. Examples of polymers used
in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl
methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone),
poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-coglycolides)
(PLGA), and polyorthoesters. In an embodiment, the polymer used in a sustained
release formulation is inert, free of leachable impurities, stable on storage, sterile, and
biodegradable. In yet another embodiment, a controlled or sustained release system can be placed
in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the
systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2,
pp. 115-138(1984)).
Controlled release systems are discussed in the review by Langer (1990) Science 249:
1527-1533). Any technique known to one of skill in the art can be used to produce sustained
release formulations comprising one or more therapeutic agents of the present disclosure. See,
e.g., U. S. Patent No. 4,526, 938; PCT Publication Nos. WO 91/05548; WO 96/20698, Ning et al.
(1996) Radiotherap. Oncol. 39: 179-189; Song et al. (1995) PDA J. Pharma. Sci. Tech. 50:372-
397; Cleek et al. (1997) Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-854, and Lam et al.
(1997) Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759- 760.
In a specific embodiment, where the composition of the invention is a nucleic acid
encoding a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to
promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of
an appropriate nucleic acid expression vector and administering it so that it becomes intracellular,
e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use
of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cellsurface
receptors or transfecting agents, or by administering it in linkage to a homeobox-like
peptide which is known to enter the nucleus (see, e.g., Joliot et al. (1991) Proc. Natl. Acad. Sci.
USA 88:1864-1868). Alternatively, a nucleic acid can be introduced intracellularly and
incorporated within host cell DNA for expression by homologous recombination.
A pharmaceutical composition of the invention is formulated to be compatible with its
intended route of administration. Examples of routes of administration include, but are not limited
to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation),
transdermal (e.g., topical), transmucosal, and rectal administration. In a specific embodiment, the
composition is formulated in accordance with routine procedures as a pharmaceutical composition
adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to
human beings. Typically, compositions for intravenous administration are solutions in sterile
isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent
and a local anesthetic such as lignocamne to ease pain at the site of the injection.
If the compositions of the invention are to be administered topically, the compositions can
be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g.,
Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th
ed., Mack Pub. Co., Easton, Pa. (1995). In an embodiment, for non- sprayable topical dosage
forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients
compatible with topical application and having a dynamic viscosity greater than water are
employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions,
creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or
mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for
influencing various properties, such as, for example, osmotic pressure. Other suitable topical
dosage forms include sprayable aerosol preparations wherein the active ingredient, in an
embodiment, in combination with a solid or liquid inert carrier, is packaged in a mixture with a
pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers
or humectants can also be added to pharmaceutical compositions and dosage forms if desired.
Examples of such additional ingredients are well-known in the art.
If the method of the invention comprises intranasal administration of a composition, the
composition can be formulated in an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to the present invention can be
conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the
case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or
insufflator may be formulated containing a powder mix of the compound and a suitable powder
base such as lactose or starch.
If the method of the invention comprises oral administration, compositions can be
formulated orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the
like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable
excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or
hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium
hydrogen phosphate) ; lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g.,
potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The
tablets may be coated by methods well-known in the art. Liquid preparations for oral
administration may take the form of, but not limited to, solutions, syrups or suspensions, or they
may be presented as a dry product for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with pharmaceutically
acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or
hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer
salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated for slow release, controlled release, or sustained
release of a prophylactic or therapeutic agent(s).
The method of the invention may comprise pulmonary administration, e.g., by use of an
inhaler or nebulizer, of a composition formulated with an aerosolizing agent. See, e.g., U.S. Patent
Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and
4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO
98/31346; and WO 99/66903. In a specific embodiment, a binding protein of the invention,
combination therapy, and/or composition of the invention is administered using Alkermes AIR®
pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).
The method of the invention may comprise administration of a composition formulated
for parenteral administration by injection (e.g., by bolus injection or continuous infusion).
Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multidose
containers) with an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active
ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogenfree
water) before use.
The methods of the invention may additionally comprise of administration of
compositions formulated as depot preparations. Such long acting formulations may be
administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compositions may be formulated with suitable polymeric or
hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble salt).
The methods of the invention encompasse administration of compositions formulated as
neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as
those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
Generally, the ingredients of compositions are supplied either separately or mixed
together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate
in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active
agent. Where the mode of administration is infusion, composition can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or saline. Where the mode of
administration is by injection, an ampoule of sterile water for injection or saline can be provided
so that the ingredients may be mixed prior to administration.
In particular, the invention also provides that one or more of the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention is packaged in a hermetically
sealed container such as an ampoule or sachette indicating the quantity of the agent. In one
embodiment, one or more of the prophylactic or therapeutic agents, or pharmaceutical
compositions of the invention is supplied as a dry sterilized lyophilized powder or water free
concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline)
to the appropriate concentration for administration to a subject. In an embodiment, one or more of
the prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied
as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5
mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at
least 75 mg, or at least 100 mg. The lyophilized prophylactic or therapeutic agents or
pharmaceutical compositions of the invention should be stored at between 2° C. and 8° C. in its
original container and the prophylactic or therapeutic agents, or pharmaceutical compositions of
the invention should be administered within 1 week, e.g., within 5 days, within 72 hours, within
48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or
within 1 hour after being reconstituted. In an alternative embodiment, one or more of the
prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied in
liquid form in a hermetically sealed container indicating the quantity and concentration of the
agent. In an embodiment, the liquid form of the administered composition is supplied in a
hermetically sealed container at least 0.25 mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5
mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml,
at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid form should be stored at
between 2° C. and 8° C. in its original container.
The binding proteins of the invention can be incorporated into a pharmaceutical
composition suitable for parenteral administration. In an embodiment, the antibody or antibodyportions
will be prepared as an injectable solution containing 0.1-250 mg/ml binding protein. The
injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or
amber vial, ampule or pre-filled syringe. The buffer can be L-histidine (1-50 mM), optimally 5-
10mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but are not limited to,
sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride
can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150
mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form,
principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose
and lactose. Other suitable bulking agents include glycine and arginine, either of which can be
included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration
of 0.005-0.01 %). Additional surfactants include but are not limited to polysorbate 20 and BRIJ
surfactants. The pharmaceutical composition comprising the binding proteins of the invention
prepared as an injectable solution for parenteral administration, can further comprise an agent
useful as an adjuvant, such as those used to increase the absorption, or dispersion of a therapeutic
protein (e.g., antibody). A particularly useful adjuvant is hyaluronidase, such as Hylenex®
(recombinant human hyaluronidase). Addition of hyaluronidase in the injectable solution
improves human bioavailability following parenteral administration, particularly subcutaneous
administration. It also allows for greater injection site volumes (i.e., greater than 1 ml) with less
pain and discomfort, and minimum incidence of injection site reactions (see PCT Publication No.
WO 2004/078140, and U.S. Patent Publication No. 2006/104968).
The compositions of this invention may be in a variety of forms. These include, for
example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and
suppositories. The form chosen depends on the intended mode of administration and therapeutic
application. Typical compositions are in the form of injectable or infusible solutions, such as
compositions similar to those used for passive immunization of humans with other antibodies.
The chosen mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal,
intramuscular). In an embodiment, the antibody is administered by intravenous infusion or
injection. In another embodiment, the antibody is administered by intramuscular or subcutaneous
injection.
Therapeutic compositions typically must be sterile and stable under the conditions of
manufacture and storage. The composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile
injectable solutions can be prepared by incorporating the active compound (i.e., antibody or
antibody portion) in the required amount in an appropriate solvent with one or a combination of
ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions
are prepared by incorporating the active compound into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those enumerated herein. In the case of
sterile, lyophilized powders for the preparation of sterile injectable solutions, the methods of
preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously sterile-filtered solution thereof. The proper
fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion and by the use of surfactants.
Prolonged absorption of injectable compositions can be brought about by including, in the
composition, an agent that delays absorption, for example, monostearate salts and gelatin.
The binding proteins of the present invention can be administered by a variety of methods
known in the art, although for many therapeutic applications, in an embodiment, the route/mode of
administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated
by the skilled artisan, the route and/or mode of administration will vary depending upon the desired
results. In certain embodiments, the active compound may be prepared with a carrier that will
protect the compound against rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and poly lactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
In certain embodiments, a binding protein of the invention may be orally administered,
for example, with an inert diluent or an assimilable edible carrier. The compound (and other
ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed
into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration,
the compounds may be incorporated with excipients and used in the form of ingestible tablets,
buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer
a compound of the invention by other than parenteral administration, it may be necessary to coat
the compound with, or co-administer the compound with, a material to prevent its inactivation.
Supplementary active compounds can also be incorporated into the compositions. In
certain embodiments, a binding protein of the invention is coformulated with and/or
coadministered with one or more additional therapeutic agents that are useful for treating
disorders with binding protein of the invention. For example, a binding protein of the invention
may be coformulated and/or coadministered with one or more additional antibodies that bind
other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules).
Furthermore, one or more antibodies of the invention may be used in combination with two or
more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize
lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or
complications associated with the various monotherapies.
In certain embodiments, a binding protein is linked to a half-life extending vehicle
known in the art. Such vehicles include, but are not limited to, the Fc domain, polyethylene
glycol, and dextran. Such vehicles are described, e.g., in U.S. Patent No. 6,660,843 and published
PCT Publication No. WO 99/25044.
In a specific embodiment, nucleic acid sequences encoding a binding protein of the
invention or another prophylactic or therapeutic agent of the invention are administered to treat,
prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene
therapy. Gene therapy refers to therapy performed by the administration to a subject of an
expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids
produce their encoded antibody or prophylactic or therapeutic agent of the invention that mediates
a prophylactic or therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the
present invention. For general reviews of the methods of gene therapy, see Goldspiel et al. (1993)
Clinical Pharmacy 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95; Tolstoshev (1993) Ann.
Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) Science 260:926- 932; and Morgan and
Anderson (1993) Ann. Rev. Biochem. 62:191-217; May (1993) TIBTECH 11(5):155-215.
Methods commonly known in the art of recombinant DNA technology which can be used are
described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &Sons,
NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY (1990). Detailed descriptions of various methods of gene therapy are disclosed in U.S. Patent
Publication No. 20090297514.
The binding proteins of the invention are useful in treating various diseases wherein the
targets that are recognized by the binding proteins are detrimental. Such diseases include, but are
not limited to, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme
arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus,
Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes
mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft versus host
disease, organ transplant rejection, acute or chronic immune disease associated with organ
transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's
granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic
active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia,
infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse
myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary
cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease,
sporadic, polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's
syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative
arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,
enteropathic synovitis, chlamydia, yersinia and salmonella associated arthropathy,
spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune
haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous
candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis,
Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases,
Hepatitis B, Hepatitis C, common varied immunodeficiency (common variable
hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature
ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory
interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial
lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis
associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease,
systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated
lung disease, Sjogren's disease associated lung disease, ankylosing spondylitis associated lung
disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced
interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic
pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty
arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid
hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated
hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute
immune disease associated with organ transplantation, chronic immune disease associated with
organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis
type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS,
glomerulonephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all
subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute
rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's syndrome,
Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia,
autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic
uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis,
alcohol-induced liver injury, choleosatatis, idiosyncratic liver disease, Drug-Induced hepatitis,
Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci (GBS) infection, mental
disorders (e.g., depression and schizophrenia), Th2 Type and Thl Type mediated diseases, acute
and chronic pain (different forms of pain), and cancers such as lung, breast, stomach, bladder,
colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia
and lymphoma), Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic or infectious
processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),
acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial
ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic
contact dermatitis, allergic rhinitis, allograft rejection, alpha-1- antitrypsin deficiency,
amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti cd3
therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aordic and
peripheral aneuryisms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous
fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B
cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch
block, Burkitt's lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors,
cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection,
cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia,
chemotherapy associated disorders, chromic myelocytic leukemia (CML), chronic alcoholism,
chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive
pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive
heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease,
Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated
disorders, Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis,
dermatologic conditions, diabetes, diabetes mellitus, diabetic aterosclerotic disease, Diffuse
Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's
Syndrome in middle age, drug- induced movement disorders induced by drugs which block CNS
dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy,
epiglottitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders,
familial hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's
ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer,
glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive
sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallerrorden-Spatz
disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis,
hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage,
hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease,
hyperkinetic movement disorders, hypersensitity reactions, hypersensitivity pneumonitis,
hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation,
idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity,
Asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza a, ionizing
radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia- reperfusion injury, ischemic
stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney
transplant rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal system,
lipedema, liver transplant rejection, lymphederma, malaria, malignamt Lymphoma, malignant
histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, migraine
headache, mitochondrial multi.system disorder, mixed connective tissue disease, monoclonal
gammopathy, multiple myeloma, multiple systems degenerations (Mencel Dejerine- Thomas Shi-
Drager and Machado-Joseph), myasthenia gravis, mycobacterium avium intracellulare,
mycobacterium tuberculosis, myelodyplastic syndrome, myocardial infarction, myocardial
ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis,
nephrosis, neurodegenerative diseases, neurogenic I muscular atrophies , neutropenic fever, nonhodgkins
lymphoma, occlusion of the abdominal aorta and its branches, occulsive arterial
disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures,
organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic
syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory
disease, perennial rhinitis, pericardial disease, peripheral atherlosclerotic disease, peripheral
vascular disorders, peritonitis, pernicious anemia, Pneumocystis carinii pneumonia, pneumonia,
POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,
and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy
syndrome, preeclampsia, Progressive supranucleo Palsy, primary pulmonary hypertension,
radiation therapy, Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease,
regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive
cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile Dementia of Lewy body type,
seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes
syndrome, small bowel transplant rejection, solid tumors, specific arrythmias, spinal ataxia,
spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum,
Subacute sclerosing panencephalitis, Syncope, syphilis of the cardiovascular system, systemic
anaphalaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid
arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans, thrombocytopenia,
toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV
hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose
veins, .vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal
infections, vital encephalitis/aseptic meningitis, vital-associated hemaphagocytic syndrome,
Wernicke- Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue, (see
PCT Publication Nos. WO 2002/097048; WO 95/24918; and WO 00/56772).
The DVD-Igs of the invention may also treat one or more of the following
diseases: Acute coronary syndromes, Acute Idiopathic Polyneuritis, Acute Inflammatory
Demyelinating Polyradiculoneuropathy, Acute ischemia, Adult Still's Disease, Alopecia areata,
Anaphylaxis, Anti-Phospholipid Antibody Syndrome, Aplastic anemia, Arteriosclerosis, Atopic
eczema, Atopic dermatitis, Autoimmune dermatitis, Autoimmune disorder associated with
Streptococcus infection, Autoimmune hearingloss, Autoimmune Lymphoproliferative Syndrome
(ALPS), Autoimmune myocarditis, autoimmune thrombocytopenia (AITP), Blepharitis,
Bronchiectasis, Bullous pemphigoid, Cardiovascular Disease, Catastrophic Antiphospholipid
Syndrome, Celiac Disease, Cervical Spondylosis, Chronic ischemia, Cicatricial pemphigoid,
Clinically isolated Syndrome (CIS) with Risk for Multiple Sclerosis, Conjunctivitis, Childhood
Onset Psychiatric Disorder, Chronic obstructive pulmonary disease (COPD), Dacryocystitis,
dermatomyositis, Diabetic retinopathy, Diabetes mellitus, Disk herniation, Disk prolaps, Drug
induced immune hemolytic anemia, Endocarditis, Endometriosis, endophthalmitis,, Episcleritis,
Erythema multiforme, erythema multiforme major, Gestational pemphigoid, Guillain-Barre
Syndrome (GBS), Hay Fever, Hughes Syndrome , Idiopathic Parkinson's Disease, idiopathic
interstitial pneumonia, IgE-mediated Allergy, Immune hemolytic anemia, Inclusion Body
Myositis, Infectious ocular inflammatory disease, Inflammatory demyelinating disease,
Inflammatory heart disease, Inflammatory kidney disease, IPF/UIP, Iritis, Keratitis,
Keratojuntivitis sicca, Kussmaul disease or Kussmaul-Meier Disease, Landry's Paralysis,
Langerhan's Cell Histiocytosis, Livedo reticularis, Macular Degeneration, malignancies,
Microscopic Polyangiitis, Morbus Bechterev, Motor Neuron Disorders, Mucous membrane
pemphigoid, Multiple Organ failure, Myasthenia Gravis, Myelodysplastic Syndrome,
Myocarditis, Nerve Root Disorders, Neuropathy, Non-A Non-B Hepatitis, Optic Neuritis,
Osteolysis, Ovarian cancer, Pauciarticular JRA, peripheral artery occlusive disease (PAOD),
peripheral vascular disease (PVD), peripheral artery disease (PAD), Phlebitis, Polyarteritis nodosa
(or periarteritis nodosa), Polychondritis, Polymyalgia Rheumatica, Poliosis, Polyarticular JRA,
Polyendocrine Deficiency Syndrome, Polymyositis, polymyalgia rheumatica (PMR), Post-Pump
Syndrome, primary parkinsonism, prostate and rectal cancer and hematopoietic malignancies
(leukemia and lymphoma), Prostatitis, Pure red cell aplasia, Primary Adrenal Insufficiency,
Recurrent Neuromyelitis Optica, Restenosis, Rheumatic heart disease, SAPHO (synovitis, acne,
pustulosis, hyperostosis, and osteitis), Scleroderma, Secondary Amyloidosis, Shock lung,
Scleritis, Sciatica, Secondary Adrenal Insufficiency, Silicone associated connective tissue disease,
Sneddon-Wilkinson Dermatosis, spondilitis ankylosans, Stevens-Johnson Syndrome (SJS),
Systemic inflammatory response syndrome, Temporal arteritis, toxoplasmic retinitis, toxic
epidermal necrolysis, Transverse myelitis, TRAPS (Tumor Necrosis Factor Receptor, Type 1
allergic reaction, Type II Diabetes, Urticaria, Usual interstitial pneumonia (UIP), Vasculitis,
Vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), Wet
macular degeneration, and Wound healing.
The binding proteins of the invention can be used to treat humans suffering from
autoimmune diseases, in particular those associated with inflammation, including, rheumatoid
arthritis, spondylitis, allergy, autoimmune diabetes, autoimmune uveitis. In an embodiment, the
binding proteins of the invention or antigen-binding portions thereof, are used to treat rheumatoid
arthritis, Crohn's disease, multiple sclerosis, insulin dependent diabetes mellitus and psoriasis.
In an embodiment, diseases that can be treated or diagnosed with the compositions and
methods of the invention include, but are not limited to, primary and metastatic cancers, including
carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,
pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder
and urothelium), female genital tract (including cervix, uterus, and ovaries as well as
choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate,
seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal,
and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those
arising from bone and soft tissues as well as Kaposi's sarcoma), tumors of the brain, nerves, eyes,
and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,
neuroblastomas, Schwannomas, and meningiomas), solid tumors arising from hematopoietic
malignancies such as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin's
lymphomas).
In an embodiment, the antibodies of the invention or antigen-binding portions thereof, are
used to treat cancer, prevention, or inhibit metastases from the tumors described herein either
when used alone or in combination with radiotherapy and/or other chemotherapeutic agents.
The antibodies of the invention, or antigen binding portions thereof, may be combined
with agents that include but are not limited to, antineoplastic agents, radiotherapy, chemotherapy
such as DNA alkylating agents, cisplatin, carboplatin, anti-tubulin agents, paclitaxel, docetaxel,
taxol, doxorubicin, gemcitabine, gemzar, anthracyclines, adriamycin, topoisomerase I inhibitors,
topoisomerase II inhibitors, 5-fluorouracil (5-FU), leucovorin, irinotecan, receptor tyrosine kinase
inhibitors (e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g., celecoxib), kinase inhibitors, and
siRNAs.
A binding protein of the invention also can be administered with one or more additional
therapeutic agents useful in the treatment of various diseases.
A binding protein of the invention can be used alone or in combination to treat such
diseases. It should be understood that the binding proteins can be used alone or in combination
with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the
skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic
agent art-recognized as being useful to treat the disease or condition being treated by the antibody
of the present invention. The additional agent also can be an agent that imparts a beneficial
attribute to the therapeutic composition e.g., an agent which affects the viscosity of the
composition.
It should further be understood that the combinations which are to be included within this
invention are those combinations useful for their intended purpose. The agents set forth below are
illustrative and arenot intended to be limited. The combinations, which are part of this invention,
can be the antibodies of the present invention and at least one additional agent selected from the
lists below. The combination can also include more than one additional agent, e.g., two or three
additional agents if the combination is such that the formed composition can perform its intended
function.
Combinations to treat autoimmune and inflammatory diseases are non-steroidal antiinflammatory
drug(s) also referred to as NSAIDS which include drugs like ibuprofen. Other
combinations are corticosteroids including prednisolone; the well known side-effects of steroid
use can be reduced or even eliminated by tapering the steroid dose required when treating patients
in combination with the DVD Igs of this invention. Non-limiting examples of therapeutic agents
for rheumatoid arthritis with which an antibody, or antibody portion, of the invention can be
combined include the following: cytokine suppressive anti-inflammatory drug(s) (CSAIDs);
antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT,
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-23, interferons,
EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins of the invention, or antigen binding
portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3,
CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, and
CTLA or their ligands including CD154 (gp39 or CD40L).
Combinations of therapeutic agents may interfere at different points in the autoimmune
and subsequent inflammatory cascade; examples include TNF antagonists like chimeric,
humanized or human TNF antibodies, ADALIMUMAB, (PCT Publication No. WO 97/29131),
CA2 (Remicade™), CDP 571, and soluble p55 or p75 TNF receptors, derivatives, thereof,
(p75TNFRlgG (Enbrel™) or p55TNFRlgG (Lenercept), and also TNFa converting enzyme
(TACE) inhibitors; similarly IL-1 inhibitors (Interleukin-1-converting enzyme inhibitors, IL-1RA
etc.) may be effective for the same reason. Other combinations include Interleukin 11. Yet
another combination includes key players of the autoimmune response which may act parallel to,
dependent on, or in concert with, IL-12 function, especially IL-18 antagonists including IL-18
antibodies, soluble IL-18 receptors, and IL-18 binding proteins. It has been shown that IL-12 and
IL-18 have overlapping but distinct functions and a combination of antagonists to both may be
most effective. Yet another combination is non-depleting anti-CD4 inhibitors. Yet other
combinations include antagonists of the co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2)
including antibodies, soluble receptors and antagonistic ligands.
The binding proteins of the invention may also be combined with agents, such as
methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate (intramuscular and oral),
azathioprine, cochicine, corticosteroids (oral, inhaled and local injection), beta-2 adrenoreceptor
agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline),
cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporin, FK506,
rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids
such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents,
complement inhibitors, adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines, such as TNF-a or IL-1 (e.g.,IRAK, NIK, IKK , p38 or MAP kinase
inhibitors), IL-1J3 converting enzyme inhibitors, TNFa-converting enzyme (TACE) inhibitors, Tcell
signalling inhibitors, such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine
receptors and derivatives thereof (e.g.,soluble p55 or p75 TNF receptors and the derivatives
P75TNFRIgG (Enbrel™ and p55TNFRIgG (Lenercept)), sIL-lRI, sIL-lRII, and slL-6R),
antiinflammatory cytokines (e.g.,IL-4, IL-10, IL-11, IL-13 andTGFP), celecoxib, folic acid,
hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib,
sulfasalazine, methylprednisolone, meloxicam, methylprednisolone acetate, gold sodium
thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylate/apap, folate, nabumetone,
diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodone
bitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol
hcl, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium,
prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine
sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl,
misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA,
CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX740,
Roflumilast, IC-485, CDC-801, and Mesopram. Combinations include methotrexate or
leflunomide and in moderate or severe rheumatoid arthritis cases, cyclosporine.
Nonlimiting additional agents, which can also be used in combination with a binding
protein to treat rheumatoid arthritis include, but are not limited to, the following: non-steroidal
anti-inflammatory drug(s) (NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs);
CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Bayer); cA2/infliximab
(chimeric anti-TNFa antibody; Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG
fusion protein; Immunex; see e.g., (1994) Arthr. Rheum. 37: S295; (1996) J. Invest. Med. 44:
235A); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDECCE9.1/
SB 210396 (non-depleting primatized anti-CD4 antibody; IDEC/SmithKline; see e.g.,
(1995) Arthr. Rheum. 38: S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins;
Seragen; see e.g., (1993) Arthrit. Rheum. 36: 1223); Anti-Tac (humanized anti-IL-2Ra; Protein
Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;
recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4
agonists (e.g., agonist antibodies); IL-1RA (IL-l receptor antagonist; Synergen/Amgen); anakinra
(Kineret®/Amgen); TNF-bp/s-TNF (soluble TNF binding protein; see e.g., (1996) Arthr. Rheum.
39(9 (supplement)): S284; (1995) Amer. J. Physiol. - Heart and Circ. Physiol. 268: 37-42);
R973401 (phosphodiesterase Type IV inhibitor; see e.g., (1996) Arthr. Rheum. 39(9
(supplement): S282); MK-966 (COX-2 Inhibitor; see e.g., (1996) Arthr. Rheum. 39(9
(supplement): S81); Iloprost (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S82);
methotrexate; thalidomide (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S282) and
thalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatory and cytokine inhibitor;
see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S131; (1996) Inflamm. Res. 45: 103-107);
tranexamic acid (inhibitor of plasminogen activation; see e.g., (1996) Arthr. Rheum. 39(9
(supplement): S284); T-614 (cytokine inhibitor; see e.g., (1996) Arthr. Rheum. 39(9
(supplement): S282); prostaglandin El (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S282);
Tenidap (non-steroidal anti-inflammatory drug; see e.g., (1996) Arthr. Rheum. 39(9 (supplement):
S280); Naproxen (non-steroidal anti-inflammatory drug; see e.g., (1996) Neuro. Report 7: 1209-
1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal antiinflammatory
drug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal
anti-inflammatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine (see
e.g., (1996) Arthr. Rheum. 39(9 (supplement): S281); Azathioprine (see e.g., (1996) Arthr.
Rheum. 39(9 (supplement): S281); ICE inhibitor (inhibitor of the enzyme interleukin-1ß
converting enzyme); zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70 or lck);
VEGF inhibitor and/or VEGF-R inhibitor (inhibitors of vascular endothelial cell growth factor or
vascular endothelial cell growth factor receptor; inhibitors of angiogenesis); corticosteroid antiinflammatory
drugs (e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies; anti-IL-
18 antibodies; interleukin-11 (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S296);
interleukin-13 (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S308); interleukin -17
inhibitors (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S120); gold; penicillamine;
chloroquine; chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; total lymphoid
irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins; orally-administered
peptides and collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228 and
HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides
(ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;
T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate; minocycline; anti-
IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; see e.g., DeLuca et
al. (1995) Rheum. Dis. Clin. North Am. 21: 759-777); auranofin; phenylbutazone; meclofenamic
acid; flufenamic acid; intravenous immune globulin; zileuton; azaribine; mycophenolic acid (RS-
61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-
chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see Bruncko, M. et al. (2007) J. Med.
Chem. 50(4): 641-662); and antivirals and immune -modulating agents.
In one embodiment, the binding protein or antigen-binding portion thereof, is
administered in combination with one of the following agents for the treatment of rheumatoid
arthritis: small molecule inhibitor of KDR, small molecule inhibitor of Tie-2; methotrexate;
prednisone; celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept; infliximab;
leflunomide; naproxen; valdecoxib; sulfasalazine; methylprednisolone; ibuprofen; meloxicam;
methylprednisolone acetate; gold sodium thiomalate; aspirin; azathioprine; triamcinolone
acetonide; propxyphene napsylate/apap; folate; nabumetone; diclofenac; piroxicam; etodolac;
diclofenac sodium; oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenac
sodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl; salsalate; sulindac;
cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate sodium; prednisolone; morphine
sulfate; lidocaine hydrochloride; indomethacin; glucosamine sulfate/chondroitin; cyclosporine;
amitriptyline hcl; sulfadiazine; oxycodone hcl/acetaminophen; olopatadine hcl; misoprostol;
naproxen sodium; omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1
TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15; BIRB-796; SCIO-469;
VX-702; AMG-548; VX-740; Roflumilast; IC-485; CDC-801; andmesopram.
Non-limiting examples of therapeutic agents for inflammatory bowel disease with which
a binding protein of the invention can be combined include the following: budenoside; epidermal
growth factor; corticosteroids; cyclosporin, sulfasalazine; aminosalicylates; 6-mercaptopurine;
azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide;
antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-ip mAbs; anti-IL-6
mAbs; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; and antibodies to, or
antagonists of, other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6,
IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the
invention, or antigen binding portions thereof, can be combined with antibodies to cell surface
molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, and CD90
and their ligands. The antibodies of the invention, or antigen binding portions thereof, may also
be combined with agents, such as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, such as ibuprofen, corticosteroids such as prednisolone,
phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by proinflammatory cytokines such as
TNFa or IL-1 (e.g.JRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-ip converting enzyme
inhibitors, TNFa converting enzyme inhibitors, T-cell signalling inhibitors such as kinase
inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines,
angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g.,
soluble p55 or p75 TNF receptors, sIL-lRI, sIL-lRII, and sIL-6R) and antiinflammatory
cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGFP), and bcl-2 inhibitors.
Examples of therapeutic agents for Crohn's disease in which a binding protein can be
combined include the following: TNF antagonists, for example, anti-TNF antibodies,
ADALIMUMAB (PCT Publication No. WO 97/29131; HUMIRA), CA2 (REMICADE), CDP
571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT))
inhibitors and PDE4 inhibitors. Antibodies of the invention, or antigen binding portions thereof,
can be combined with corticosteroids, for example, budenoside and dexamethasone. Binding
proteins of the invention, or antigen binding portions thereof, may also be combined with agents
such as sulfasalazine, 5-aminosalicylic acid and olsalazine, and agents which interfere with
synthesis or action of proinflammatory cytokines such as IL-1, for example, IL-1 $ converting
enzyme inhibitors and IL-lra. Antibodies of the invention or antigen binding portion thereof may
also be used with T cell signaling inhibitors, for example, tyrosine kinase inhibitors 6-
mercaptopurines. Binding proteins of the invention, or antigen binding portions thereof, can be
combined with IL-11. Binding proteins of the invention, or antigen binding portions thereof, can
be combined with mesalamine, prednisone, azathioprine, mercaptopurine, infliximab,
methylprednisolone sodium succinate, diphenoxylate/atrop sulfate, loperamide hydrochloride,
methotrexate, omeprazole, folate, ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap,
tetracycline hydrochloride, fluocinonide, metronidazole, thimerosal/boric acid,
cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyamine sulfate, meperidine
hydrochloride, midazolam hydrochloride, oxycodone hcl/acetaminophen, promethazine
hydrochloride, sodium phosphate, sulfamethoxazole/trimethoprim, celecoxib, polycarbophil,
propoxyphene napsylate, hydrocortisone, multivitamins, balsalazide disodium, codeine
phosphate/apap, colesevelam hcl, cyanocobalamin, folic acid, levofloxacin, methylprednisolone,
natalizumab and interferon-gamma
Non-limiting examples of therapeutic agents for multiple sclerosis with which binding
proteins of the invention can be combined include the following: corticosteroids; prednisolone;
methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-
aminopyridine; tizanidine; interferon-pia (AVONEX; Biogen); interferon-pib (BETASERON;
Chiron/Berlex); interferon a-n3) (Interferon Sciences/Fujimoto), interferon-a (Alfa
Wassermann/J&J), interferon pi A-IF (Serono/Inhale Therapeutics), Peginterferon a 2b
(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; Teva Pharmaceutical Industries,
Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine; antibodies to or antagonists of
other human cytokines or growth factors and their receptors, for example, TNF, LT, IL-1, IL-2,
IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Binding
proteins of the invention can be combined with antibodies to cell surface molecules such as CD2,
CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90
or their ligands. Binding proteins of the invention, may also be combined with agents, such as
methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs,
for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents
which interfere with signalling by proinflammatory cytokines such as TNFa or IL-1 (e.g.,IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-1 (3 converting enzyme inhibitors, TACE inhibitors,
T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine
receptors and derivatives thereof (e.g.,soluble p55 or p75 TNF receptors, sIL-lRI, sIL-lRII, and
sIL-6R), antiinflammatory cytokines (e.g.,IL-4, IL-10, IL-13 and TGFP) and bcl-2 inhibitors.
Examples of therapeutic agents for multiple sclerosis in which binding proteins of the
invention can be combined include interferon-P, for example, IFNpia and IFNpib; Copaxone,
corticosteroids, caspase inhibitors, for example inhibitors of caspase-1, IL-1 inhibitors, TNF
inhibitors, and antibodies to CD40 ligand and CD80.
The binding proteins of the invention, may also be combined with agents, such as
alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone, xaliproden hydrochloride,
fampridine, glatiramer acetate, natalizumab, sinnabidol, a-immunokine NNS03, ABR-215062,
AnergiX.MS, chemokine receptor antagonists, BBR-2778, calagualine, CPI-1189, LEM
(liposome encapsulated mitoxantrone), THC.CBD (cannabinoid agonist) MBP-8298, mesopram
(PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258
(RDP-1258), sTNF-Rl, talampanel, teriflunomide,TGF-beta2, tiplimotide, VLA-4 antagonists
(for example, TR-14035, VLA4 Ultrahaler, Antegran-ELAN/Biogen), interferon gamma
antagonists, and IL-4 agonists.
Non-limiting examples of therapeutic agents for Angina with which binding proteins of
the invention can be combined include the following: aspirin, nitroglycerin, isosorbide
mononitrate, metoprolol succinate, atenolol, metoprolol tartrate, amlodipine besylate, diltiazem
hydrochloride, isosorbide dinitrate, clopidogrel bisulfate, nifedipine, atorvastatin calcium,
potassium chloride, furosemide, simvastatin, verapamil hcl, digoxin, propranolol hydrochloride,
carvedilol, lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate, nadolol, ramipril,
enoxaparin sodium, heparin sodium, valsartan, sotalol hydrochloride, fenofibrate, ezetimibe,
bumetanide, losartan potassium, lisinopril/hydrochlorothiazide, felodipine, captopril, and
bisoprolol fumarate.
Non-limiting examples of therapeutic agents for Ankylosing Spondylitis with which
binding proteins of the invention can be combined include the following: ibuprofen, diclofenac
and misoprostol, naproxen, meloxicam, indomethacin, diclofenac, celecoxib, rofecoxib,
Sulfasalazine, Methotrexate, azathioprine, minocyclin, prednisone, etanercept, and infliximab.
Non-limiting examples of therapeutic agents for Asthma with which binding proteins of
the invention can be combined include the following: albuterol, salmeterol/fluticasone,
montelukast sodium, fluticasone propionate, budesonide, prednisone, salmeterol xinafoate,
levalbuterol hcl, albuterol sulfate/ipratropium, prednisolone sodium phosphate, triamcinolone
acetonide, beclomethasone dipropionate, ipratropium bromide, azithromycin, pirbuterol acetate,
prednisolone, theophylline anhydrous, methylprednisolone sodium succinate, clarithromycin,
zaflrlukast, formoterol fumarate, influenza virus vaccine, methylprednisolone, amoxicillin
trihydrate, flunisolide, allergy injection, cromolyn sodium, fexofenadine hydrochloride,
flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhaler assist device, guaifenesin,
dexamethasone sodium phosphate, moxifloxacin hcl, doxycycline hyclate, guaifenesin/dmethorphan,
p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride, mometasone
furoate, salmeterol xinafoate, benzonatate, cephalexin, pe/hydrocodone/chlorphenir, cetirizine
hcl/pseudoephed, phenylephrine/cod/promethazine, codeine/promethazine, cefprozil,
dexamethasone, guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone, nedocromil
sodium, terbutaline sulfate, epinephrine, methylprednisolone, and metaproterenol sulfate.
Non-limiting examples of therapeutic agents for COPD with which binding proteins of
the invention can be combined include the following: albuterol sulfate/ipratropium, ipratropium
bromide, salmeterol/fluticasone, albuterol, salmeterol xinafoate, fluticasone propionate,
prednisone, theophylline anhydrous, methylprednisolone sodium succinate, montelukast sodium,
budesonide, formoterol fumarate, triamcinolone acetonide, levofloxacin, guaifenesin,
azithromycin, beclomethasone dipropionate, levalbuterol hcl, flunisolide, ceftriaxone sodium,
amoxicillin trihydrate, gatifloxacin, zafirlukast, amoxicillin/clavulanate, flunisolide/menthol,
chlorpheniramine/hydrocodone, metaproterenol sulfate, methylprednisolone, mometasone furoate,
p-ephedrine/cod/chlorphenir, pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate,
tiotropium bromide, (R,R)-formoterol, TgAAT, Cilomilast, and Roflumilast.
Non-limiting examples of therapeutic agents for HCV with which binding proteins of the
invention can be combined include the following: Interferon-alpha-2a, Interferon-alpha-2b,
Interferon-alpha conl, Interferon-alpha-nl, Pegylated interferon-alpha-2a, Pegylated interferonalpha-
2b, ribavirin, Peginterferon alfa-2b + ribavirin, Ursodeoxycholic Acid, Glycyrrhizic Acid,
Thymalfasin, Maxamine, VX-497 and any compounds that are used to treat HCV through
intervention with the following targets: HCV polymerase, HCV protease, HCV helicase, and
HCV IRES (internal ribosome entry site).
Non-limiting examples of therapeutic agents for Idiopathic Pulmonary Fibrosis with
which binding proteins of the invention can be combined include the following: prednisone,
azathioprine, albuterol, colchicine, albuterol sulfate, digoxin, gamma interferon,
methylprednisolone sod succ, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone,
cyclophosphamide, ipratropium bromide, actinomycin d, alteplase, fluticasone propionate,
levofloxacin, metaproterenol sulfate, morphine sulfate, oxycodone hcl, potassium chloride,
triamcinolone acetonide, tacrolimus anhydrous, calcium, interferon-alpha, methotrexate,
mycophenolate mofetil, and Interferon-gamma-1ß.
Non-limiting examples of therapeutic agents for Myocardial Infarction with which
binding proteins of the invention can be combined include the following: aspirin, nitroglycerin,
metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate, carvedilol,
atenolol, morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril, isosorbide
mononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate,
torsemide, retavase, losartan potassium, quinapril hc1/mag carb, bumetanide, alteplase, enalaprilat,
amiodarone hydrochloride, tirofiban hcl m-hydrate, diltiazem hydrochloride, captopril, irbesartan,
valsartan, propranolol hydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide,
cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone, interferon, sotalol
hydrochloride, potassium chloride, docusate sodium, dobutamine hcl, alprazolam, pravastatin
sodium, atorvastatin calcium, midazolam hydrochloride, meperidine hydrochloride, isosorbide
dinitrate, epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin, ezetimibe/simvastatin,
avasimibe, and cariporide.
Non-limiting examples of therapeutic agents for Psoriasis with which binding proteins of
the invention can be combined include the following: small molecule inhibitor of KDR, small
molecule inhibitor of Tie-2, calcipotriene, clobetasol propionate, triamcinolone acetonide,
halobetasol propionate, tazarotene, methotrexate, fluocinonide, betamethasone diprop augmented,
fluocinolone acetonide, acitretin, tar shampoo, betamethasone valerate, mometasone furoate,
ketoconazole, pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea,
betamethasone, clobetasol propionate/emoll, fluticasone propionate, azithromycin,
hydrocortisone, moisturizing formula, folic acid, desonide, pimecrolimus, coal tar, diflorasone
diacetate, etanercept folate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor,
methylprednisolone acetate, prednisone, sunscreen, halcinonide, salicylic acid, anthralin,
clocortolone pivalate, coal extract, coal tar/salicylic acid, coal tar/salicylic acid/sulfur,
desoximetasone, diazepam, emollient, fluocinonide/emollient, mineral oil/castor oil/na lact,
mineral oil/peanut oil, petroleum/isopropyl myristate, psoralen, salicylic acid, soap/tribromsalan,
thimerosal/boric acid, celecoxib, infliximab, cyclosporine, alefacept, efalizumab, tacrolimus,
pimecrolimus, PUVA, UVB, and sulfasalazine.
Non-limiting examples of therapeutic agents for Psoriatic Arthritis with which binding
proteins of the invention can be combined include the following: methotrexate, etanercept,
rofecoxib, celecoxib, folic acid, sulfasalazine, naproxen, leflunomide, methylprednisolone acetate,
indomethacin, hydroxychloroquine sulfate, prednisone, sulindac, betamethasone diprop
augmented, infliximab, methotrexate, folate, triamcinolone acetonide, diclofenac,
dimethylsulfoxide, piroxicam, diclofenac sodium, ketoprofen, meloxicam, methylprednisolone,
nabumetone, tolmetin sodium, calcipotriene, cyclosporine, diclofenac sodium/misoprostol,
fluocinonide, glucosamine sulfate, gold sodium thiomalate, hydrocodone bitartrate/apap,
ibuprofen, risedronate sodium, sulfadiazine, thioguanine, valdecoxib, alefacept, efalizumab and
bcl-2 inhibitors.
Non-limiting examples of therapeutic agents for Restenosis with which binding proteins
of the invention can be combined include the following: sirolimus, paclitaxel, everolimus,
tacrolimus, Zotarolimus, and acetaminophen.
Non-limiting examples of therapeutic agents for Sciatica with which binding proteins of
the invention can be combined include the following: hydrocodone bitartrate/apap, rofecoxib,
cyclobenzaprine hcl, methylprednisolone, naproxen, ibuprofen, oxycodone hcl/acetaminophen,
celecoxib, valdecoxib, methylprednisolone acetate, prednisone, codeine phosphate/apap, tramadol
hcl/acetaminophen, metaxalone, meloxicam, methocarbamol, lidocaine hydrochloride, diclofenac
sodium, gabapentin, dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin,
acetaminophen, diazepam, nabumetone, oxycodone hcl, tizanidine hcl, diclofenac
sodium/misoprostol, propoxyphene napsylate/apap, asa/oxycod/oxycodone ter,
ibuprofen/hydrocodone bit, tramadol hcl, etodolac, propoxyphene hcl, amitriptyline hcl,
carisoprodol/codeine phos/asa, morphine sulfate, multivitamins, naproxen sodium, orphenadrine
citrate, and temazepam.
Examples of therapeutic agents for SLE (Lupus) in which binding proteins of the
invention can be combined include the following: NSAIDS, for example, diclofenac, naproxen,
ibuprofen, piroxicam, indomethacin; COX2 inhibitors, for example, Celecoxib, rofecoxib,
valdecoxib; anti-malarials, for example, hydroxychloroquine; Steroids, for example, prednisone,
prednisolone, budenoside, dexamethasone; Cytotoxics, for example, azathioprine,
cyclophosphamide, mycophenolate mofetil, methotrexate; and inhibitors of PDE4 or a purine
synthesis inhibitor, for example Cellcept. Binding proteins of the invention, may also be
combined with agents such as sulfasalazine, 5-aminosalicylic acid, olsalazine, Imuran and agents
which interfere with synthesis, production or action of proinflammatory cytokines such as IL-1,
for example, caspase inhibitors like IL-ip converting enzyme inhibitors and IL-lra. Binding
proteins of the invention may also be used with T cell signaling inhibitors, for example, tyrosine
kinase inhibitors; or molecules that target T cell activation molecules, for example, CTLA-4-IgG
or anti-B7 family antibodies, and anti-PD-1 family antibodies. Binding proteins of the invention,
can be combined with IL-11 or anti-cytokine antibodies, for example, fonotolizumab (anti-IFNg
antibody), or anti-receptor receptor antibodies, for example, anti-IL-6 receptor antibody and
antibodies to B-cell surface molecules. Antibodies of the invention or antigen binding portion
thereof may also be used with LJP 394 (abetimus), agents that deplete or inactivate B-cells, for
example, Rituximab (anti-CD20 antibody), lymphostat-B (anti-BlyS antibody), TNF antagonists,
for example, anti-TNF antibodies, Adalimumab (PCT Publication No. WO 97/29131; HUMIRA),
CA2 (REMICADE), CDP 571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL) and
p55TNFRIgG (LENERCEPT)) and bcl-2 inhibitors, because bcl-2 overexpression in transgenic
mice has been demonstrated to cause a lupus like phenotype (see Marquina, R. et al. (2004) J.
Immunol. 172(11): 7177-7185), therefore inhibition is expected to have therapeutic effects.
The pharmaceutical compositions of the invention may include a "therapeutically
effective amount" or a "prophylactically effective amount" of a binding protein of the invention.
A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of
time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of
the binding protein may be determined by a person skilled in the art and may vary according to
factors such as the disease state, age, sex, and weight of the individual, and the ability of the
binding protein to elicit a desired response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody, or antibody portion, are
outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers
to an amount effective, at dosages and for periods of time necessary, to achieve the desired
prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be less than the therapeutically
effective amount.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a
therapeutic or prophylactic response). For example, a single bolus may be administered, several
divided doses may be administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous
to formulate parenteral compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as
unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined
quantity of active compound calculated to produce the desired therapeutic effect in association
with the required pharmaceutical carrier. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on (a) the unique characteristics of the active
compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active compound for the treatment of
sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically or prophylactically effective
amount of a binding protein of the invention is 0.1-20 mg/kg, for example, 1-10 mg/kg. It is to be
noted that dosage values may vary with the type and severity of the condition to be alleviated. It
is to be further understood that for any particular subject, specific dosage regimens should be
adjusted over time according to the individual need and the professional judgment of the person
administering or supervising the administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the scope or practice of the claimed
composition.
It will be readily apparent to those skilled in the art that other suitable modifications and
adaptations of the methods of the invention described herein are obvious and may be made using
suitable equivalents without departing from the scope of the invention or the embodiments
disclosed herein. Having now described the present invention in detail, the same will be more
clearly understood by reference to the following examples, which are included for purposes of
illustration only and are not intended to be limiting of the invention.
V. Diagnostics
The disclosure herein also provides diagnostic applications. This is further elucidated below.
I. Method of Assay
The present disclosure also provides a method for determining the presence, amount or
concentration of an analyte (or a fragment thereof) in a test sample using at least one DVD-Ig as
described herein. Any suitable assay as is known in the art can be used in the method. Examples
include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal,
polyclonal and/or DVD-Ig sandwich immunoassays or any variation thereof (e.g.,
monoclonal/DVD-Ig, DVD-Ig/polyclonal, etc.), including radioisotope detection
(radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked
immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis,
MN))), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization
immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), bioluminescence
resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc. In a SELDIbased
immunoassay, a capture reagent that specifically binds an analyte (or a fragment thereof) of
interest is attached to the surface of a mass spectrometry probe, such as a pre-activated protein
chip array. The analyte (or a fragment thereof) is then specifically captured on the biochip, and
the captured analyte (or a fragment thereof) is detected by mass spectrometry. Alternatively, the
analyte (or a fragment thereof) can be eluted from the capture reagent and detected by traditional
MALDI (matrix-assisted laser desorption/ionization) or by SELDI. A chemiluminescent
microparticle immunoassay, in particular one employing the ARCHITECT® automated analyzer
(Abbott Laboratories, Abbott Park, IL), is an example of a preferred immunoassay.
Methods well-known in the art for collecting, handling and processing urine, blood,
serum and plasma, and other body fluids, are used in the practice of the present disclosure, for
instance, when a DVD-Ig as described herein is employed as an immunodiagnostic reagent and/or
in an analyte immunoassay kit. The test sample can comprise further moieties in addition to the
analyte of interest, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors,
proteins, peptides, polypeptides, oligonucleotides and/or polynucleotides. For example, the
sample can be a whole blood sample obtained from a subject. It can be necessary or desired that a
test sample, particularly whole blood, be treated prior to immunoassay as described herein, e.g.,
with a pretreatment reagent. Even in cases where pretreatment is not necessary (e.g., most urine
samples), pretreatment optionally can be done (e.g., as part of a regimen on a commercial
platform).
The pretreatment reagent can be any reagent appropriate for use with the immunoassay
and kits of the invention. The pretreatment optionally comprises: (a) one or more solvents (e.g.,
methanol and ethylene glycol) and optionally, salt, (b) one or more solvents and salt, and
optionally, detergent, (c) detergent, or (d) detergent and salt. Pretreatment reagents are known in
the art, and such pretreatment can be employed, e.g., as used for assays on Abbott TDx,
AxSYM®, and ARCHITECT® analyzers (Abbott Laboratories, Abbott Park, IL), as described in
the literature (see, e.g., Yatscoff et al., (1990) Clin. Chem. 36: 1969-1973 and Wallemacq et al.
(1999) Clin. Chem. 45: 432-435), and/or as commercially available. Additionally, pretreatment
can be done as described in U.S. Patent No. 5,135,875, EU Patent Pubublication No. EU0471293,
U.S. Patent No. 6,660,843, and U.S. Patent Application No. 20080020401. The pretreatment
reagent can be a heterogeneous agent or a homogeneous agent.
With use of a heterogeneous pretreatment reagent, the pretreatment reagent precipitates
analyte binding protein (e.g., protein that can bind to an analyte or a fragment thereof) present in
the sample. Such a pretreatment step comprises removing any analyte binding protein by
separating from the precipitated analyte binding protein the supernatant of the mixture formed by
addition of the pretreatment agent to sample. In such an assay, the supernatant of the mixture
absent any binding protein is used in the assay, proceeding directly to the antibody capture step.
With use of a homogeneous pretreatment reagent there is no such separation step. The
entire mixture of test sample and pretreatment reagent are contacted with a labeled specific
binding partner for analyte (or a fragment thereof), such as a labeled anti-analyte antibody (or an
antigenically reactive fragment thereof). The pretreatment reagent employed for such an assay
typically is diluted in the pretreated test sample mixture, either before or during capture by the
first specific binding partner. Despite such dilution, a certain amount of the pretreatment reagent
is still present (or remains) in the test sample mixture during capture. According to the invention,
the labeled specific binding partner can be a DVD-Ig (or a fragment, a variant, or a fragment of a
variant thereof).
In a heterogeneous format, after the test sample is obtained from a subject, a first mixture
is prepared. The mixture contains the test sample being assessed for an analyte (or a fragment
thereof) and a first specific binding partner, wherein the first specific binding partner and any
analyte contained in the test sample form a first specific binding partner-analyte complex.
Preferably, the first specific binding partner is an anti-analyte antibody or a fragment thereof. The
first specific binding partner can be a DVD-Ig (or a fragment, a variant, or a fragment of a variant
thereof) as described herein. The order in which the test sample and the first specific binding
partner are added to form the mixture is not critical. Preferably, the first specific binding partner
is immobilized on a solid phase. The solid phase used in the immunoassay (for the first specific
binding partner and, optionally, the second specific binding partner) can be any solid phase
known in the art, such as, but not limited to, a magnetic particle, a bead, a test tube, a microtiter
plate, a cuvette, a membrane, a scaffolding molecule, a film, a filter paper, a disc and a chip.
After the mixture containing the first specific binding partner-analyte complex is formed,
any unbound analyte is removed from the complex using any technique known in the art. For
example, the unbound analyte can be removed by washing. Desirably, however, the first specific
binding partner is present in excess of any analyte present in the test sample, such that all analyte
that is present in the test sample is bound by the first specific binding partner.
After any unbound analyte is removed, a second specific binding partner is added to the
mixture to form a first specific binding partner-analyte-second specific binding partner complex.
The second specific binding partner is preferably an anti-analyte antibody that binds to an epitope
on analyte that differs from the epitope on analyte bound by the first specific binding partner.
Moreover, also preferably, the second specific binding partner is labeled with or contains a
detectable label as described above. The second specific binding partner can be a DVD-Ig (or a
fragment, a variant, or a fragment of a variant thereof) as described herein.
Any suitable detectable label as is known in the art can be used. For example, the
detectable label can be a radioactive label (such as 3H, 1251,35S, 14C, 32P, and 33P), an enzymatic
label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase,
and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides;
luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as
fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-car^oxyfluorescein, 5(6)-
carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein
isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g.,
zinc sulfide-capped cadmium selenide), a thermometric label, or an immuno-polymerase chain
reaction label. An introduction to labels, labeling procedures and detection of labels is found in
Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y.
(1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996),
which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene,
Oregon. A fluorescent label can be used in FPIA (see, e.g., U.S. Patent Nos. 5,593,896;
5,573,904; 5,496,925; 5,359,093; and 5,352,803. An acridinium compound can be used as a
detectable label in a homogeneous or heterogeneous chemiluminescent assay (see, e.g.,
Adamczyk et al. (2006) Bioorg. Med. Chem. Lett. 16: 1324-1328; Adamczyk et al. (2004)
Bioorg. Med. Chem. Lett. 4: 2313-2317; Adamczyk et al. (2004) Biorg. Med. Chem. Lett. 14:
3917-3921; and Adamczyk et al. (2003) Org. Lett. 5: 3779-3782).
A preferred acridinium compound is an acridinium-9-carboxamide. Methods for
preparing acridinium 9-carboxamides are described in Mattingly (1991) J. Biolumin.
Chemilumin. 6: 107-114; Adamczyk et al. (1998) J. Org. Chem. 63: 5636-5639; Adamczyk et al.
(1999) Tetrahedron 55: 10899-10914; Adamczyk et al. (1999) Org. Lett. 1: 779-781; Adamczyk
et al. (2000) Biocon. Chem. 11: 714-724; Mattingly et al., In Luminescence Biotechnology:
Instruments and Applications; Dyke, K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002);
Adamczyk et al. (2003) Org. Lett. 5: 3779-3782; and U.S. Patent Nos. 5,468,646; 5,543,524; and
5,783,699. Another preferred acridinium compound is an acridinium-9-carboxylate aryl ester.
An example of an acridinium-9-carboxylate aryl ester is 10-methyl-9-
(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor,
MI). Methods for preparing acridinium 9-carboxylate aryl esters are described in McCapra et al.
(1965) Photochem. Photobiol. 4: 1111-21; Razavi et al. (2000) Luminescence 15: 245-249;
Razavi et al. (2000) Luminescence 15: 239-244; and U.S. Patent No. 5,241,070. Further details
regarding acridinium-9-carboxylate aryl ester and its use are set forth in US Patent Publication
No. 20080248493.
Chemiluminescent assays (e.g., using acridinium as described above or other
chemiluminescent agents) can be performed in accordance with the methods described in
Adamczyk et al. (2006) Anal. Chim. Acta 579(1): 61-67. While any suitable assay format can be
used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, Oak
Ridge, TN) enables the assay of multiple samples of small volumes rapidly.
The order in which the test sample and the specific binding partners) are added to form
the mixture for chemiluminescent assay is not critical. If the first specific binding partner is
detectably labeled with a chemiluminescent agent such as an acridinium compound, detectably
labeled first specific binding partner-analyte complexes form. Alternatively, if a second specific
binding partner is used and the second specific binding partner is detectably labeled with a
chemiluminescent agent such as an acridinium compound, detectably labeled first specific binding
partner-analyte-second specific binding partner complexes form. Any unbound specific binding
partner, whether labeled or unlabeled, can be removed from the mixture using any technique
known in the art, such as washing.
Hydrogen peroxide can be generated in situ in the mixture or provided or supplied to the
mixture (e.g., the source of the hydrogen peroxide being one or more buffers or other solutions
that are known to contain hydrogen peroxide) before, simultaneously with, or after the addition of
an above-described acridinium compound. Hydrogen peroxide can be generated in situ in a
number of ways such as would be apparent to one skilled in the art.
Upon the simultaneous or subsequent addition of at least one basic solution to the sample,
a detectable signal, namely, a chemiluminescent signal, indicative of the presence of analyte is
generated. The basic solution contains at least one base and has a pH greater than or equal to 10,
preferably, greater than or equal to 12. Examples of basic solutions include, but are not limited
to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide,
magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium
carbonate, and calcium bicarbonate. The amount of basic solution added to the sample depends
on the concentration of the basic solution. Based on the concentration of the basic solution used,
one skilled in the art can easily determine the amount of basic solution to add to the sample.
The chemiluminescent signal that is generated can be detected using routine techniques
known to those skilled in the art. Based on the intensity of the signal generated, the amount of
analyte in the sample can be quantified. Specifically, the amount of analyte in the sample is
proportional to the intensity of the signal generated. The amount of analyte present can be
quantified by comparing the amount of light generated to a standard curve for analyte or by
comparison to a reference standard. The standard curve can be generated using serial dilutions or
solutions of known concentrations of analyte by mass spectroscopy, gravimetric methods, and
other techniques known in the art. While the above is described with emphasis on use of an
acridinium compound as the chemiluminescent agent, one of ordinary skill in the art can readily
adapt this description for use of other chemiluminescent agents.
Analyte immunoassays generally can be conducted using any format known in the art,
such as, but not limited to, a sandwich format. Specifically, in one immunoassay format, at least
two antibodies are employed to separate and quantify analyte, such as human analyte, or a
fragment thereof in a sample. More specifically, the at least two antibodies bind to different
epitopes on an analyte (or a fragment thereof) forming an immune complex, which is referred to
as a "sandwich." Generally, in the immunoassays one or more antibodies can be used to capture
the analyte (or a fragment thereof) in the test sample (these antibodies are frequently referred to as
a "capture" antibody or "capture" antibodies) and one or more antibodies can be used to bind a
detectable (namely, quantifiable) label to the sandwich (these antibodies are frequently referred to
as the "detection antibody," the "detection antibodies," the "conjugate," or the "conjugates").
Thus, in the context of a sandwich immunoassay format, a binding protein or a DVD-Ig (or a
fragment, a variant, or a fragment of a variant thereof) as described herein can be used as a
capture antibody, a detection antibody, or both. For example, one binding protein or DVD-Ig
having a domain that can bind a first epitope on an analyte (or a fragment thereof) can be used as
a capture agent and/or another binding protein or DVD-Ig having a domain that can bind a second
epitope on an analyte (or a fragment thereof) can be used as a detection agent. In this regard, a
binding protein or a DVD-Ig having a first domain that can bind a first epitope on an analyte (or a
fragment thereof) and a second domain that can bind a second epitope on an analyte (or a
fragment thereof) can be used as a capture agent and/or a detection agent. Alternatively, one
binding protein or DVD-Ig having a first domain that can bind an epitope on a first analyte (or a
fragment thereof) and a second domain that can bind an epitope on a second analyte (or a
fragment thereof) can be used as a capture agent and/or a detection agent to detect, and optionally
quantify, two or more analytes. In the event that an analyte can be present in a sample in more
than one form, such as a monomeric form and a dimeric/multimeric form, which can be
homomeric or heteromeric, one binding protein or DVD-Ig having a domain that can bind an
epitope that is only exposed on the monomeric form and another binding protein or DVD-Ig
having a domain that can bind an epitope on a different part of a dimeric/multimeric form can be
used as capture agents and/or detection agents, thereby enabling the detection, and optional
quantification, of different forms of a given analyte. Furthermore, employing binding proteins or
DVD-Igs with differential affinities within a single binding protein or DVD-Ig and/or between
binding proteins or DVD-Igs can provide an avidity advantage. In the context of immunoassays
as described herein, it generally may be helpful or desired to incorporate one or more linkers
within the structure of a binding protein or a DVD-Ig. When present, optimally the linker should
be of sufficient length and structural flexibility to enable binding of an epitope by the inner
domains as well as binding of another epitope by the outer domains. In this regard, when a
binding protein or a DVD-Ig can bind two different analytes and one analyte is larger than the
other, desirably the larger analyte is bound by the outer domains.
Generally speaking, a sample being tested for (for example, suspected of containing)
analyte (or a fragment thereof) can be contacted with at least one capture agent (or agents) and at
least one detection agent (which can be a second detection agent or a third detection agent or even
a successively numbered agent, e.g., as where the capture and/or detection agent comprises
multiple agents) either simultaneously or sequentially and in any order. For example, the test
sample can be first contacted with at least one capture agent and then (sequentially) with at least
one detection agent. Alternatively, the test sample can be first contacted with at least one
detection agent and then (sequentially) with at least one capture agent. In yet another alternative,
the test sample can be contacted simultaneously with a capture agent and a detection agent.
In the sandwich assay format, a sample suspected of containing analyte (or a fragment
thereof) is first brought into contact with at least one first capture agent under conditions that
allow the formation of a first agent/analyte complex. If more than one capture agent is used, a
first capture agent/analyte complex comprising two or more capture agents is formed. In a
sandwich assay, the agents, i.e., preferably, the at least one capture agent, are used in molar excess
amounts of the maximum amount of analyte (or a fragment thereof) expected in the test sample.
For example, from about 5 μg to about 1 mg of agent per mL of buffer (e.g., microparticle coating
buffer) can be used.
Competitive inhibition immunoassays, which are often used to measure small analytes
because binding by only one antibody (i.e., a binding protein and/or a DVD-Ig in the context of
the present disclosure) is required, comprise sequential and classic formats. In a sequential
competitive inhibition immunoassay a capture agent to an analyte of interest is coated onto a well
of a microtiter plate or other solid support. When the sample containing the analyte of interest is
added to the well, the analyte of interest binds to the capture agent. After washing, a known
amount of labeled (e.g., biotin or horseradish peroxidase (HRP)) analyte capable of binding the
capture antibody is added to the well. A substrate for an enzymatic label is necessary to generate
a signal. An example of a suitable substrate for HRP is 3,3',5,5'-tetramethylbenzidine (TMB).
After washing, the signal generated by the labeled analyte is measured and is inversely
proportional to the amount of analyte in the sample. In a classic competitive inhibition
immunoassay typically an antibody (i.e., a binding protein and/or a DVD-Ig in the context of the
present disclosure) to an analyte of interest is coated onto a solid support (e.g., a well of a
microtiter plate). However, unlike the sequential competitive inhibition immunoassay, the sample
and the labeled analyte are added to the well at the same time. Any analyte in the sample
competes with labeled analyte for binding to the capture agent. After washing, the signal
generated by the labeled analyte is measured and is inversely proportional to the amount of
analyte in the sample. Of course, there are many variations of these formats - - e.g., such as when
binding to the solid substrate takes place, whether the format is one-step, two-step, delayed twostep,
and the like - - and these would be recognized by one of ordinary skill in the art.
Optionally, prior to contacting the test sample with the at least one capture agent (for
example, the first capture agent), the at least one capture agent can be bound to a solid support,
which facilitates the separation of the first agent/analyte (or a fragment thereof) complex from the
test sample. The substrate to which the capture agent is bound can be any suitable solid support
or solid phase that facilitates separation of the capture agent-analyte complex from the sample.
Examples include a well of a plate, such as a microtiter plate, a test tube, a porous gel
(e.g., silica gel, agarose, dextran, or gelatin), a polymeric film (e.g., polyacrylamide), beads (e.g.,
polystyrene beads or magnetic beads), a strip of a filter/membrane (e.g., nitrocellulose or nylon),
microparticles (e.g., latex particles, magnetizable microparticles (e.g., microparticles having ferric
oxide or chromium oxide cores and homo- or hetero-polymeric coats and radii of about 1-10
microns). The substrate can comprise a suitable porous material with a suitable surface affinity to
bind antigens and sufficient porosity to allow access by detection antibodies. A microporous
material is generally preferred, although a gelatinous material in a hydrated state can be used.
Such porous substrates are preferably in the form of sheets having a thickness of about 0.01 to
about 0.5 mm, preferably about 0.1 mm. While the pore size may vary quite a bit, preferably the
pore size is from about 0.025 to about 15 microns, more preferably from about 0.15 to about 15
microns. The surface of such substrates can be passively coated or activated by chemical
processes that cause covalent linkage of an antibody to the substrate. Irreversible binding,
generally by adsorption through hydrophobic forces, of the antigen or the antibody to the
substrate results; alternatively, a chemical coupling agent or other means can be used to bind
covalently the antibody to the substrate, provided that such binding does not interfere with the
ability of the antibody to bind to analyte. Alternatively, the antibody (i.e., binding protein and/or
DVD-Ig in the context of the present disclosure) can be bound with microparticles, which have
been previously coated with streptavidin (e.g., DYNAL® Magnetic Beads, Invitrogen, Carlsbad,
CA) or biotin (e.g., using Power-BindTM-SA-MP streptavidin-coated microparticles (Seradyn,
Indianapolis, IN)) or anti-species-specific monoclonal antibodies (i.e., binding proteins and/or
DVD-Igs in the context of the present disclosure). If necessary or desired, the substrate (e.g., for
the label) can be derivatized to allow reactivity with various functional groups on the antibody
(i.e., binding protein or DVD-Ig in the context of the present disclosure). Such derivatization
requires the use of certain coupling agents, examples of which include, but are not limited to,
maleic anhydride, N-hydroxysuccinimide, and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
If desired, one or more capture agents, such as antibodies (or fragments thereof) (i.e., binding
proteins and/or DVD-Igs in the context of the present disclosure), each of which is specific for
analyte(s) can be attached to solid phases in different physical or addressable locations (e.g., such
as in a biochip configuration (see, e.g., U.S. Patent No. 6,225,047; PCT Publication No. WO
99/51773; U.S. Patent No. 6,329,209; PCT Publication No. WO 00/56934, and U.S. Patent No.
5,242,828). If the capture agent is attached to a mass spectrometry probe as the solid support, the
amount of analyte bound to the probe can be detected by laser desorption ionization mass
spectrometry. Alternatively, a single column can be packed with different beads, which are
derivatized with the one or more capture agents, thereby capturing the analyte in a single place
(see, antibody-derivatized, bead-based technologies, e.g., the xMAP technology of Luminex
(Austin, TX)).
After the test sample being assayed for analyte (or a fragment thereof) is brought into
contact with the at least one capture agent (for example, the first capture agent), the mixture is
incubated in order to allow for the formation of a first capture agent (or multiple capture agent)-
analyte (or a fragment thereof) complex. The incubation can be carried out at a pH of from about
4.5 to about 10.0, at a temperature of from about 2°C to about 45°C, and for a period from at least
about one (1) minute to about eighteen (18) hours, preferably from about 1 to about 24 minutes,
most preferably for about 4 to about 18 minutes. The immunoassay described herein can be
conducted in one step (meaning the test sample, at least one capture agent and at least one
detection agent are all added sequentially or simultaneously to a reaction vessel) or in more than
one step, such as two steps, three steps, etc.
After formation of the (first or multiple) capture agent/analyte (or a fragment thereof)
complex, the complex is then contacted with at least one detection agent under conditions which
allow for the formation of a (first or multiple) capture agent/analyte (or a fragment
thereof)/second detection agent complex). While captioned for clarity as the "second" agent (e.g.,
second detection agent), in fact, where multiple agents are used for capture and/or detection, the at
least one detection agent can be the second, third, fourth, etc., agents used in the immunoassay. If
the capture agent/analyte (or a fragment thereof) complex is contacted with more than one
detection agent, then a (first or multiple) capture agent/analyte (or a fragment thereof)/(multiple)
detection agent complex is formed. As with the capture agent (e.g., the first capture agent), when
the at least one (e.g., second and any subsequent) detection agent is brought into contact with the
capture agent/analyte (or a fragment thereof) complex, a period of incubation under conditions
similar to those described above is required for the formation of the (first or multiple) capture
agent/analyte (or a fragment thereof)/(second or multiple) detection agent complex. Preferably, at
least one detection agent contains a detectable label. The detectable label can be bound to the at
least one detection agent (e.g., the second detection agent) prior to, simultaneously with, or after
the formation of the (first or multiple) capture agent/analyte (or a fragment thereof)/(second or
multiple) detection agent complex. Any detectable label known in the art can be used (see
discussion above, including of the Polak and Van Noorden (1997) and Haugland (1996)
references).
The detectable label can be bound to the agents either directly or through a coupling
agent. An example of a coupling agent that can be used is EDAC (l-ethyl-3-(3-
dimethylaminopropyl) carbodiimide, hydrochloride), which is commercially available from
Sigma-Aldrich, St. Louis, MO. Other coupling agents that can be used are known in the art.
Methods for binding a detectable label to an antibody are known in the art. Additionally, many
detectable labels can be purchased or synthesized that already contain end groups that facilitate
the coupling of the detectable label to the agent, such as CPSP-Acridinium Ester (i.e., 9-[Ntosyl-
N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridinium carboxamide) or SPSP-Acridinium
Ester (i.e., N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).
The (first or multiple) capture agent/analyte/(second or multiple) detection agent complex
can be, but does not have to be, separated from the remainder of the test sample prior to
quantification of the label. For example, if the at least one capture agent (e.g., the first capture
agent, such as a binding protein and/or a DVD-Ig in accordance with the present disclosure) is
bound to a solid support, such as a well or a bead, separation can be accomplished by removing
the fluid (of the test sample) from contact with the solid support. Alternatively, if the at least first
capture agent is bound to a solid support, it can be simultaneously contacted with the analytecontaining
sample and the at least one second detection agent to form a first (multiple)
agent/analyte/second (multiple) agent complex, followed by removal of the fluid (test sample)
from contact with the solid support. If the at least one first capture agent is not bound to a solid
support, then the (first or multiple) capture agent/analyte/(second or multiple) detection agent
complex does not have to be removed from the test sample for quantification of the amount of the
label.
After formation of the labeled capture agent/analyte/detection agent complex (e.g., the
first capture agent/analyte/second detection agent complex), the amount of label in the complex is
quantified using techniques known in the art. For example, if an enzymatic label is used, the
labeled complex is reacted with a substrate for the label that gives a quantifiable reaction such as
the development of color. If the label is a radioactive label, the label is quantified using
appropriate means, such as a scintillation counter. If the label is a fluorescent label, the label is
quantified by stimulating the label with a light of one color (which is known as the "excitation
wavelength") and detecting another color (which is known as the "emission wavelength") that is
emitted by the label in response to the stimulation. If the label is a chemiluminescent label, the
label is quantified by detecting the light emitted either visually or by using luminometers, x-ray
film, high speed photographic film, a CCD camera, etc. Once the amount of the label in the
complex has been quantified, the concentration of analyte or a fragment thereof in the test sample
is determined by appropriate means, such as by use of a standard curve that has been generated
using serial dilutions of analyte or a fragment thereof of known concentration. Other than using
serial dilutions of analyte or a fragment thereof, the standard curve can be generated
gravimetrically, by mass spectroscopy and by other techniques known in the art.
In a chemiluminescent microparticle assay employing the ARCHITECT® analyzer, the
conjugate diluent pH should be about 6.0 +/- 0.2, the microparticle coating buffer should be
maintained at about room temperature (i.e., at from about 17 to about 27 °C), the microparticle
coating buffer pH should be about 6.5 +/- 0.2, and the microparticle diluent pH should be about
7.8 +/- 0.2. Solids preferably are less than about 0.2%, such as less than about 0.15%, less than
about 0.14%, less than about 0.13%, less than about 0.12%, or less than about 0.11%, such as
about 0.10%.
FPIAs are based on competitive binding immunoassay principles. A fluorescently labeled
compound, when excited by a linearly polarized light, will emit fluorescence having a degree of
polarization inversely proportional to its rate of rotation. When a fluorescently labeled tracerantibody
complex is excited by a linearly polarized light, the emitted light remains highly
polarized because the fluorophore is constrained from rotating between the time light is absorbed
and the time light is emitted. When a "free" tracer compound (i.e., a compound that is not bound
to an antibody) is excited by linearly polarized light, its rotation is much faster than the
corresponding tracer-antibody conjugate (or tracer-binding protein and/or tracer-DVD-Ig in
accordance with the present disclosure) produced in a competitive binding immunoassay. FPIAs
are advantageous over RIAs inasmuch as there are no radioactive substances requiring special
handling and disposal. In addition, FPIAs are homogeneous assays that can be easily and rapidly
performed.
In view of the above, a method of determining the presence, amount, or concentration of
analyte (or a fragment thereof) in a test sample is provided. The method comprises assaying the
test sample for an analyte (or a fragment thereof) by an assay (i) employing (i') at least one of an
antibody, a fragment of an antibody that can bind to an analyte, a variant of an antibody that can
bind to an analyte, a fragment of a variant of an antibody that can bind to an analyte, a binding
protein as disclosed herein, and a DVD-Ig (or a fragment, a variant, or a fragment of a variant
thereof) that can bind to an analyte, and (ii') at least one detectable label and (ii) comprising
comparing a signal generated by the detectable label as a direct or indirect indication of the
presence, amount or concentration of analyte (or a fragment thereof) in the test sample to a signal
generated as a direct or indirect indication of the presence, amount or concentration of analyte (or
a fragment thereof) in a control or calibrator. The calibrator is optionally part of a series of
calibrators, in which each of the calibrators differs from the other calibrators by the concentration
of analyte.
The method can comprise (i) contacting the test sample with at least one first specific
binding partner for analyte (or a fragment thereof) selected from the group consisting of an
antibody, a fragment of an antibody that can bind to an analyte, a variant of an antibody that can
bind to an analyte, a fragment of a variant of an antibody that can bind to an analyte, a binding
protein as disclosed herein, and a DVD-Ig (or a fragment, a variant, or a fragment of a variant
thereof) that can bind to an analyte so as to form a first specific binding partner/analyte (or
fragment thereof) complex, (ii) contacting the first specific binding partner/analyte (or fragment
thereof) complex with at least one second specific binding partner for analyte (or fragment
thereof) selected from the group consisting of a detectably labeled anti-analyte antibody, a
detectably labeled fragment of an anti-analyte antibody that can bind to analyte, a detectably
labeled variant of an anti-analyte antibody that can bind to analyte, a detectably labeled fragment
of a variant of an anti-analyte antibody that can bind to analyte, a detectably labeled binding
protein as disclosed herein that can bind to analyte, and a detectably labeled DVD-Ig (or a
fragment, a variant, or a fragment of a variant thereof) so as to form a first specific binding
partner/analyte (or fragment thereof)/second specific binding partner complex, and (iii)
determining the presence, amount or concentration of analyte in the test sample by detecting or
measuring the signal generated by the detectable label in the first specific binding partner/analyte
(or fragment thereof)/second specific binding partner complex formed in (ii). A method in which
at least one first specific binding partner for analyte (or a fragment thereof) and/or at least one
second specific binding partner for analyte (or a fragment thereof) is a binding protein as
disclosed herein or a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) as
described herein can be preferred.
Alternatively, the method can comprise contacting the test sample with at least one first
specific binding partner for analyte (or a fragment thereof) selected from the group consisting of
an antibody, a fragment of an antibody that can bind to an analyte, a variant of an antibody that
can bind to an analyte, a fragment of a variant of an antibody that can bind to an analyte, a
binding protein as disclosed herein, and a DVD-Ig (or a fragment, a variant, or a fragment of a
variant thereof) and simultaneously or sequentially, in either order, contacting the test sample
with at least one second specific binding partner, which can compete with analyte (or a fragment
thereof) for binding to the at least one first specific binding partner and which is selected from the
group consisting of a detectably labeled analyte, a detectably labeled fragment of analyte that can
bind to the first specific binding partner, a detectably labeled variant of analyte that can bind to
the first specific binding partner, and a detectably labeled fragment of a variant of analyte that can
bind to the first specific binding partner. Any analyte (or a fragment thereof) present in the test
sample and the at least one second specific binding partner compete with each other to form a first
specific binding partner/analyte (or fragment thereof) complex and a first specific binding
partner/second specific binding partner complex, respectively. The method further comprises
determining the presence, amount or concentration of analyte in the test sample by detecting or
measuring the signal generated by the detectable label in the first specific binding partner/second
specific binding partner complex formed in (ii), wherein the signal generated by the detectable
label in the first specific binding partner/second specific binding partner complex is inversely
proportional to the amount or concentration of analyte in the test sample.
The above methods can further comprise diagnosing, prognosticating, or assessing the
efficacy of a therapeutic/prophylactic treatment of a patient from whom the test sample was
obtained. If the method further comprises assessing the efficacy of a therapeutic/prophylactic
treatment of the patient from whom the test sample was obtained, the method optionally further
comprises modifying the therapeutic/prophylactic treatment of the patient as needed to improve
efficacy. The method can be adapted for use in an automated system or a semi-automated system.
More specifically, a method of determining the presence, amount or concentration of an
antigen (or a fragment thereof) in a test sample is provided. The method comprises assaying the
test sample for the antigen (or a fragment thereof) by an immunoassay. The immunoassay (i)
employs at least one binding protein and at least one detectable label and (ii) comprises
comparing a signal generated by the detectable label as a direct or indirect indication of the
presence, amount or concentration of the antigen (or a fragment thereof) in the test sample to a
signal generated as a direct or indirect indication of the presence, amount or concentration of the
antigen (or a fragment thereof) in a control or a calibrator. The calibrator is optionally part of a
series of calibrators in which each of the calibrators differs from the other calibrators in the series
by the concentration of the antigen (or a fragment thereof). One of the at least one binding protein
(i') comprises a polypeptide chain comprising VDl-(Xl)n-VD2-C-(X2)n, in which VD1 is a first
heavy chain variable domain obtained from a first parent antibody (or antigen binding portion
thereof), VD2 is a second heavy chain variable domain obtained from a second parent antibody
(or antigen binding portion thereof), which can be the same as or different from the first parent
antibody, C is a heavy chain constant domain, (Xl)n is a linker, which is optionally present and,
when present, is other than CHI, and (X2)n is an Fc region, which is optionally present, and (ii')
can bind a pair of antigens. The method can comprise (i) contacting the test sample with at least
one capture agent, which binds to an epitope on the antigen (or a fragment thereof) so as to form a
capture agent/antigen (or a fragment thereof) complex, (ii) contacting the capture agent/antigen
(or a fragment thereof) complex with at least one detection agent, which comprises a detectable
label and binds to an epitope on the antigen (or a fragment thereof) that is not bound by the
capture agent, to form a capture agent/antigen (or a fragment thereof)/detection agent complex,
and (iii) determining the presence, amount or concentration of the antigen (or a fragment thereof)
in the test sample based on the signal generated by the detectable label in the capture
agent/antigen (or a fragment thereof)/detection agent complex formed in (ii), wherein at least one
capture agent and/or at least one detection agent is the at least one binding protein. Alternatively,
the method can comprise (i) contacting the test sample with at least one capture agent, which
binds to an epitope on the antigen (or a fragment thereof) so as to form a capture agent/antigen (or
a fragment thereof) complex, and simultaneously or sequentially, in either order, contacting the
test sample with detectably labeled antigen (or a fragment thereof), which can compete with any
antigen (or a fragment thereof) in the test sample for binding to the at least one capture agent,
wherein any antigen (or a fragment thereof) present in the test sample and the detectably labeled
antigen compete with each other to form a capture agent/antigen (or a fragment thereof) complex
and a capture agent/detectably labeled antigen (or a fragment thereof) complex, respectively, and
(ii) determining the presence, amount or concentration of the antigen (or a fragment thereof) in
the test sample based on the signal generated by the detectable label in the capture
agent/detectably labeled antigen (or a fragment thereof) complex formed in (ii), wherein at least
one capture agent is the at least one binding protein and wherein the signal generated by the
detectable label in the capture agent/detectably labeled antigen (or a fragment thereof) complex is
inversely proportional to the amount or concentration of antigen (or a fragment thereof) in the test
sample. The test sample can be from a patient, in which case the method can further comprise
diagnosing, prognosticating, or assessing the efficacy of therapeutic/prophylactic treatment of the
patient. If the method further comprises assessing the efficacy of therapeutic/prophylactic
treatment of the patient, the method optionally further comprises modifying the
therapeutic/prophylactic treatment of the patient as needed to improve efficacy. The method can
be adapted for use in an automated system or a semi-automated system.
Another method of determining the presence, amount or concentration of an antigen (or a
fragment thereof) in a test sample is provided. The method comprises assaying the test sample for
the antigen (or a fragment thereof) by an immunoassay. The immunoassay (i) employs at least
one binding protein and at least one detectable label and (ii) comprises comparing a signal
generated by the detectable label as a direct or indirect indication of the presence, amount or
concentration of the antigen (or a fragment thereof) in the test sample to a signal generated as a
direct or indirect indication of the presence, amount or concentration of the antigen (or a fragment
thereof) in a control or a calibrator. The calibrator is optionally part of a series of calibrators in
which each of the calibrators differs from the other calibrators in the series by the concentration of
the antigen (or a fragment thereof)- One of the at least one binding protein (i') comprises a
polypeptide chain comprising VDl-(Xl)n-VD2-C-(X2)n, in which VD1 is a first light chain
variable domain obtained from a first parent antibody (or antigen binding portion thereof), VD2 is
a second light chain variable domain obtained from a second parent antibody (or antigen binding
portion thereof), which can be the same as or different from the first parent antibody, C is a light
chain constant domain, (Xl)n is a linker, which is optionally present and, when present, is other
than CHl, and (X2)n is an Fc region, which is optionally present, and (ii') can bind a pair of
antigens. The method can comprise (i) contacting the test sample with at least one capture agent,
which binds to an epitope on the antigen (or a fragment thereof) so as to form a capture
agent/antigen (or a fragment thereof) complex, (ii) contacting the capture agent/antigen (or a
fragment thereof) complex with at least one detection agent, which comprises a detectable label
and binds to an epitope on the antigen (or a fragment thereof) that is not bound by the capture
agent, to form a capture agent/antigen (or a fragment thereof)/detection agent complex, and (iii)
determining the presence, amount or concentration of the antigen (or a fragment thereof) in the
test sample based on the signal generated by the detectable label in the capture agent/antigen (or a
fragment thereof)/detection agent complex formed in (ii), wherein at least one capture agent
and/or at least one detection agent is the at least one binding protein. Alternatively, the method
can comprise (i) contacting the test sample with at least one capture agent, which binds to an
epitope on the antigen (or a fragment thereof) so as to form a capture agent/antigen (or a fragment
thereof) complex, and simultaneously or sequentially, in either order, contacting the test sample
with detectably labeled antigen (or a fragment thereof), which can compete with any antigen (or a
fragment thereof) in the test sample for binding to the at least one capture agent, wherein any
antigen (or a fragment thereof) present in the test sample and the detectably labeled antigen
compete with each other to form a capture agent/antigen (or a fragment thereof) complex and a
capture agent/detectably labeled antigen (or a fragment thereof) complex, respectively, and
(ii) determining the presence, amount or concentration of the antigen (or a fragment thereof) in
the test sample based on the signal generated by the detectable label in the capture
agent/detectably labeled antigen (or a fragment thereof) complex formed in (ii), wherein at least
one capture agent is the at least one binding protein and wherein the signal generated by the
detectable label in the capture agent/detectably labeled antigen (or a fragment thereof) complex is
inversely proportional to the amount or concentration of antigen (or a fragment thereof) in the test
sample. If the test sample is from a patient, the method can further comprise diagnosing,
prognosticating, or assessing the efficacy of therapeutic/prophylactic treatment of the patient. If
the method further comprises assessing the efficacy of therapeutic/prophylactic treatment of the
patient, the method optionally further comprises modifying the therapeutic/prophylactic treatment
of the patient as needed to improve efficacy. The method can be adapted for use in an automated
system or a semi-automated system.
Yet another method of determining the presence, amount or concentration of an antigen
(or a fragment thereof) in a test sample is provided. The method comprises assaying the test
sample for the antigen (or a fragment thereof) by an immunoassay. The immunoassay (i) employs
at least one binding protein and at least one detectable label and (ii) comprises comparing a signal
generated by the detectable label as a direct or indirect indication of the presence, amount or
concentration of the antigen (or a fragment thereof) in the test sample to a signal generated as a
direct or indirect indication of the presence, amount or concentration of the antigen (or a fragment
thereof) in a control or a calibrator. The calibrator is optionally part of a series of calibrators in
which each of the calibrators differs from the other calibrators in the series by the concentration of
the antigen (or a fragment thereof). One of the at least one binding protein (i') comprises a first
polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a
first VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first heavy chain variable domain obtained
from a first parent antibody (or antigen binding portion thereof), VD2 is a second heavy chain
variable domain obtained from a second parent antibody (or antigen binding portion thereof),
which can be the same as or different from the first parent antibody, C is a heavy chain constant
domain, (X1)n is a linker, which is optionally present and, when present, is other than CH1, and
(X2)n is an Fc region, which is optionally present, and wherein the second polypeptide chain
comprises a second VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first light chain variable
domain obtained from a first parent antibody (or antigen binding portion thereof), VD2 is a
second light chain variable domain obtained from a second parent antibody (or antigen binding
portion thereof), which can be the same as or different from the first parent antibody, C is a light
chain constant domain, (X1 )n is a linker, which is optionally present and, when present, is other
than CH1, and (X2)n is an Fc region, which is optionally present, and (ii') can bind a pair of
antigens. The method can comprise (i) contacting the test sample with at least one capture agent,
which binds to an epitope on the antigen (or a fragment thereof) so as to form a capture
agent/antigen (or a fragment thereof) complex, (ii) contacting the capture agent/antigen (or a
fragment thereof) complex with at least one detection agent, which comprises a detectable label
and binds to an epitope on the antigen (or a fragment thereof) that is not bound by the capture
agent, to form a capture agent/antigen (or a fragment thereof)/detection agent complex, and (iii)
determining the presence, amount or concentration of the antigen (or a fragment thereof) in the
test sample based on the signal generated by the detectable label in the capture agent/antigen (or a
fragment thereof)/detection agent complex formed in (ii), wherein at least one capture agent
and/or at least one detection agent is the at least one binding protein. Alternatively, the method
can comprise (i) contacting the test sample with at least one capture agent, which binds to an
epitope on the antigen (or a fragment thereof) so as to form a capture agent/antigen (or a fragment
thereof) complex, and simultaneously or sequentially, in either order, contacting the test sample
with detectably labeled antigen (or a fragment thereof), which can compete with any antigen (or a
fragment thereof) in the test sample for binding to the at least one capture agent, wherein any
antigen (or a fragment thereof) present in the test sample and the detectably labeled antigen
compete with each other to form a capture agent/antigen (or a fragment thereof) complex and a
capture agent/detectably labeled antigen (or a fragment thereof) complex, respectively, and (ii)
determining the presence, amount or concentration of the antigen (or a fragment thereof) in the
test sample based on the signal generated by the detectable label in the capture agent/detectably
labeled antigen (or a fragment thereof) complex formed in (ii), wherein at least one capture agent
is the at least one binding protein and wherein the signal generated by the detectable label in the
capture agent/detectably labeled antigen (or a fragment thereof) complex is inversely proportional
to the amount or concentration of antigen (or a fragment thereof) in the test sample. If the test
sample is from a patient, the method can further comprise diagnosing, prognosticating, or
assessing the efficacy of therapeutic/prophylactic treatment of the patient. If the method further
comprises assessing the efficacy of therapeutic/prophylactic treatment of the patient, the method
optionally further comprises modifying the therapeutic/prophylactic treatment of the patient as
needed to improve efficacy. The method can be adapted for use in an automated system or a
semi-automated system.
Still yet another method of determining the presence, amount or concentration of an
antigen (or a fragment thereof) in a test sample is provided. The method comprises assaying the
test sample for the antigen (or a fragment thereof) by an immunoassay. The immunoassay (i)
employs at least one DVD-Ig that can bind two antigens and at least one detectable label and (ii)
comprises comparing a signal generated by the detectable label as a direct or indirect indication of
the presence, amount or concentration of the antigen (or a fragment thereof) in the test sample to a
signal generated as a direct or indirect indication of the presence, amount or concentration of the
antigen (or a fragment thereof) in a control or a calibrator. The calibrator is optionally part of a
series of calibrators in which each of the calibrators differs from the other calibrators in the series
by the concentration of the antigen (or a fragment thereof). One of the at least one DVD-Ig (i')
comprises four polypeptide chains, wherein the first and third polypeptide chains comprise a first
VD1-(Xl)n-VD2-C-(X2)n, in which VD1 is a first heavy chain variable domain obtained from a
first parent antibody (or antigen binding portion thereof), VD2 is a second heavy chain variable
domain obtained from a second parent antibody (or antigen binding portion thereof), which can be
the same as or different from the first parent antibody, C is a heavy chain constant domain, (Xl)n
is a linker, which is optionally present and, when present, is other than CH1, and (X2)n is an Fc
region, which is optionally present, and wherein the second and fourth polypeptide chains
comprise a second VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first light chain variable domain
obtained from a first parent antibody (or antigen binding portion thereof), VD2 is a second light
chain variable domain obtained from a second parent antibody (or antigen binding portion
thereof), which can be the same as or different from the first parent antibody, C is a light chain
constant domain, (X1)n is a linker, which is optionally present and, when present, is other than
CH1, and (X2)n is an Fc region, which is optionally present, and (ii') can bind two antigens (or
fragments thereof). The method can comprise (i) contacting the test sample with at least one
capture agent, which binds to an epitope on the antigen (or a fragment thereof) so as to form a
capture agent/antigen (or a fragment thereof) complex, (ii) contacting the capture agent/antigen
(or a fragment thereof) complex with at least one detection agent, which comprises a detectable
label and binds to an epitope on the antigen (or a fragment thereof) that is not bound by the
capture agent, to form a capture agent/antigen (or a fragment thereof)/detection agent complex,
and (iii) determining the presence, amount or concentration of the antigen (or a fragment thereof)
in the test sample based on the signal generated by the detectable label in the capture
agent/antigen (or a fragment thereof)/detection agent complex formed in (ii), wherein at least one
capture agent and/or at least one detection agent is the at least one DVD-Ig. Alternatively, the
method can comprise (i) contacting the test sample with at least one capture agent, which binds to
an epitope on the antigen (or a fragment thereof) so as to form a capture agent/antigen (or a
fragment thereof) complex, and simultaneously or sequentially, in either order, contacting the test
sample with detectably labeled antigen (or a fragment thereof), which can compete with any
antigen (or a fragment thereof) in the test sample for binding to the at least one capture agent,
wherein any antigen (or a fragment thereof) present in the test sample and the detectably labeled
antigen compete with each other to form a capture agent/antigen (or a fragment thereof) complex
and a capture agent/detectably labeled antigen (or a fragment thereof) complex, respectively, and
(ii) determining the presence, amount or concentration of the antigen (or a fragment thereof) in
the test sample based on the signal generated by the detectable label in the capture
agent/detectably labeled antigen (or a fragment thereof) complex formed in (ii), wherein at least
one capture agent is the at least one DVD-Ig and wherein the signal generated by the detectable
label in the capture agent/detectably labeled antigen (or a fragment thereof) complex is inversely
proportional to the amount or concentration of antigen (or a fragment thereof) in the test sample.
If the test sample is from a patient, the method can further comprise diagnosing, prognosticating,
or assessing the efficacy of therapeutic/prophylactic treatment of the patient. If the method
further comprises assessing the efficacy of therapeutic/prophylactic treatment of the patient, the
method optionally further comprises modifying the therapeutic/prophylactic treatment of the
patient as needed to improve efficacy. The method can be adapted for use in an automated system
or a semi-automated system.
With regard to the methods of assay (and kit therefor), it may be possible to employ
commercially available anti-analyte antibodies or methods for production of anti-analyte as
described in the literature. Commercial supplies of various antibodies include, but are not limited
to, Santa Cruz Biotechnology Inc. (Santa Cruz, CA), Gen Way Biotech, Inc. (San Diego, CA), and
R&D Systems (RDS; Minneapolis, MN).
Generally, a predetermined level can be employed as a benchmark against which to assess
results obtained upon assaying a test sample for analyte or a fragment thereof, e.g., for detecting
disease or risk of disease. Generally, in making such a comparison, the predetermined level is
obtained by running a particular assay a sufficient number of times and under appropriate
conditions such that a linkage or association of analyte presence, amount or concentration with a
particular stage or endpoint of a disease, disorder or condition or with particular clinical indicia
can be made. Typically, the predetermined level is obtained with assays of reference subjects (or
populations of subjects). The analyte measured can include fragments thereof, degradation
products thereof, and/or enzymatic cleavage products thereof.
In particular, with respect to a predetermined level as employed for monitoring disease
progression and/or treatment, the amount or concentration of analyte or a fragment thereof may be
"unchanged," "favorable" (or "favorably altered"), or "unfavorable" (or "unfavorably altered").
"Elevated" or "increased" refers to an amount or a concentration in a test sample that is higher
than a typical or normal level or range (e.g., predetermined level), or is higher than another
reference level or range (e.g., earlier or baseline sample). The term "lowered" or "reduced" refers
to an amount or a concentration in a test sample that is lower than a typical or normal level or
range (e.g., predetermined level), or is lower than another reference level or range (e.g., earlier or
baseline sample). The term "altered" refers to an amount or a concentration in a sample that is
altered (increased or decreased) over a typical or normal level or range (e.g., predetermined level),
or over another reference level or range (e.g., earlier or baseline sample).
The typical or normal level or range for analyte is defined in accordance with standard
practice. Because the levels of analyte in some instances will be very low, a so-called altered
level or alteration can be considered to have occurred when there is any net change as compared
to the typical or normal level or range, or reference level or range, that cannot be explained by
experimental error or sample variation. Thus, the level measured in a particular sample will be
compared with the level or range of levels determined in similar samples from a so-called normal
subject. In this context, a "normal subject" is an individual with no detectable disease, for
example, and a "normal" (sometimes termed "control") patient or population is/are one(s) that
exhibit(s) no detectable disease, respectively, for example. Furthermore, given that analyte is not
routinely found at a high level in the majority of the human population, a "normal subject" can be
considered an individual with no substantial detectable increased or elevated amount or
concentration of analyte, and a "normal" (sometimes termed "control") patient or population
is/are one(s) that exhibit(s) no substantial detectable increased or elevated amount or
concentration of analyte. An "apparently normal subject" is one in which analyte has not yet been
or currently is being assessed. The level of an analyte is said to be "elevated" when the analyte is
normally undetectable (e.g., the normal level is zero, or within a range of from about 25 to about
75 percentiles of normal populations), but is detected in a test sample, as well as when the analyte
is present in the test sample at a higher than normal level. Thus, inter alia, the disclosure
provides a method of screening for a subject having, or at risk of having, a particular disease,
disorder, or condition. The method of assay can also involve the assay of other markers and the
like.
Accordingly, the methods described herein also can be used to determine whether or not a
subject has or is at risk of developing a given disease, disorder or condition. Specifically, such a
method can comprise the steps of:
(a) determining the concentration or amount in a test sample from a subject of analyte (or
a fragment thereof) (e.g., using the methods described herein, or methods known in the art); and
(b) comparing the concentration or amount of analyte (or a fragment thereof) determined
in step (a) with a predetermined level, wherein, if the concentration or amount of analyte
determined in step (a) is favorable with respect to a predetermined level, then the subject is
determined not to have or be at risk for a given disease, disorder or condition. However, if the
concentration or amount of analyte determined in step (a) is unfavorable with respect to the
predetermined level, then the subject is determined to have or be at risk for a given disease,
disorder or condition.
Additionally, provided herein is method of monitoring the progression of disease in a
subject. Optimally the method comprising the steps of:
(a) determining the concentration or amount in a test sample from a subject of analyte;
(b) determining the concentration or amount in a later test sample from the subject of
analyte; and
(c) comparing the concentration or amount of analyte as determined in step (b) with the
concentration or amount of analyte determined in step (a), wherein if the concentration or amount
determined in step (b) is unchanged or is unfavorable when compared to the concentration or
amount of analyte determined in step (a), then the disease in the subject is determined to have
continued, progressed or worsened. By comparison, if the concentration or amount of analyte as
determined in step (b) is favorable when compared to the concentration or amount of analyte as
determined in step (a), then the disease in the subject is determined to have discontinued,
regressed or improved.
Optionally, the method further comprises comparing the concentration or amount of
analyte as determined in step (b), for example, with a predetermined level. Further, optionally the
method comprises treating the subject with one or more pharmaceutical compositions for a period
of time if the comparison shows that the concentration or amount of analyte as determined in step
(b), for example, is unfavorably altered with respect to the predetermined level.
Still further, the methods can be used to monitor treatment in a subject receiving
treatment with one or more pharmaceutical compositions. Specifically, such methods involve
providing a first test sample from a subject before the subject has been administered one or more
pharmaceutical compositions. Next, the concentration or amount in a first test sample from a
subject of analyte is determined (e.g., using the methods described herein or as known in the art).
After the concentration or amount of analyte is determined, optionally the concentration or
amount of analyte is then compared with a predetermined level. If the concentration or amount of
analyte as determined in the first test sample is lower than the predetermined level, then the
subject is not treated with one or more pharmaceutical compositions. However, if the
concentration or amount of analyte as determined in the first test sample is higher than the
predetermined level, then the subject is treated with one or more pharmaceutical compositions for
a period of time. The period of time that the subject is treated with the one or more
pharmaceutical compositions can be determined by one skilled in the art (for example, the period
of time can be from about seven (7) days to about two years, preferably from about fourteen (14)
days to about one (1) year).
During the course of treatment with the one or more pharmaceutical compositions, second
and subsequent test samples are then obtained from the subject. The number of test samples and
the time in which said test samples are obtained from the subject are not critical. For example, a
second test sample could be obtained seven (7) days after the subject is first administered the one
or more pharmaceutical compositions, a third test sample could be obtained two (2) weeks after
the subject is first administered the one or more pharmaceutical compositions, a fourth test sample
could be obtained three (3) weeks after the subject is first administered the one or more
pharmaceutical compositions, a fifth test sample could be obtained four (4) weeks after the subject
is first administered the one or more pharmaceutical compositions, etc.
After each second or subsequent test sample is obtained from the subject, the
concentration or amount of analyte is determined in the second or subsequent test sample is
determined (e.g., using the methods described herein or as known in the art). The concentration
or amount of analyte as determined in each of the second and subsequent test samples is then
compared with the concentration or amount of analyte as determined in the first test sample (e.g.,
the test sample that was originally optionally compared to the predetermined level). If the
concentration or amount of analyte as determined in step (c) is favorable when compared to the
concentration or amount of analyte as determined in step (a), then the disease in the subject is
determined to have discontinued, regressed or improved, and the subject should continue to be
administered the one or pharmaceutical compositions of step (b). However, if the concentration
or amount determined in step (c) is unchanged or is unfavorable when compared to the
concentration or amount of analyte as determined in step (a), then the disease in the subject is
determined to have continued, progressed or worsened, and the subject should be treated with a
higher concentration of the one or more pharmaceutical compositions administered to the subject
in step (b) or the subject should be treated with one or more pharmaceutical compositions that are
different from the one or more pharmaceutical compositions administered to the subject in step
(b). Specifically, the subject can be treated with one or more pharmaceutical compositions that
are different from the one or more pharmaceutical compositions that the subject had previously
received to decrease or lower said subject's analyte level.
Generally, for assays in which repeat testing may be done (e.g., monitoring disease
progression and/or response to treatment), a second or subsequent test sample is obtained at a
period in time after the first test sample has been obtained from the subject. Specifically, a second
test sample from the subject can be obtained minutes, hours, days, weeks or years after the first
test sample has been obtained from the subject. For example, the second test sample can be
obtained from the subject at a time period of about 1 minute, about 5 minutes, about 10 minutes,
about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,
about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours,
about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,
about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5
days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks,
about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17
weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about
23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks,
about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34
weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about
40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks,
about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51
weeks , about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5
years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0 years, about
6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about
9.5 years or about 10.0 years after the first test sample from the subject is obtained.
When used to monitor disease progression, the above assay can be used to monitor the
progression of disease in subjects suffering from acute conditions. Acute conditions, also known
as critical care conditions, refer to acute, life-threatening diseases or other critical medical
conditions involving, for example, the cardiovascular system or excretory system. Typically,
critical care conditions refer to those conditions requiring acute medical intervention in a hospitalbased
setting (including, but not limited to, the emergency room, intensive care unit, trauma
center, or other emergent care setting) or administration by a paramedic or other field-based
medical personnel. For critical care conditions, repeat monitoring is generally done within a
shorter time frame, namely, minutes, hours or days (e.g., about 1 minute, about 5 minutes, about
10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2
hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,
about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,
about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about
4 days, about 5 days, about 6 days or about 7 days), and the initial assay likewise is generally
done within a shorter timeframe, e.g., about minutes, hours or days of the onset of the disease or
condition.
The assays also can be used to monitor the progression of disease in subjects suffering
from chronic or non-acute conditions. Non-critical care or, non-acute conditions, refers to
conditions other than acute, life-threatening disease or other critical medical conditions involving,
for example, the cardiovascular system and/or excretory system. Typically, non-acute conditions
include those of longer-term or chronic duration. For non-acute conditions, repeat monitoring
generally is done with a longer timeframe, e.g., hours, days, weeks, months or years (e.g., about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours,
about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours,
about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days,
about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3
weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about
15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks,
about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26
weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about
32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks,
about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43
weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about
49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about
2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about
5.5. years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years,
about 8.5 years, about 9.0 years, about 9.5 years or about 10.0 years), and the initial assay
likewise generally is done within a longer time frame, e.g., about hours, days, months or years of
the onset of the disease or condition.
Furthermore, the above assays can be performed using a first test sample obtained from a
subject where the first test sample is obtained from one source, such as urine, serum or plasma.
Optionally, the above assays can then be repeated using a second test sample obtained from the
subject where the second test sample is obtained from another source. For example, if the first
test sample was obtained from urine, the second test sample can be obtained from serum or
plasma. The results obtained from the assays using the first test sample and the second test
sample can be compared. The comparison can be used to assess the status of a disease or
condition in the subject.
Moreover, the present disclosure also relates to methods of determining whether a subject
predisposed to or suffering from a given disease, disorder or condition will benefit from
treatment. In particular, the disclosure relates to analyte companion diagnostic methods and
products. Thus, the method of "monitoring the treatment of disease in a subject" as described
herein further optimally also can encompass selecting or identifying candidates for therapy.
Thus, in particular embodiments, the disclosure also provides a method of determining
whether a subject having, or at risk for, a given disease, disorder or condition is a candidate for
therapy. Generally, the subject is one who has experienced some symptom of a given disease,
disorder or condition or who has actually been diagnosed as having, or being at risk for, a given
disease, disorder or condition, and/or who demonstrates an unfavorable concentration or amount
of analyte or a fragment thereof, as described herein.
The method optionally comprises an assay as described herein, where analyte is assessed
before and following treatment of a subject with one or more pharmaceutical compositions (e.g.,
particularly with a pharmaceutical related to a mechanism of action involving analyte), with
immunosuppressive therapy, or by immunoabsorption therapy, or where analyte is assessed
following such treatment and the concentration or the amount of analyte is compared against a
predetermined level. An unfavorable concentration of amount of analyte observed following
treatment confirms that the subject will not benefit from receiving further or continued treatment,
whereas a favorable concentration or amount of analyte observed following treatment confirms
that the subject will benefit from receiving further or continued treatment. This confirmation
assists with management of clinical studies, and provision of improved patient care.
It goes without saying that, while certain embodiments herein are advantageous when
employed to assess a given disease, disorder or condition as discussed herein, the assays and kits
can be employed to assess analyte in other diseases, disorders and conditions. The method of
assay can also involve the assay of other markers and the like.
The method of assay also can be used to identify a compound that ameliorates a given
disease, disorder or condition. For example, a cell that expresses analyte can be contacted with a
candidate compound. The level of expression of analyte in the cell contacted with the compound
can be compared to that in a control cell using the method of assay described herein.
B. Kit
A kit for assaying a test sample for the presence, amount or concentration of an analyte
(or a fragment thereof) in a test sample is also provided. The kit comprises at least one
component for assaying the test sample for the analyte (or a fragment thereof) and instructions for
assaying the test sample for the analyte (or a fragment thereof). The at least one component for
assaying the test sample for the analyte (or a fragment thereof) can include a composition
comprising a binding protein as disclosed herein and/or an anti-analyte DVD-Ig (or a fragment, a
variant, or a fragment of a variant thereof), which is optionally immobilized on a solid phase.
The kit can comprise at least one component for assaying the test sample for an analyte
by immunoassay, e.g., chemiluminescent microparticle immunoassay, and instructions for
assaying the test sample for an analyte by immunoassay, e.g., chemiluminescent microparticle
immunoassay. For example, the kit can comprise at least one specific binding partner for an
analyte, such as an anti-analyte, monoclonal/polyclonal antibody (or a fragment thereof that can
bind to the analyte, a variant thereof that can bind to the analyte, or a fragment of a variant that
can bind to the analyte) a binding protein as disclosed herein or an anti-analyte DVD-Ig (or a
fragment, a variant, or a fragment of a variant thereof), either of which can be detectably labeled.
Alternatively or additionally, the kit can comprise detectably labeled analyte (or a fragment
thereof that can bind to an anti-analyte, monoclonal/polyclonal antibody a binding protein as
disclosed herein,or an anti-analyte DVD-Ig (or a fragment, a variant, or a fragment of a variant
thereof)), which can compete with any analyte in a test sample for binding to an anti-analyte,
monoclonal/polyclonal antibody (or a fragment thereof that can bind to the analyte, a variant
thereof that can bind to the analyte, or a fragment of a variant that can bind to the analyte), a
binding protein as disclosed herein, or an anti-analyte DVD-Ig (or a fragment, a variant, or a
fragment of a variant thereof), either of which can be immobilized on a solid support. The kit can
comprise a calibrator or control, e.g., isolated or purified analyte. The kit can comprise at least
one container (e.g., tube, microtiter plates or strips, which can be already coated with a first
specific binding partner, for example) for conducting the assay, and/or a buffer, such as an assay
buffer or a wash buffer, either one of which can be provided as a concentrated solution, a
substrate solution for the detectable label (e.g., an enzymatic label), or a stop solution. Preferably,
the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are
necessary to perform the assay. The instructions can be in paper form or computer-readable form,
such as a disk, CD, DVD, or the like.
More specifically, provided is a kit for assaying a test sample for an antigen (or a
fragment thereof). The kit comprises at least one component for assaying the test sample for an
antigen (or a fragment thereof) and instructions for assaying the test sample for an antigen (or a
fragment thereof), wherein the at least one component includes at least one composition
comprising a binding protein, which (i') comprises a polypeptide chain comprising VDl-(Xl)n-
VD2-C-(X2)n, in which VD1 is a first heavy chain variable domain obtained from a first parent
antibody (or antigen binding portion thereof), VD2 is a second heavy chain variable domain
obtained from a second parent antibody (or antigen binding portion thereof), which can be same
as or different from the first parent antibody, C is a heavy chain constant domain, (X1)n is a
linker, which is optionally present and, when present, is other than CH1, and (X2)n is an Fc
region, which is optionally present, and (ii') can bind a pair of antigens, wherein the binding
protein is optionally detectably labeled.
Further provided is another kit for assaying a test sample for an antigen (or a fragment
thereof). The kit comprises at least one component for assaying the test sample for an antigen (or
a fragment thereof) and instructions for assaying the test sample for an antigen (or a fragment
thereof), wherein the at least one component includes at least one composition comprising a
binding protein, which (i') comprises a polypeptide chain comprising VD1-(X1)n-VD2-C-(X2)n,
in which VD1 is a first light chain variable domain obtained from a first parent antibody (or
antigen binding portion thereof), VD2 is a second light chain variable domain obtained from a
second parent antibody (or antigen binding portion thereof), which can be the same as or different
from the first parent antibody, C is a light chain constant domain, (X1)n is a linker, which is
optionally present and, when present, is other than CH1, and (X2)n is an Fc region, which is
optionally present, and (ii') can bind a pair of antigens, wherein the binding protein is optionally
detectably labeled.
Still further provided is another kit for assaying a test sample for an antigen (or a
fragment thereof). The kit comprises at least one component for assaying the test sample for an
antigen (or a fragment thereof) and instructions for assaying the test sample for an antigen (or a
fragment thereof), wherein the at least one component includes at least one composition
comprising a binding protein, which (i') comprises a first polypeptide chain and a second
polypeptide chain, wherein the first polypeptide chain comprises a first VD1-(Xl)n-VD2-C-
(X2)n, in which VD1 is a first heavy chain variable domain obtained from a first parent antibody
(or antigen binding portion thereof), VD2 is a second heavy chain variable domain obtained from
a second parent antibody (or antigen binding portion thereof), which can be the same as or
different from the first parent antibody, C is a heavy chain constant domain, (X1)n is a linker,
which is optionally present and, when present, is other than CH1, and (X2)n is an Fc region,
which is optionally present, and wherein the second polypeptide chain comprises a second VD1-
(Xl)n-VD2-C-(X2)n, in which VD1 is a first light chain variable domain obtained from a first
parent antibody (or antigen binding portion thereof), VD2 is a second light chain variable domain
obtained from a second parent antibody (or antigen binding portion thereof), which can be the
same as or different from the first parent antibody, C is a light chain constant domain, (X1)n is a
linker, which is optionally present and, when present, is other than CH1, and (X2)n is an Fc
region, which is optionally present, and (ii') can bind a pair of antigens, wherein the binding
protein is optionally detectably labeled.
Even still further provided is another kit for assaying a test sample for an antigen (or a
fragment thereof). The kit comprises at least one component for assaying the test sample for an
antigen (or a fragment thereof) and instructions for assaying the test sample for an antigen (or a
fragment thereof), wherein the at least one component includes at least one composition
comprising a DVD-Ig, which (i') comprises four polypeptide chains, wherein the first and third
polypeptide chains comprise a first VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first heavy
chain variable domain obtained from a first parent antibody (or antigen binding portion thereof),
VD2 is a second heavy chain variable domain obtained from a second parent antibody (or antigen
binding portion thereof), which can be the same as or different from the first parent antibody, C is
a heavy chain constant domain, (X1)n is a linker, which is optionally present and, when present, is
other than CH1, and (X2)n is an Fc region, which is optionally present, and wherein the second
and fourth polypeptide chains comprise a second VDl-(Xl)n-VD2-C-(X2)n, in which VD1 is a
first light chain variable domain obtained from a first parent antibody (or antigen binding portion
thereof), VD2 is a second light chain variable domain obtained from a second parent antibody (or
antigen binding portion thereof), which can be the same as or different from the first parent
antibody, C is a light chain constant domain, (Xl)n is a linker, which is optionally present and,
when present, is other than CH1, and (X2)n is an Fc region, which is optionally present, and (ii')
can bind two antigens (or fragments thereof), wherein the DVD-Ig is optionally detectably
labeled.
Any antibodies, such as an anti-analyte antibody, any binding proteins as disclosed
herein, any anti-analyte DVD-Igs, or tracers can incorporate a detectable label as described
herein, such as a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a
chromophore, a chemiluminescent label, or the like, or the kit can include reagents for carrying
out detectable labeling. The antibodies, calibrators and/or controls can be provided in separate
containers or pre-dispensed into an appropriate assay format, for example, into microtiter plates.
Optionally, the kit includes quality control components (for example, sensitivity panels,
calibrators, and positive controls). Preparation of quality control reagents is well-known in the art
and is described on insert sheets for a variety of immunodiagnostic products. Sensitivity panel
members optionally are used to establish assay performance characteristics, and further optionally
are useful indicators of the integrity of the immunoassay kit reagents, and the standardization of
assays.
The kit can also optionally include other reagents required to conduct a diagnostic assay
or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors,
enzyme substrates, detection reagents, and the like. Other components, such as buffers and
solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also can
be included in the kit. The kit can additionally include one or more other controls. One or more
of the components of the kit can be lyophilized, in which case the kit can further comprise
reagents suitable for the reconstitution of the lyophilized components.
The various components of the kit optionally are provided in suitable containers as
necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a
sample (e.g., a container or cartridge for a urine sample). Where appropriate, the kit optionally
also can contain reaction vessels, mixing vessels, and other components that facilitate the
preparation of reagents or the test sample. The kit can also include one or more instruments for
assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the
like.
If the detectable label is at least one acridinium compound, the kit can comprise at least
one acridinium-9-carboxamide, at least one acridinium-9-carboxylate aryl ester, or any
combination thereof. If the detectable label is at least one acridinium compound, the kit also can
comprise a source of hydrogen peroxide, such as a buffer, a solution, and/or at least one basic
solution. If desired, the kit can contain a solid phase, such as a magnetic particle, bead, test tube,
microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc or chip.
C. Adaptation of Kit and Method
The kit (or components thereof), as well as the method of determining the presence,
amount or concentration of an analyte in a test sample by an assay, such as an immunoassay as
described herein, can be adapted for use in a variety of automated and semi-automated systems
(including those wherein the solid phase comprises a microparticle), as described, e.g., in U.S.
Patent Nos. 5,089,424 and 5,006,309, and as commercially marketed, e.g., by Abbott Laboratories
(Abbott Park, IL) as ARCHITECT®.
Some of the differences between an automated or semi-automated system as compared to
a non-automated system (e.g., ELISA) include the substrate to which the first specific binding
partner (e.g., an anti-analyte, monoclonal/polyclonal antibody (or a fragment thereof, a variant
thereof, or a fragment of a variant thereof), a binding protein as disclosed herein, or an antianalyte
DVD-Ig (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) is
attached; either way, sandwich formation and analyte reactivity can be impacted), and the length
and timing of the capture, detection and/or any optional wash steps. Whereas a non-automated
format, such as an ELISA, may require a relatively longer incubation time with sample and
capture reagent (e.g., about 2 hours), an automated or semi-automated format (e.g.,
ARCHITECT®, Abbott Laboratories) may have a relatively shorter incubation time (e.g.,
approximately 18 minutes for ARCHITECT®). Similarly, whereas a non-automated format, such
as an ELISA, may incubate a detection antibody, such as the conjugate reagent, for a relatively
longer incubation time (e.g., about 2 hours), an automated or semi-automated format (e.g.,
ARCHITECT®) may have a relatively shorter incubation time (e.g., approximately 4 minutes for
the ARCHITECT®).
Other platforms available from Abbott Laboratories include, but are not limited to,
AxSYM®, IMx® (see, e.g., U.S. Patent No. 5,294,404), PRISM®, EIA (bead), and Quantum™
II, as well as other platforms. Additionally, the assays, kits and kit components can be employed
in other formats, for example, on electrochemical or other hand-held or point-of-care assay
systems. The present disclosure is, for example, applicable to the commercial Abbott Point of
Care (i-STAT®, Abbott Laboratories) electrochemical immunoassay system that performs
sandwich immunoassays. Immunosensors and their methods of manufacture and operation in
single-use test devices are described, for example in, U.S. Patent No. Nos. 5,063,081; 7,419,821;
and 7,682,833; and U.S. Patent Publication Nos. 20040018577 and 20060160164.
In particular, with regard to the adaptation of an analyte assay to the I-STAT® system,
the following configuration is preferred. A microfabricated silicon chip is manufactured with a
pair of gold amperometric working electrodes and a silver-silver chloride reference electrode. On
one of the working electrodes, polystyrene beads (0.2 mm diameter) with immobilized antianalyte,
monoclonal/polyclonal antibody (or a fragment thereof, a variant thereof, or a fragment
of a variant thereof), a binding protein as disclosed herein, or anti-analyte DVD-Ig (or a fragment
thereof, a variant thereof, or a fragment of a variant thereof), are adhered to a polymer coating of
patterned polyvinyl alcohol over the electrode. This chip is assembled into an I-STAT® cartridge
with a fluidics format suitable for immunoassay. On a portion of the wall of the sample-holding
chamber of the cartridge there is a layer comprising a specific binding partner for an analyte, such
as an anti-analyte, monoclonal/polyclonal antibody (or a fragment thereof, a variant thereof, or a
fragment of a variant thereof that can bind the analyte), a binding protein as disclosed herein, or
an anti-analyte DVD-Ig (or a fragment thereof, a variant thereof, or a fragment of a variant thereof
that can bind the analyte), either of which can be detectably labeled. Within the fluid pouch of the
cartridge is an aqueous reagent that includes p-aminophenol phosphate.
In operation, a sample suspected of containing an analyte is added to the holding chamber
of the test cartridge, and the cartridge is inserted into the I-STAT® reader. After the specific
binding partner for an analyte has dissolved into the sample, a pump element within the cartridge
forces the sample into a conduit containing the chip. Here it is oscillated to promote formation of
the sandwich. In the penultimate step of the assay, fluid is forced out of the pouch and into the
conduit to wash the sample off the chip and into a waste chamber. In the final step of the assay,
the alkaline phosphatase label reacts with p-aminophenol phosphate to cleave the phosphate group
and permit the liberated p-aminophenol to be electrochemically oxidized at the working electrode.
Based on the measured current, the reader is able to calculate the amount of analyte in the sample
by means of an embedded algorithm and factory-determined calibration curve.
It further goes without saying that the methods and kits as described herein necessarily
encompass other reagents and methods for carrying out the immunoassay. For instance,
encompassed are various buffers such as are known in the art and/or which can be readily
prepared or optimized to be employed, e.g., for washing, as a conjugate diluent, microparticle
diluent, and/or as a calibrator diluent. An exemplary conjugate diluent is ARCHITECT®
conjugate diluent employed in certain kits (Abbott Laboratories, Abbott Park, IL) and containing
2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, an antimicrobial agent, and
a detergent. An exemplary calibrator diluent is ARCHITECT® human calibrator diluent
employed in certain kits (Abbott Laboratories, Abbott Park, IL), which comprises a buffer
containing MES, other salt, a protein blocker, and an antimicrobial agent. Additionally, as
described in U.S. Patent Application No. 61/142,048 filed December 31,2008, improved signal
generation may be obtained, e.g., in an I-Stat cartridge format, using a nucleic acid sequence
linked to the signal antibody as a signal amplifier.
EXEMPLIFICATION
It will be readily apparent to those skilled in the art that other suitable modifications and
adaptations of the methods described herein are obvious and may be made using suitable
equivalents without departing from the scope of the claimed invention or the embodiments
disclosed herein. Having now described the present invention in detail, the same will be more
clearly understood by reference to the following examples, which are included for purposes of
illustration only and are not intended to be limiting of the claimed invention.
EXAMPLES
Example 1: Design. Construction, and Analysis of a DVD-Ig
Example 1.1: Assays Used to Identify and Characterize Parent Antibodies and DVD-Ig
The following assays are used throughout the Examples to identify and characterize
parent antibodies and DVD-Ig, unless otherwise stated.
Example 1.1.1: Assays Used To Determine Binding and Affinity of Parent Antibodies and
DVD-Ig for Their Target Antigen(s)
Example 1.1.1A: Direct Bind ELISA
Enzyme Linked Immunosorbent Assays to screen for antibodies that bind a desired target
antigen are performed as follows. High bind ELISA plates (Corning Costar # 3369, Acton, MA)
are coated with 100μL/well of 10μg/ml of desired target antigen (R&D Systems, Minneapolis,
MN) or desired target antigen extra-cellular domain / FC fusion protein (R&D Systems,
Minneapolis, MN) or monoclonal mouse anti-polyHistidine antibody (R&D Systems # MAB050,
Minneapolis, MN) in phosphate buffered saline (10X PBS, Abbott Bioresearch Center, Media
Prep# MPS-073, Worcester, MA) overnight at 4°C. Plates are washed four times with PBS
containing 0.02% Tween 20. Plates are blocked by the addition of 300 μL/well blocking solution
(non-fat dry milk powder, various retail suppliers, diluted to 2% in PBS) for 1/2 hour at room
temperature. Plates are washed four times after blocking with PBS containing 0.02% Tween 20.
Alternatively, one hundred microliters per well of 10 μg/ml of Histidine (His) tagged
desired target antigen (R&D Systems, Minneapolis, MN) are added to ELISA plates coated with
monoclonal mouse anti-polyHistidine antibody as described above and incubated for 1 hour at
room temperature. Wells are washed four times with PBS containing 0.02% Tween 20.
One hundred microliters of antibody or DVD-Ig preparations diluted in blocking solution
as described above is added to the desired target antigen plate or desired target antigen / FC fusion
plate or the anti-polyHistidine antibody / His tagged desired target antigen plate prepared as
described above and incubated for 1 hour at room temperature. Wells are washed four times with
PBS containing 0.02% Tween 20.
One hundred microliters of 10ng/mL goat anti-human IgG -FC specific HRP conjugated
antibody (Southern Biotech # 2040-05, Birmingham, AL) is added to each well of the desired
target antigen plate or anti-polyHistidine antibody / Histidine tagged desired target antigen plate.
Alternatively, one hundred microliters of 10 ng/mL goat anti-human IgG -kappa light chain
specific HRP conjugated antibody (Southern Biotech # 2060-05 Birmingham, AL) is added to
each well of the desired target antigen / FC fusion plate and incubated for 1 hour at room
temperature. Plates are washed 4 times with PBS containing 0.02% Tween 20.
One hundred microliters of enhanced TMB solution (Neogen Corp. #308177, K Blue,
Lexington, KY) is added to each well and incubated for 10 minutes at room temperature. The
reaction is stopped by the addition of 50 μL 1N sulphuric acid. Plates are read
spectrophotometrically at a wavelength of 450 nm.
Example l.l.l.B: Capture ELISA
ELISA plates (Nunc, MaxiSorp, Rochester, NY) are incubated overnight at 4°C with antihuman
Fc antibody (5 μ g/ml in PBS, Jackson Immunoresearch, West Grove, PA). Plates are
washed three times in washing buffer (PBS containing 0.05% Tween 20), and blocked for 1 hour
at 25°C in blocking buffer (PBS containing 1% BSA). Wells are washed three times, and serial
dilutions of each antibody or DVD-Ig in PBS containing 0.1% BSA are added to the wells and
incubated at 25°C for 1 hour. The wells are washed three times, and biotinylated antigen (2nM) is
added to the plates and incubated for 1 hour at 25°C. The wells are three times, and then incubated
for 1 hour at 25°C with streptavidin-HRP (KPL #474-3000, Gaithersburg, MD). The wells are
washed three times, and 100 μ 1 of ULTRA-TMB ELISA (Pierce, Rockford, IL) are added per
well. Following color development the reaction is stopped with 1N HCL and absorbance at
450nM is measured.
Example l.l.l.C: IgG-Fc Capture ELISA
96-well Nunc-Immuno plates are coated with 2μg/mL goat-anti-human IgG Fc specific
antibody (Jackson Immunoresearch # 109-055-098, West Grove, PA, 50μL/well) in PBS (Gibco
#10010-023 from Invitrogen,Grand Island, NY), and incubated overnight at 4°C. Plates are
washed three times with washing buffer (PBS, 0.05% Tween 20) and subsequently blocked with
100μL/well of blocking buffer (PBS, 2% BSA) for one hour at room temperature. Plates are
washed three times and incubated with 50μL/well of a 1μg/mL solution of the appropriate
antibody or DVD-Ig for one hour at room temperature. After the one hour incubation, the plates
are washed three times and incubated with 50μL/well of his-tagged, recombinant antigen protein
(R&D Systems , Minneapolis, MN, 1000nM to OnM final dose range) for one hour at room
temperature. Plates are washed three times, and 50μL/well of a rabbit-anti-His tag-HRP antibody
(Abeam ab1187, Cambridge, MA, diluted at 1:10,000 in 2% BSA/PBS solution) is added and
plates are incubated at room temperature for one hour. After the final wash, 50μl/well of TMB
substrate (Pierce #34028, Rockford, IL) is added, and the reaction is terminated after five minutes
using 50μl/well of 2N H2SC4. The absorbance is read at 450 nm (Spectra Max Plus plate reader,
Molecular Devices, Sunnyvale, CA). EC50s are calculated in GraphPad Prism 4.03.
Example 1.1.1.D: Affinity Determination using BIACORE Technology
BIACORE Methods:
The BIACORE assay (Biacore, Inc, Piscataway, NJ) determines the affinity of antibodies
or DVD-Ig with kinetic measurements of on-rate and off-rate constants. Binding of antibodies or
DVD-Ig to a target antigen (for example, a purified recombinant target antigen) is determined by
surface plasmon resonance-based measurements with a Biacore® 1000 or 3000 instrument
(Biacore® AB, Uppsala, Sweden) using running HBS-EP (10 mM HEPES [pH 7.4], 150 mM
NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C. All chemicals are obtained from
Biacore® AB (Uppsala, Sweden) or otherwise from a different source as described in the text.
For example, approximately 5000 RU of goat anti-mouse IgG, (Fcγ), fragment specific polyclonal
antibody (Pierce Biotechnology Inc, Rockford, IL) diluted in 10 mM sodium acetate (pH 4.5) is
directly immobilized across a CM5 research grade biosensor chip using a standard amine
coupling kit according to manufacturer's instructions and procedures at 25 μg/ml. Unreacted
moieties on the biosensor surface are blocked with ethanolamine. Modified carboxymethyl
dextran surface in flowcell 2 and 4 is used as a reaction surface. Unmodified carboxymethyl
dextran without goat anti-mouse IgG in flow cell 1 and 3 is used as the reference surface. For
kinetic analysis, rate equations derived from the 1:1 Langmuir binding model are fitted
simultaneously to association and dissociation phases of all eight injections (using global fit
analysis) with the use of Biaevaluation 4.0.1 software. Purified antibodies or DVD-Ig are diluted
in HEPES-buffered saline for capture across goat anti-mouse IgG specific reaction surfaces.
Antibodies or DVD-Ig to be captured as a ligand (25 ng/ml) are injected over reaction matrices at
a flow rate of 5 μl/min. The association and dissociation rate constants, kon (M-1s-1) and koff (s-1)
are determined under a continuous flow rate of 25 μl/min. Rate constants are derived by making
kinetic binding measurements at different antigen concentrations ranging from 10 - 200 nM. The
equilibrium dissociation constant (M) of the reaction between antibodies or DVD-Igs and the
target antigen is then calculated from the kinetic rate constants by the following formula: KD =
kofl/kon. Binding is recorded as a function of time and kinetic rate constants are calculated. In this
assay, on-rates as fast as 106M-1s-1 and off-rates as slow as 10-6 s-1 can be measured.
Example 1.1.2: Assays Used To Determine the Functional Activity Of Parent Antibodies
And PVD-Ig
Example 1.1.2.A: Cytokine Bioassav
The ability of an anti-cytokine or an anti-growth factor parent antibody or DVD-Ig
containing anti-cytokine or anti-growth factor sequences to inhibit or neutralize a target cytokine
or growth factor bioactivity is analyzed by determining the inhibitory potential of the antibody or
DVD-Ig. For example, the ability of an anti-IL-4 antibody to inhibit IL-4 mediated IgE
production may be used. For example, human naive B cells are isolated from peripheral blood,
respectively, buffy coats by Ficoll-paque density centrifugation, followed by magnetic separation
with MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) specific for human slgD FITC
labeled goat F(ab)2 antibodies followed by anti-FITC MACS beads. Magnetically sorted naive B
cells are adjusted to 3 x 105 cells per ml in XV15 and plated out in 100 μl per well of 96-well
plates in a 6 x 6 array in the center of the plate, surrounded by PBS filled wells during the 10 days
of culture at 37° C in the presence of 5% CO2. One plate each is prepared per antibody to be
tested, consisting of 3 wells each of un-induced and induced controls and quintuplicate repeats of
antibody titrations starting at 7 μg/ml and running in 3-fold dilution down to 29 ng/ml final
concentrations added in 50μl four times concentrated pre-dilution. To induce IgE production,
rhIL-4 at 20 ng/ml plus anti-CD40 monoclonal antibody (Novartis, Basel, Switzerland) at 0.5
μg/ml final concentrations in 50 μl each are added to each well, and IgE concentrations are
determined at the end of the culture period by a standard sandwich ELISA method.
Example 1.1.2.B: Cytokine Release Assay
The ability of a parent antibody or DVD-Ig to cause cytokine release is analyzed.
Peripheral blood is withdrawn from three healthy donors by venipuncture into heparized
vacutainer tubes. Whole blood is diluted 1:5 with RPMI-1640 medium and placed in 24-well
tissue culture plates at 0.5 mL per well. The anti-cytokine antibodies (e.g., anti-IL-4) are diluted
into RPMI-1640 and placed in the plates at 0.5 mL/well to give final concentrations of 200, 100,
50, 10, and 1 μg/mL. The final dilution of whole blood in the culture plates is 1:10. LPS and
PHA are added to separate wells at 2μg/mL and 5μg/mL final concentration as a positive control
for cytokine release. Polyclonal human IgG is used as negative control antibody. The experiment
is performed in duplicate. Plates are incubated at 37°C at 5% CO2. Twenty-four hours later the
contents of the wells are transferred into test tubes and spun for 5 minutes at 1200 rpm. Cell-free
supernatants are collected and frozen for cytokine assays. Cells left over on the plates and in the
tubes are lysed with 0.5 mL of lysis solution, and placed at -20°C and thawed. 0.5 mL of medium
is added (to bring the volume to the same level as the cell-free supernatant samples) and the cell
preparations are collected and frozen for cytokine assays. Cell-free supernatants and cell lysates
are assayed for cytokine levels by ELISA, for example, for levels of IL-8, IL-6, IL-1ß, IL-1RA,
or TNF-α.
Example 1.1.2.C: Cytokine Cross-Reactivity Study
The ability of an anti-cytokine parent antibody or DVD-Ig directed to a cytokine(s) of
interest to cross react with other cytokines is analyzed. Parent antibodies or DVD-Ig are
immobilized on a Biacore biosensor matrix. An anti-human Fc mAb is covalently linked via free
amine groups to the dextran matrix by first activating carboxyl groups on the matrix with 100mM
N-hydroxysuccinimide (NHS) and 400mM N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC). Approximately 50μL of each antibody or DVD-Ig preparation at a
concentration of 25μg/mL, diluted in sodium acetate, pH 4.5, is injected across the activated
biosensor and free amines on the protein are bound directly to the activated carboxyl groups.
Typically, 5000 Resonance Units (RU's) are immobilized. Unreacted matrix EDC-esters are
deactivated by an injection of 1 M ethanolamine. A second flow cell is prepared as a reference
standard by immobilizing human IgG1/K using the standard amine coupling kit. SPR
measurements are performed using the CM biosensor chip. All antigens to be analyzed on the
biosensor surface are diluted in HBS-EP running buffer containing 0.01% P20.
To examine the cytokine binding specificity, excess cytokine of interest (100nM, e.g.,
soluble recombinant human) is injected across the anti-cytokine parent antibody or DVD-Ig
immobilized biosensor surface (5 minute contact time). Before injection of the cytokine of
interest and immediately afterward, HBS-EP buffer alone flows through each flow cell. The net
difference in the signals between the baseline and the point corresponding to approximately 30
seconds after completion of cytokine injection are taken to represent the final binding value.
Again, the response is measured in Resonance Units. Biosensor matrices are regenerated using
10mM HC1 before injection of the next sample where a binding event is observed, otherwise
running buffer was injected over the matrices. Human cytokines (e.g., IL-1α, IL-1ß, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19,
IL-20, IL-22, IL-23, IL-27, TNF-a, TNF-p, and IFN-y, for example) are also simultaneously
injected over the immobilized mouse IgGl/K reference surface to record any nonspecific binding
background. By preparing a reference and reaction surface, Biacore can automatically subtract
the reference surface data from the reaction surface data in order to eliminate the majority of the
refractive index change and injection noise. Thus, it is possible to ascertain the true binding
response attributed to an anti-cytokine antibody or DVD-Ig binding reaction.
When a cytokine of interest is injected across immobilized anti-cytokine antibody,
significant binding is observed. 10mM HC1 regeneration completely removes all non-covalently
associated proteins. Examination of the sensorgram shows that immobilized anti-cytokine
antibody or DVD-Ig binding to soluble cytokine is strong and robust. After confirming the
expected result with the cytokine of interest, the panel of remaining recombinant human cytokines
is tested, for each antibody or DVD-Ig separately. The amount of anti-cytokine antibody or
DVD-Ig bound or unbound cytokine for each injection cycle is recorded. The results from three
independent experiments are used to determine the specificity profile of each antibody or DVDIg.
Antibodies or DVD-Ig with the expected binding to the cytokine of interest and no binding to
any other cytokine are selected.
Example 1.1.2.D: Tissue Cross Reactivity
Tissue cross reactivity studies are done in three stages, with the first stage including
cryosections of 32 tissues, second stage including up to 38 tissues, and the 3rd stage including
additional tissues from 3 unrelated adults as described below. Studies are done typically at two
dose levels.
Stage 1: Cryosections (about 5 μm) of human tissues (32 tissues (typically:
Adrenal Gland, Gastrointestinal Tract, Prostate, Bladder, Heart, Skeletal Muscle, Blood Cells,
Kidney, Skin, Bone Marrow, Liver, Spinal Cord, Breast, Lung, Spleen, Cerebellum, Lymph
Node, Testes, Cerebral Cortex, Ovary, Thymus, Colon, Pancreas, Thyroid, Endothelium,
Parathyroid, Ureter, Eye, Pituitary, Uterus, Fallopian Tube and Placenta) from one human donor
obtained at autopsy or biopsy) are fixed and dried on object glass. The peroxidase staining of
tissue sections is performed, using the avidin-biotin system.
Stage 2:Cryosections (about 5 μm) of human tissues 38 tissues (including adrenal, blood,
blood vessel, bone marrow, cerebellum, cerebrum, cervix, esophagus, eye, heart, kidney, large
intestine, liver, lung, lymph node, breast mammary gland, ovary, oviduct, pancreas, parathyroid,
peripheral nerve, pituitary, placenta, prostate, salivary gland, skin, small intestine, spinal cord,
spleen, stomach, striated muscle, testis, thymus, thyroid, tonsil, ureter, urinary bladder, and
uterus) from 3 unrelated adults obtained at autopsy or biopsy) are fixed and dried on object glass.
The peroxidase staining of tissue sections is performed, using the avidin-biotin system.
Stage 3: Cryosections (about 5 μm) of cynomolgus monkey tissues (38 tissues (including
adrenal, blood, blood vessel, bone marrow, cerebellum, cerebrum, cervix, esophagus, eye, heart,
kidney, large intestine, liver, lung, lymph node, breast mammary gland, ovary, oviduct, pancreas,
parathyroid, peripheral nerve, pituitary, placenta, prostate, salivary gland, skin, small intestine,
spinal cord, spleen, stomach, striated muscle, testis, thymus, thyroid, tonsil, ureter, urinary
bladder, and uterus) from 3 unrelated adult monkeys obtained at autopsy or biopsy) are fixed and
dried on object glass. The peroxidase staining of tissue sections is performed, using the avidinbiotin
system.
The antibody or DVD-Ig is incubated with the secondary biotinylated anti-human IgG
and developed into immune complex. The immune complex at the final concentrations of 2 and
10 ng/mL of antibody or DVD-Ig is added onto tissue sections on object glass and then the tissue
sections are reacted for 30 minutes with a avidin-biotin-peroxidase kit. Subsequently, DAB (3,3'-
diaminobenzidine), a substrate for the peroxidase reaction, is applied for 4 minutes for tissue
staining. Antigen-Sepharose beads are used as positive control tissue sections. Target antigen
and human serum blocking studies serve as additional controls. The immune complex at the final
concentrations of 2 and 10 μg/mL of antibody or DVD-Ig is pre-incubated with target antigen
(final concentration of 100 μg/ml) or human serum (final concentration 10%) for 30 minutes, and
then added onto the tissue sections on object glass and then the tissue sections are reacted for 30
minutes with a avidin-biotin-peroxidase kit. Subsequently, DAB (3,3'-diaminobenzidine), a
substrate for the peroxidase reaction, is applied for 4 minutes for tissue staining.
Any specific staining is judged to be either an expected (e.g., consistent with antigen
expression) or unexpected reactivity based upon known expression of the target antigen in
question. Any staining judged specific is scored for intensity and frequency. The tissue staining
between stage 2 (human tissue) and stage 3 (cynomolgus monkey tissue) is either judged to be
similar or different.
Example 1.1.2.E: Growth Inhibitory Effect of an EGFR Monoclonal Antibody or DVD-Igs
In Vitro
Parent antibodies or DVD-Ig that bind to target antigens on tumor cells may be analysed
for tumoricidal activity. Briefly, parent antibodies or DVD-Ig are diluted in D-PBS-BSA
(Dulbecco's phosphate buffered saline with 0.1%BSA) 20μL are added to human tumor cells at
final concentrations of 0.01 μg/mL to 100 μg/mL in 180uL. The plates are incubated at 37 °C in a
humidified, 5% CO2 atmosphere for 3 days. The number of live cells in each well is quantified
using MTS reagents according to the manufacturer's instructions (Promega, Madison, WI) to
determine the percent of tumor growth inhibition. Wells without antibody treatment are used as
controls of 0% inhibition whereas wells without cells are considered to show 100% inhibition.
Example 1.1.2.F: Tumoricidal Effect of A Parent or DVD-Ig Antibody In Vitro
Parent antibodies or DVD-Ig that bind to target antigens on tumor cells may be analyzed
for tumoricidal activity. Briefly, parent antibodies or DVD-Ig are diluted in D-PBS-BSA
(Dulbecco's phosphate buffered saline with 0.1%BSA) and added to human tumor cells at final
concentrations of 0.01 μg/mL to 100 μg/mL in 200μL. The plates are incubated at 37 °C in a
humidified, 5% CO2 atmosphere for 3 days. The number of live cells in each well is quantified
using MTS reagents according to the manufacturer's instructions (Promega, Madison, WI) to
determine the percent of tumor growth inhibition. Wells without antibody treatment are used as
controls of 0% inhibition whereas wells without cells were considered to show 100% inhibition.
For assessment of apoptosis, caspase-3 activation is determined by the following
protocol: antibody-treated cells in 96 well plates are lysed in 120 μl of 1x lysis buffer (1.67mM
Hepes, pH 7.4, 7mM KC1,0.83mM MgCl2,0.11mM EDTA, 0.1 ImM EGTA, 0.57% CHAPS,
ImM DTT, 1x protease inhibitor cocktail tablet; EDTA-free; Roche Pharmaceuticals, Nutley, NJ)
at room temperature with shaking for 20 minutes. After cell lysis, 80 μl of a caspase-3 reaction
buffer (48mM Hepes, pH 7.5, 252mM sucrose, 0.1% CHAPS, 4mM DTT, and 20 μM Ac-DEVDAMC
substrate; Biomol Research Labs, Inc., Plymouth Meeting, PA) is added and the plates are
incubated for 2 hours at 37°C. The plates are read on a 1420 VICTOR Multilabel Counter (Perkin
Elmer Life Sciences, Downers Grove, IL) using the following settings: excitation= 360/40,
emission= 460/40. An increase of fluorescence units from antibody-treated cells relative to the
isotype antibody control-treated cells is seen, which is indicative of apoptosis.
Example 1.1.2.G: Inhibition Of Receptor Activation bv Antibody Or DVD-Ig Constructs In
Vitro
Parent antibodies or DVD-Ig that bind to cell receptors or their ligands may be tested for
inhibition of receptor activation. Parent antibodies or DVD-Ig diluted in D-PBS-BSA (Dulbecco's
phosphate buffered saline with 0.1%BSA) are added to human carcinoma cells at final
concentrations of 0.01 μg/mL to 100 μg/mL (180μL). The plates are incubated at 37 °C in a
humidified, 5% CO2 atmosphere for 1 hour. Growth factors (e.g., EGF) at a final concentration of
1-100ng/mL (20nL) are added to the cells for 5-15 minutes to stimulate receptor (e.g., EGFR)
autophosphorylation. Wells without antibody treatment are used as controls of 0% inhibition
whereas wells without growth factor stimulation are considered to show 100% inhibition. Cell
lysates are made by incubation with cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 10%
Glycerol, 0.1% SDS, and protease inhibitor cocktail). For example, phospho-EGFR in these cell
lysates is determined using the p-EGFR ELISA kit from R&D Systems (#DYC1095,
Minneapolis, MN) according to the manufacturer's instructions.
Example 1.1.2.H: Efficacy Of An Anti-Tumor Cell Antigen Antibody or DVD-Ig By Itself
Or In Combination with Chemotherapy On The Growth Of Human Carcinoma Xenografts
(Subcutaneous Flank. Orthotopic. Or Spontaneous Metastases)
Human cancer cells are grown in vitro to 99% viability, 85% confluence in tissue culture
flasks. SCID mice (Charles Rivers Labs) at 19-25 grams are ear tagged and shaved. Mice are
then inoculated subcutaneously into the right flank with 0.2ml of 2 x 106 human tumor cells (1:1
matrigel) on study day 0. Administration (IP, Q3D/ week) of vehicle (PBS), antibody or DVD-Ig,
and/or chemotherapy is initiated after mice are size matched into separate ages of mice with mean
tumor volumes of approximately 150 to 200 mm3. The tumors are measured by a pair of calipers
twice a week starting on approximately day 10 post inoculation and the tumor volumes calculated
according to the formula V = L xW2/2 (V: volume, mm3; L: length, mm; W: width, mm).
Reduction in tumor volume is seen in animals treated with the antibody or DVD-Ig alone or in
combination with chemotherapy relative to tumors in animals that received only vehicle or an
isotype control mAb.
Example 1.1.2.1: Binding of Monoclonal Antibodies to the Surface of Human Tumor Cell
Lines as Assessed bv Flow Cytometry
Stable cell lines overexpressing a cell-surface antigen of interest or human tumor cell
lines are harvested from tissue culture flasks and resuspended in phosphate buffered saline (PBS)
containing 5% fetal bovine serum (PBS/FBS). Prior to staining, human tumor cells are incubated
on ice with (100μ1) human IgG at 5μg/ml in PBS/FCS. 1-5 x105 cells are incubated with antibody
or DVD-Ig (2 μg/mL) in PBS/FBS for 30-60 minutes on ice. Cells are washed twice and 100μl of
F(ab')2 goat anti human IgG, Fcγ- phycoerythrin (1:200 dilution in PBS) (Jackson
ImmunoResearch, West Grove, PA, Cat.#109-116-170) is added. After 30 minutes incubation on
ice, cells are washed twice and resuspended in PBS/FBS. Fluorescence is measured using a
Becton Dickinson FACSCalibur (Becton Dickinson, San Jose, CA).
Example 1.1.2.K: Binding of Monoclonal Antibodies to the Surface of Activated NK Cells
as Assessed bv Flow Cytometry
Activated NK cells were plated at 0.5x105 cells/well on a 96 well round bottom plate.
Antibodies and DVD-Igs were diluted to 10 μg/ml in FACS buffer (1% FBS in PBS pH 7.4). The
supernatant was removed from the cells and 30 μL of diluted antibodies or DVD-Igs was added to
the wells. Cells were incubated with the antibodies at 4°C for 30 minutes. Following incubation,
the cells were washed three times with 150 μL FACS buffer. The cells were resuspended in
50 uL FACS buffer with 1:125 diluted R-PE conjugated anti-human IgG F(Ab')2 (Jackson
ImmunoResearch, West Grove, PA, Cat.# 109-116-170), anti-CD56-APC (eBioscience, San
Diego, CA, Cat.#l 7-0569), or anti-CD3-488 (eBioscience, San Diego, CA, Cat.#53-0037) and
incubated at 4°C for 30 minutes. Cells were washed three times, and finally resuspended in 100
μL FACS buffer. Samples were run on a FACSCalibur machine (Becton Dickinson, San Jose,
CA). FACSCalibur settings for FL1, FL2, and FL4 were adjusted such that a non-antibody-treated
control sample had a GMFI of 3. Experimental samples were run subsequently. Flow Jo software
(Treestar, Inc, Ashland, OR) was used to analyze the data and determine R-PE GMFI on CD56
positive, live cells as designated by a forward and side scatter gate.
The Table 3 contains the FACS geometric mean of parent antibodies and DVD-Ig
constructs on both stable cell lines or NK cells.
Table 3: Fluorescent Activated Cell Sorting (FACS) of DVD-Ig Constructs
All DVD-Igs showed binding to their cell surface targets. The N-terminal domains of
DVD-Igs bound their targets on the cell surface as well as, or better than, the parent antibody.
Binding can be restored or improved by adjusting linker length.
Example 1.1.2.L: Binding of Monoclonal Antibodies to the Surface of Human Tumor Cell
Lines as Assessed bv Flow Cytometry using FACSCanto
Stable cell lines overexpressing a cell-surface antigen of interest or human tumor cell
lines were harvested from tissue culture flasks and resuspended in phosphate buffered saline
(PBS) containing 5% fetal bovine serum (PBS/FBS). Prior to staining, human tumor cells were
incubated on ice with (100μl) human IgG at 5μg/ml in PBS/FCS. 1-5 x105 cells were incubated
with antibody or DVD-Ig (2 μg/mL) in PBS/FBS for 30-60 minutes on ice. Cells were washed
twice and 100μl of F(ab')2 goat anti human IgG, Fcγ- Dylight488 (1:200 dilution in PBS)
(Jackson ImmunoResearch, West Grove, PA, Cat.# 109-486-098) was added. After 30 minutes
incubation on ice, cells were washed twice and resuspended in PBS/FBS. Fluorescence was
measured using a Becton Dickinson FACSCanto machine (Becton Dickinson, San Jose, CA).
Table 4 contains the FACS geometric mean of parent antibodies and DVD-Ig constructs.
Table 4: Fluorescent Activated CeU Sorting (FACS) of DVD-Ig Constructs using FACS
Canto
All DVD-Igs showed binding to their cell surface targets. The N-terminal domains of
DVD-Igs bound their targets on the cell surface as well as or better than the parent antibody.
Binding can be restored or improved by adjusting linker length.
Example 1.2: Generation Of Parent Monoclonal Antibodies to a Human Antigen of Interest
Parent mouse mAbs able to bind to and neutralize a human antigen of interest and a
variant thereof are obtained as follows:
Example 1.2.A: Immunization Of Mice With a Human Antigen of Interest
Twenty micrograms of recombinant purified human antigen (e.g., IGF1,2) mixed with
complete Freund's adjuvant or Immunoeasy adjuvant (Qiagen, Valencia, CA) is injected
subcutaneously into five 6-8 week-old Balb/C, five C57B/6 mice, and five AJ mice on Day 1. On
days 24, 38, and 49, twenty micrograms of recombinant purified human antigen variant mixed
with incomplete Freund's adjuvant or Immunoeasy adjuvant is injected subcutaneously into the
same mice. On day 84 or day 112 or day 144, mice are injected intravenously with 1μg
recombinant purified human antigen of interest.
Example 1.2.B: Generation of a Hvbridoma
Splenocytes obtained from the immunized mice described in Example 1.2.A are fused
with SP2/0-Ag-l4 cells at a ratio of 5:1 according to the established method described in Kohler,
G. and Milstein (1975) Nature, 256:495 to generate hybridomas. Fusion products are plated in
selection media containing azaserine and hypoxanthine in 96-well plates at a density of 2.5x106
spleen cells per well. Seven to ten days post fusion, macroscopic hybridoma colonies are
observed. Supernatant from each well containing hybridoma colonies is tested by ELISA for the
presence of antibody to the antigen of interest (as described in Example 1.1.LA). Supernatants
displaying antigen-specific activity are then tested for activity (as described in the assays of
Example 1.1.2), for example, the ability to neutralize the antigen of interest in a bioassay such as
that described in Example 1.1.2.1).
Example 1.2.C: Identification And Characterization Of Parent Monoclonal Antibodies to a
Human Target Antigen of Interest
Example 1.2.C.1: Analyzing Parent Monoclonal Antibody Neutralizing Activity
Hybridoma supernatants are assayed for the presence of parent antibodies that bind an
antigen of interest, generated according to Examples 1.2.A and 1.2.B, and a variant of the antigen
of interest ("antigen variant"). Supernatants with antibodies positive in both assays are then
tested for their antigen neutralization potency, for example, in the cytokine bioassay of Example
1.1.2.1. The hybridomas producing antibodies with IC50 values in the bioassay less than l,000pM,
in an embodiment, less than lOOpM are scaled up and cloned by limiting dilution. Hybridoma
cells are expanded into media containing 10% low IgG fetal bovine serum (Hyclone #SH30151,
Logan, UT). On average, 250 mL of each hybridoma supernatant (derived from a clonal
population) is harvested, concentrated and purified by protein A affinity chromatography, as
described in Harlow, E. and Lane, D. 1988 "Antibodies: A Laboratory Manual". The ability of
purified mAbs to inhibit the activity of its target antigen is determined, for example, using the
cytokine bioassay as described in Example 1.1.2.1.
Example 1.2.C.2: Analyzing Parent Monoclonal Antibody Cross-Reactivity To Cynomolgus
Target Antigen Of Interest
To determine whether the selected mAbs described herein recognize cynomolgus antigen
of interest, BIACORE analysis is conducted as described herein (Example 1.1.1.B) using
recombinant cynomolgus target antigen. In addition, neutralization potencies of mAbs against
recombinant cvnomolgus antigen of interest may also be measured in the cytokine bioassay
(Example 1.1.2.A). MAbs with good cyno cross-reactivity (in an embodiment, within 5-fold of
reactivity for human antigen) are selected for future characterization.
Example 1.2.D: Determination Of The Amino Acid Sequence Of The Variable Region For
Each Murine Anti-Human Monoclonal Antibody
Isolation of the cDNAs, expression and characterization of the recombinant anti-human
mouse mAbs is conducted as follows. For each amino acid sequence determination,
approximately 1 x 106 hybridoma cells are isolated by centrifugation and processed to isolate
total RNA with Trizol (Gibco BRL/Invitrogen, Carlsbad, CA.) following manufacturer's
instructions. Total RNA is subjected to first strand DNA synthesis using the Superscript First-
Strand Synthesis System (Invitrogen, Carlsbad, CA) per the manufacturer's instructions.
Oligo(dT) is used to prime first-strand synthesis to select for poly(A)+ RNA. The first-strand
cDNA product is then amplified by PCR with primers designed for amplification of murine
immunoglobulin variable regions (Ig-Primer Sets, Novagen, Madison, WI). PCR products are
resolved on an agarose gel, excised, purified, and then subcloned with the TOPO Cloning kit into
pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 chemically
competent E. coli (Invitrogen, Carlsbad, CA). Colony PCR is performed on the transformants to
identify clones containing insert. Plasmid DNA is isolated from clones containing insert using a
QIAprep Miniprep kit (Qiagen, Valencia, CA). Inserts in the plasmids are sequenced on both
strands to determine the variable heavy or variable light chain DNA sequences using M13
forward and M13 reverse primers (Fermentas Life Sciences, Hanover MD). Variable heavy and
variable light chain sequences of the mAbs are identified. In an embodiment, the selection criteria
for a panel of lead mAbs for next step development (humanization) includes the following:
• The antibody does not contain any N-linked glycosylation sites (NXS), except from the
standard one in CH2
• The antibody does not contain any extra cysteines in addition to the normal cysteines in
every antibody
• The antibody sequence is aligned with the closest human germline sequences for VH and
VL and any unusual amino acids should be checked for occurrence in other natural
human antibodies
• N-terminal Glutamine (Q) is changed to Glutamic acid (E) if it does not affect the activity
of the antibody. This will reduce heterogeneity due to cyclization of Q
• Efficient signal sequence cleavage is confirmed by Mass Spectrophotometry. This can be
done with COS cell or 293 cell material
• The protein sequence is checked for the risk of deamidation of Asn that could result in
loss of activity
• The antibody has a low level of aggregation
• The antibody has solubility >5-l 0 mg/ml (in research phase); >25 mg/ml
• The antibody has a normal size (5-6 nm) by Dynamic Light Scattering (DLS)
• The antibody has a low charge heterogeneity
• The antibody lacks cytokine release (see Example 1.1.2.B)
• The antibody has specificity for the intended cytokine (see Example 1.1.2.C)
• The antibody lacks unexpected tissue cross reactivity (see Example 1.1.2.D)
• The antibody has similarity between human and cynomolgus tissue cross reactivity (see
Example 1.1.2.D)
Example 1.2.2: Recombinant Humanized Parent Antibodies
Example 1.2.2.1: Construction And Expression Of Recombinant Chimeric Anti Human
Parent Antibodies
The DNA encoding the heavy chain constant region of murine anti-human parent mAbs is
replaced by a cDNA fragment encoding the human IgGl constant region containing 2 hingeregion
amino acid mutations by homologous recombination in bacteria. These mutations are a
leucine to alanine change at position 234 (EU numbering) and a leucine to alanine change at
position 235 (Lund et al. (1991) J. Immunol.: 147:2657). The light chain constant region of each
of these antibodies is replaced by a human kappa constant region. Full-length chimeric antibodies
are transiently expressed in COS cells by co-transfection of chimeric heavy and light chain
cDNAs ligated into the pBOS expression plasmid (Mizushima and Nagata (1990) Nucl. Acids
Res. 18: 5322). Cell supernatants containing recombinant chimeric antibody are purified by
Protein A Sepharose chromatography and bound antibody is eluted by addition of acid buffer.
Antibodies are neutralized and dialyzed into PBS.
The heavy chain cDNA encoding a chimeric mAb is co-transfected with its chimeric light
chain cDNA (both ligated in the pBOS vector) into COS cells. Cell supernatant containing
recombinant chimeric antibody is purified by Protein A Sepharose chromatography and bound
antibody is eluted by addition of acid buffer. Antibodies are neutralized and dialyzed into PBS.
The purified chimeric anti-human parent mAbs are then tested for their ability to bind (by
Biacore) and for functional activity, e.g., to inhibit the cytokine induced production of IgE as
described in Examples 1.1.1 and 1.1.2. Chimeric mAbs that maintain the activity of the parent
hybridoma mAbs are selected for future development.
Example 1.2.2.2: Construction And Expression Of Humanized Anti Human Parent
Antibodies
Example 1.2.2.2.A: Selection Of Human Antibody Frameworks
Each murine variable heavy and variable light chain gene sequence is separately aligned
against 44 human immunoglobulin germline variable heavy chain or 46 germline variable light
chain sequences (derived from NCBI Ig Blast website at
http://www.ncbi.nlm.nih.gov/igblast/retrieveig.html.) using Vector NTI software.
Humanization is based on amino acid sequence homology, CDR cluster analysis,
frequency of use among expressed human antibodies, and available information on the crystal
structures of human antibodies. Taking into account possible effects on antibody binding, VHVL
pairing, and other factors, murine residues are mutated to human residues where murine and
human framework residues are different, with a few exceptions. Additional humanization
strategies are designed based on an analysis of human germline antibody sequences, or a
subgroup thereof, that possessed a high degree of homology, i.e., sequence similarity, to the actual
amino acid sequence of the murine antibody variable regions.
Homology modeling is used to identify residues unique to the murine antibody sequences
that are predicted to be critical to the structure of the antibody combining site, the CDRs.
Homology modeling is a computational method whereby approximate three dimensional
coordinates are generated for a protein. The source of initial coordinates and guidance for their
further refinement is a second protein, the reference protein, for which the three dimensional
coordinates are known and the sequence of which is related to the sequence of the first protein.
The relationship among the sequences of the two proteins is used to generate a correspondence
between the reference protein and the protein for which coordinates are desired, the target protein.
The primary sequences of the reference and target proteins are aligned with coordinates of
identical portions of the two proteins transferred directly from the reference protein to the target
protein. Coordinates for mismatched portions of the two proteins, e.g., from residue mutations,
insertions, or deletions, are constructed from generic structural templates and energy refined to
insure consistency with the already transferred model coordinates. This computational protein
structure may be further refined or employed directly in modeling studies. The quality of the
model structure is determined by the accuracy of the contention that the reference and target
proteins are related and the precision with which the sequence alignment is constructed.
For the murine mAbs, a combination of BLAST searching and visual inspection is used to
identify suitable reference structures. Sequence identity of 25% between the reference and target
amino acid sequences is considered the minimum necessary to attempt a homology modeling
exercise. Sequence alignments are constructed manually and model coordinates are generated
with the program Jackal (see Petrey, D. et al. (2003) Proteins 53 (Suppl. 6): 430-435).
The primary sequences of the murine and human framework regions of the selected
antibodies share significant identity. Residue positions that differ are candidates for inclusion of
the murine residue in the humanized sequence in order to retain the observed binding potency of
the murine antibody. A list of framework residues that differ between the human and murine
sequences is constructed manually. Table 5 shows the framework sequences chosen for this
study.
Table 5: Sequence Of Human IgG Heavy Chain Constant Domain And Light Chain
Constant Domain
The likelihood that a given framework residue would impact the binding properties of the
antibody depends on its proximity to the CDR residues. Therefore, using the model structures,
the residues that differ between the murine and human sequences are ranked according to their
distance from any atom in the CDRs. Those residues that fell within 4.5 A of any CDR atom are
identified as most important and are recommended to be candidates for retention of the murine
residue in the humanized antibody (i.e., back mutation).
In silico constructed humanized antibodies are constructed using oligonucleotides. For
each variable region cDNA, 6 oligonucleotides of 60-80 nucleotides each are designed to overlap
each other by 20 nucleotides at the 5' and/or 3' end of each oligonucleotide. In an annealing
reaction, all 6 oligonulceotides are combined, boiled, and annealed in the presence of dNTPs.
DNA polymerase I, Large (Klenow) fragment (New England Biolabs #M0210, Beverley, MA.) is
added to fill-in the approximately 40bp gaps between the overlapping oligonucleotides. PCR is
performed to amplify the entire variable region gene using two outermost primers containing
overhanging sequences complementary to the multiple cloning site in a modified pBOS vector
(Mizushima, S. and Nagata, S. (1990) Nucleic Acids Res. 18: 17). The PCR products derived
from each cDNA assembly are separated on an agarose gel and the band corresponding to the
predicted variable region cDNA size is excised and purified. The variable heavy region is
inserted in-frame onto a cDNA fragment encoding the human IgGl constant region containing 2
hinge-region amino acid mutations by homologous recombination in bacteria. These mutations
are a leucine to alanine change at position 234 (EU numbering) and a leucine to alanine change at
position 235 (Lund et al. (1991) J. Immunol. 147:2657). The variable light chain region is
inserted in-frame with the human kappa constant region by homologous recombination. Bacterial
colonies are isolated and plasmid DNA extracted. cDNA inserts are sequenced in their entirety.
Correct humanized heavy and light chains corresponding to each antibody are co-transfected into
COS cells to transiently produce full-length humanized anti-human antibodies. Cell supernatants
containing recombinant chimeric antibody are purified by Protein A Sepharose chromatography
and bound antibody is eluted by addition of acid buffer. Antibodies are neutralized and dialyzed
into PBS.
Example 1.2.2.3: Characterization Of Humanized Antibodies
The ability of purified humanized antibodies to inhibit a functional activity is determined,
e.g., using the cytokine bioassay as described in Examples I.I.2.A. The binding affinities of the
humanized antibodies to recombinant human antigen are determined using surface plasmon
resonance (Biacore®) measurement as described in Example 1.1.1 .B. The IC50 values from the
bioassays and the affinity of the humanized antibodies are ranked. The humanized mAbs that
fully maintain the activity of the parent hybridoma mAbs are selected as candidates for future
development. The top 2-3 most favorable humanized mAbs are further characterized.
Example 1.2.2.3.A: Pharmacokinetic Analysis Of Humanized Antibodies
Pharmacokinetic studies are carried out in Sprague-Dawley rats and cynomolgus
monkeys. Male and female rats and cynomolgus monkeys are dosed intravenously or
subcutaneously with a single dose of 4mg/kg mAb and samples are analyzed using antigen
capture ELISA, and pharmacokinetic parameters are determined by noncompartmental analysis.
Briefly, ELISA plates are coated with goat anti-biotin antibody (5 mg/ml, 4°C, overnight),
blocked with Superblock (Pierce), and incubated with biotinylated human antigen at 50 ng/ml in
10% Superblock TTBS at room temperature for 2 hours. Serum samples are serially diluted
(0.5% serum, 10% Superblock in TTBS) and incubated on the plate for 30 minutes at room
temperature. Detection is carried out with HRP-labeled goat anti human antibody and
concentrations are determined with the help of standard curves using the four parameter logistic
fit. Values for the pharmacokinetic parameters are determined by non-compartmental model
using WinNonlm software (Pharsight Corporation, Mountain View, CA). Humanized mAbs with
good pharmacokinetics profile (Tl/2 is 8-13 days or better, with low clearance and excellent
bioavailability 50-100%) are selected.
Example 1.2.2.3.B: Physicochemical And In Vitro Stability Analysis Of Humanized
Monoclonal Antibodies
Size exclusion chromatography
Antibodies are diluted to 2.5 mg/mL with water and 20 mL is analyzed on a Shimadzu
HPLC system using a TSK gel G3000 SWXL column (Tosoh Bioscience, cat# k5539-05k).
Samples are eluted from the column with 211 mM sodium sulfate, 92 mM sodium phosphate, pH
7.0, at a flow rate of 0.3 mL/minutes. The HPLC system operating conditions are the following:
Mobile phase: 211 mM Na2SO4, 92 mM Na2HPO4*7H20, pH 7.0
Gradient: Isocratic
Flow rate: 0.3 mL/minute
Detector wavelength: 280 nm
Autosampler cooler temp: 4°C
Column oven temperature: Ambient
Run time: 50 minutes
Table 6: Purity of Parent Antibodies and D VD-Ig Constructs as Determined by Size
Exclusion Chromatography (SEC)
DVD-Igs showed an excellent SEC profile with most DVD-Ig showing >90% monomer.
This DVD-Ig profile is similar to that observed for parent antibodies.
SDS-PAGE
Antibodies are analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis
(SDS-PAGE) under both reducing and non-reducing conditions. Adalimumab lot AFP04C is used
as a control. For reducing conditions, the samples are mixed 1:1 with 2X tris glycine SDS-PAGE
sample buffer (Invitrogen, cat# LC2676, lot# 1323208) with 100 mM DTT, and heated at 60°C
for 30 minutes. For non-reducing conditions, the samples are mixed 1:1 with sample buffer and
heated at 100°C for 5 minutes. The reduced samples (10 mg per lane) are loaded on a 12% precast
tris-glycine gel (Invitrogen, cat# EC6005box, lot# 6111021), and the non-reduced samples
(10 mg per lane) are loaded on an 8%-16% pre-cast tris-glycine gel (Invitrogen, cat# EC6045box,
lot# 6111021). SeeBlue Plus 2 (Invitrogen, cat#LC5925, lot# 1351542) is used as a molecular
weight marker. The gels are run in a XCell SureLock mini cell gel box (Invitrogen, cat# EI0001)
and the proteins are separated by first applying a voltage of 75 to stack the samples in the gel,
followed by a constant voltage of 125 until the dye front reached the bottom of the gel. The
running buffer used is IX tris glycine SDS buffer, prepared from a 10X tris glycine SDS buffer
(ABC, MPS-79-080106)). The gels are stained overnight with colloidal blue stain (Invitrogen
cat# 46-7015, 46-7016) and destained with Milli-Q water until the background is clear. The
stained gels are then scanned using an Epson Expression scanner (model 1680, S/N
DASX003641).
Sedimentation Velocity Analysis
Antibodies are loaded into the sample chamber of each of three standard two-sector
carbon epon centerpieces. These centerpieces have a 1.2 cm optical path length and are built with
sapphire windows. PBS is used for a reference buffer and each chamber contained 140 uL. All
samples are examined simultaneously using a 4-hole (AN-60Ti) rotor in a Beckman ProteomeLab
XL-I analytical ultracentrifuge (serial # PL106C01).
Run conditions are programmed and centrifuge control is performed using ProteomeLab
(v5.6). The samples and rotor are allowed to thermally equilibrate for one hour prior to analysis
(20.0 ± 0.1 °C). Confirmation of proper cell loading is performed at 3000 rpm and a single scan is
recorded for each cell. The sedimentation velocity conditions are the following:
Sample Cell Volume: 420 mL
Reference Cell Volume: 420 mL
Temperature: 20°C
Rotor Speed: 35,000 rpm
Time: 8:00 hours
UV Wavelength: 280 nm
Radial Step Size: 0.003 cm
Data Collection: One data point per step without signal averaging.
Total Number of Scans: 100
LC-MS molecular weight measurement of intact antibodies
Molecular weights of intact antibodies are analyzed by LC-MS. Each antibody is diluted
to approximately 1 mg/mL with water. An 1100 HPLC (Agilent) system with a protein microtrap
(Michrom Bioresources, Inc, cat# 004/25109/03) is used to desalt and introduce 5 mg of the
sample into an API Qstar pulsar i mass spectrometer (Applied Biosystems). A short gradient is
used to elute the samples. The gradient is run with mobile phase A (0.08% FA, 0.02% TFA in
HPLC water) and mobile phase B (0.08% FA and 0.02% TFA in acetonitrile) at a flow rate of 50
mL/minute. The mass spectrometer is operated at 4.5 kvolts spray voltage with a scan range from
2000 to 3500 mass to charge ratio.
LC-MS molecular weight measurement of antibody light and heavy chains
Molecular weight measurement of antibody light chain (LC), heavy chain (HC) and
deglycosylated HC are analyzed by LC-MS. Aantibody is diluted to 1 mg/mL with water and the
sample is reduced to LC and HC with a final concentration of 10 mM DTT for 30 minutes at
37°C. To deglycosylate the antibody, 100 mg of the antibody is incubated with 2 mL of PNGase
F, 5 mL of 10% N-octylglucoside in a total volume of 100 mL overnight at 37 °C. After
deglycosylation the sample is reduced with a final concentration of 10 mM DTT for 30 minutes at
37°C. An Agilent 1100 HPLC system with a C4 column (Vydac, cat# 214TP5115, S/N
060206537204069) is used to desalt and introduce the sample (5 mg) into an API Qstar pulsar i
mass spectrometer (Applied Biosystems). A short gradient is used to elute the sample. The
gradient is run with mobile phase A (0.08% FA, 0.02% TFA in HPLC water) and mobile phase B
(0.08% FA and 0.02% TFA in acetonitrile) at a flow rate of 50 mL/minute. The mass
spectrometer is operated at 4.5 kvolts spray voltage with a scan range from 800 to 3500 mass to
charge ratio.
Peptide mapping
Antibody is denatured for 15 minutes at room temperature with a final concentration of 6
M guanidine hydrochloride in 75 mM ammonium bicarbonate. The denatured samples are
reduced with a final concentration of 10 mM DTT at 37°C for 60 minutes, followed by alkylation
with 50 mM iodoacetic acid (IAA) in the dark at 37°C for 30 minutes. Following alkylation, the
sample is dialyzed overnight against four liters of 10 mM ammonium bicarbonate at 4°C. The
dialyzed sample is diluted to 1 mg/mL with 10 mM ammonium bicarbonate, pH 7.8 and 100 mg
of antibody is either digested with trypsin (Promega, cat# V5111) or Lys-C (Roche, cat# 11 047
825 001) at a 1:20 (w/w) trypsin/Lys-C:antibody ratio at 37°C for 4 hrs. Digests are quenched
with 1 mL of 1 N HC1. For peptide mapping with mass spectrometer detection, 40 mL of the
digests are separated by reverse phase high performance liquid chromatography (RPHPLC) on a
C18 column (Vydac, cat# 218TP51, S/N NE9606 10.3.5) with an Agilent 1100 HPLC system.
The peptide separation is run with a gradient using mobile phase A (0.02% TFA and 0.08% FA in
HPLC grade water) and mobile phase B (0.02% TFA and 0.08% FA in acetonitrile) at a flow rate
of 50 mL/minutes. The API QSTAR Pulsar i mass spectromer is operated in positive mode at 4.5
kvolts spray voltage and a scan range from 800 to 2500 mass to charge ratio.
Disulfide Bond Mapping
To denature the antibody, 100 mL of the antibody is mixed with 300 mL of 8 M
guanidine HC1 in 100 mM ammonium bicarbonate. The pH is checked to ensure that it is
between 7 and 8 and the samples are denatured for 15 minutes at room temperature in a final
concentration of 6 M guanidine HC1. A portion of the denatured sample (100 mL) is diluted to
600 mL with Milli-Q water to give a final guanidine-HCl concentration of 1 M. The sample (220
mg) is digested with either trypsin (Promega, cat # V5111, lot# 22265901) or Lys-C (Roche, cat#
11047825001, lot# 12808000) at a 1:50 trypsin or 1:50 Lys-C: antibody (w/w) ratios (4.4 mg
enzyme: 220 mg sample) at 37°C for approximately 16 hours. An additional 5 mg of trypsin or
Lys-C is added to the samples and digestion is allowed to proceed for an additional 2 hours at
37°C. Digestions are stopped by adding 1 mL of TFA to each sample. Digested samples are
separated by RPHPLC using a CI8 column (Vydac, cat# 218TP51 S/N NE020630-4-1A) on an
Agilent HPLC system. The separation is run with the same gradient used for peptide mapping
using mobile phase A (0.02% TFA and 0.08% FA in HPLC grade water) and mobile phase B
(0.02% TFA and 0.08% FA in acetonitrile) at a flow rate of 50 mL/minute. The HPLC operating
conditions are the same as those used for peptide mapping. The API QSTAR Pulsar i mass
spectromer is operated in positive mode at 4.5 kvolts spray voltage and a scan range from 800 to
2500 mass-to-charge ratio. Disulfide bonds are assigned by matching the observed MWs of
peptides with the predicted MWs of tryptic or Lys-C peptides linked by disulfide bonds.
Free sulfhydryl determination
The method used to quantify free cysteines in an antibody is based on the reaction of
Ellman's reagent, 5,50- dithio-bis (2-nitrobenzoic acid) (DTNB), with sulfhydryl groups (SH)
which gives rise to a characteristic chromophoric product, 5-thio-(2-nitrobenzoic acid) (TNB).
The reaction is illustrated in the formula:
DTNB + RSH ® RS-TNB + TNB- + H+
The absorbance of the TNB- is measured at 412 nm using a Cary 50 spectrophotometer.
An absorbance curve is plotted using dilutions of 2 mercaptoethanol (b-ME) as the free SH
standard and the concentrations of the free sulfhydryl groups in the protein are determined from
absorbance at 412 nm of the sample.
The b-ME standard stock is prepared by a serial dilution of 14.2 M b-ME with HPLC
grade water to a final concentration of 0.142 mM. Then standards in triplicate for each
concentration are prepared. Antibody is concentrated to 10 mg/mL using an amicon ultra 10,000
MWCO centrifugal filter (Millipore, cat# UFC801096, lot# L3KN5251) and the buffer is changed
to the formulation buffer used for adalimumab (5.57 mM sodium phosphate monobasic, 8.69 mM
sodium phosphate dibasic, 106.69 mM NaCl, 1.07 mM sodium citrate, 6.45 mM citric acid, 66.68
mM mannitol, pH 5.2, 0.1 % (w/v) Tween). The samples are mixed on a shaker at room
temperature for 20 minutes. Then 180 mL of 100 mM Tris buffer, pH 8.1 is added to each sample
and standard followed by the addition of 300 mL of 2 mM DTNB in 10 mM phosphate buffer, pH
8.1. After thorough mixing, the samples and standards are measured for absorption at 412 nm on
a Cary 50 spectrophotometer. The standard curve is obtained by plotting the amount of free SH
and OD412 nm of the b-ME standards. Free SH content of samples are calculated based on this
curve after subtraction of the blank.
Weak Cation Exchange Chromatography
Antibody is diluted to 1 mg/mL with 10 mM sodium phosphate, pH 6.0. Charge
heterogeneity is analyzed using a Shimadzu HPLC system with a WCX-10 ProPac analytical
column (Dionex, cat# 054993, S/N 02722). The samples are loaded on the column in 80% mobile
phase A (10 mM sodium phosphate, pH 6.0) and 20% mobile phase B (10 mM sodium phosphate,
500 mM NaCl, pH 6.0) and eluted at a flow rate of 1.0 mL/minute.
Oligosaccharide Profiling
Oligosaccharides released after PNGase F treatment of antibody are derivatized with 2-
aminobenzamide (2-AB) labeling reagent. The fluorescent-labeled oligosaccharides are separated
by normal phase high performance liquid chromatography (NPHPLC) and the different forms of
oligosaccharides are characterized based on retention time comparison with known standards.
The antibody is first digested with PNGaseF to cleave N-linked oligosaccharides from the
Fc portion of the heavy chain. The antibody (200 mg) is placed in a 500 mL Eppendorf tube
along with 2 mL PNGase F and 3 mL of 10% N-octylglucoside. Phosphate buffered saline is
added to bring the final volume to 60 mL. The sample is incubated overnight at 37°C in an
Eppendorf thermomixer set at 700 RPM. Adalimumab lot AFP04C is also digested with PNGase
F as a control.
After PNGase F treatment, the samples are incubated at 95 °C for 5 minutes in an
Eppendorf thermomixer set at 750 RPM to precipitate out the proteins, then the samples are
placed in an Eppendorf centrifuge for 2 minutes at 10,000 RPM to spin down the precipitated
proteins. The supernatent containing the oligosaccharides are transferred to a 500 mL Eppendorf
tube and dried in a speed-vac at 65°C.
The oligosaccharides are labeled with 2AB using a 2AB labeling kit purchased from
Prozyme (cat# GKK-404, lot# 132026). The labeling reagent is prepared according to the
manufacturer's instructions. Acetic acid (150 mL, provided in kit) is added to the DMSO vial
(provided in kit) and mixed by pipeting the solution up and down several times. The acetic
acid/DMSO mixture (100 mL) is transferred to a vial of 2-AB dye (just prior to use) and mixed
until the dye is fully dissolved. The dye solution is then added to a vial of reductant (provided in
kit) and mixed well (labeling reagent). The labeling reagent (5 mL) is added to each dried
oligosaccharide sample vial, and mixed thoroughly. The reaction vials are placed in an Eppendorf
thermomixer set at 65°C and 700-800 RPM for 2 hours of reaction.
After the labeling reaction, the excess fluorescent dye is removed using GlycoClean S
Cartridges from Prozyme (cat# GKI-4726). Prior to adding the samples, the cartridges are
washed with 1 mL of milli-Q water followed with 5 washes of 1 mL 30% acetic acid solution.
Just prior to adding the samples, 1 mL of acetonitrile (Burdick and Jackson, cat# AH015-4) is
added to the cartridges.
After all of the acetonitrile passed through the cartridge, the sample is spotted onto the
center of the freshly washed disc and allowed to adsorb onto the disc for 10 minutes. The disc is
washed with 1 mL of acetonitrile followed by five washes of 1 mL of 96% acetonitrile. The
cartridges are placed over a 1.5 mL Eppendorf tube and the 2-AB labeled oligosaccharides are
eluted with 3 washes (400 mL each wash) of milli Q water.
The oligosaccharides are separated using a Glycosep N HPLC (cat# GKI-4728) column
connected to a Shimadzu HPLC system. The Shimadzu HPLC system consisted of a system
controller, degasser, binary pumps, autosampler with a sample cooler, and a fluorescent detector.
Stability at Elevated Temperatures
The buffer of antibody is either 5.57 mM sodium phosphate monobasic, 8.69 mM sodium
phosphate dibasic, 106.69 mM NaCl, 1.07 mM sodium citrate, 6.45 mM citric acid, 66.68 mM
mannitol, 0.1% (w/v) Tween, pH 5.2; or 10 mM histidine, 10 mM methionine, 4% mannitol, pH
5.9 using Amicon ultra centrifugal filters. The final concentration of the antibodies is adjusted to
2 mg/mL with the appropriate buffers. The antibody solutions are then filter sterized and 0.25 mL
aliquots are prepared under sterile conditions. The aliquots are left at either -80°C, 5°C, 25°C, or
40°C for 1, 2 or 3 weeks. At the end of the incubation period, the samples are analyzed by size
exclusion chromatography and SDS-PAGE.
The stability samples are analyzed by SDS-PAGE under both reducing and non-reducing
conditions. The procedure used is the same as described herein. The gels are stained overnight
with colloidal blue stain (Invitrogen cat# 46-7015,46-7016) and destained with Milli-Q water
until the background is clear. The stained gels are then scanned using an Epson Expression
scanner (model 1680, S/N DASX003641). To obtain more sensitivity, the same gels are silver
stained using silver staining kit (Owl Scientific) and the recommended procedures given by the
manufacturer is used.
Example 1.2.2.3.C: Efficacy Of A Humanized Monoclonal Antibody By Itself Or In
Combination With Chemotherapy On The Growth Of Human Carcinoma Xenografts
Human cancer cells are grown in vitro to 99% viability, 85% confluence in tissue culture
flasks. SCID female or male mice (Charles Rivers Labs) at 19-25 grams, are ear tagged and
shaved. Mice are then inoculated subcutaneously into the right flank with 0.2 ml of 2 x 106
human tumor cells (1:1 matrigel) on study day 0. Administration (IP, Q3D/ week) of vehicle
(PBS), humanized antibody, and/or chemotherapy is initiated after mice are size matched into
separate cages of mice with mean tumor volumes of approximately 150 to 200 mm3. The tumors
are measured by a pair of calipers twice a week starting on approximately day 10 post inoculation
and the tumor volumes calculated according to the formula V = L x W2/2 (V: volume, mm3; L:
length, mm; W: width, mm). Reduction in tumor volume is seen in animals treated with mAb
alone or in combination with chemotherapy relative to tumors in animals that received only
vehicle or an isotype control mAb.
Example 1.2.2.3.D: FACS Based Redirected Cytotoxicity (rCTL) Assay
Human CD3+ T cells qre isolated from previously frozen isolated peripheral blood
mononuclear cells (PBMC) by a negative selection enrichment column (R&D Systems,
Minneapolis, MN; Cat.#HTCC-525). T cells are stimulated for 4 days in flasks (vent cap,
Corning, Acton, MA) coated with 10ng/mL anti-CD3 (OKT-3, eBioscience, Inc., San Diego, CA)
and 2μg/mL anti-CD28 (CD28.2, eBioscience, Inc., San Diego, CA) in D-PBS (Invitrogen,
Carlsbad, CA) and cultured in 30U/mL IL-2 (Roche) in complete RPMI 1640 media (Invitrogen,
Carlsbad, CA) with L-glutamine, 55mM (3-ME, Pen/Strep, 10% FBS). T cells are then rested
overnight in 30U/mL IL-2 before using in assay. DoHH2 or Raji target cells are labeled with
PKH26 (Sigma-Aldrich, St. Louis, MO) according to manufacturer's instructions. RPMI 1640
media (no phenol, Invitrogen, Carlsbad, CA) containing L-glutamine and 10% FBS (Hyclone,
Logan, UT) is used throughout the rCTL assay. (See Dreier et al. (2002) Int J Cancer 100:690).
Effector T cells (E) and targets (T) are plated at a final cell concentration of 105 and 104
cells/well in 96-well plates (Costar #3799, Acton, MA), respectively to give an E:T ratio of 10:1.
DVD-Ig molecules are diluted to obtain concentration-dependent titration curves. After an
overnight incubation cells are pelleted and washed with D-PBS once before resuspending in
FACS buffer containing 0.1% BSA (Invitrogen, Carlsbad, CA), 0.1% sodium azide and 0.5μg/mL
propidium iodide (BD) in D-PBS. FACS data is collected on a FACS Canto II machine (Becton
Dickinson, San Jose, CA) and analyzed in Flowjo (Treestar). The percent live targets in the
DVD-Ig treated samples divided by the percent total targets (control, no treatment) is calculated to
determine percent specific lysis. IC50s were calculated in Prism (Graphpad).
Example 1.2.2.3.E: Antibody-Dependent Cell Mediated Cytotoxicity (ADCC) Method
Target (DoHH2, A431, and U87MGde2-7) cells were harvested and washed with 10 mL
RPMI no phenol red medium (Invitrogen, Carlsbad, CA, Cat.#l 1835) and incubated in calcein-
AM (eBioscience, San Diego, CA, Cat.# 65-0853) at a concentration of 4xl06 cells/mL for 30
minutes. Target cells were washed 3 times with 10mL RPMI no phenol red 10% FBS (Thermo
Scientific HyClone, Logan, UT, Cat.#SH30070.03) and aliquoted at 180,000 cells/well in a 96-
well round bottom plate. Antibodies and DVD-Igs were diluted in RPMI no phenol red 10% FBS
to 10 μg/mL. The supernatant was removed from the target cells, and 30 μL/well of the diluted
antibodies and DVD-Igs were added. Cells were incubated on ice for 1 hour and then washed 3
times with 150 μL/well RPMI no phenol red 10% FBS. Cells were transferred to a 2 mL assay
block and resuspended at a concentration of 1.33 x 105 cells/mL. Unactivated human NK cells
(Astarte Biologies, Redmond, WA, Cat.#1027) or activated human NK cells (In-house blood
donor program PBMCs activated 2 weeks using kit, Myltenyi Biotech, Auburn, CA, Cat.# 130-
094-483) were thawed, washed with 10 mL RPMI no phenol red 10% FBS twice, and
resuspended at 1.2 x 106 cells/mL. NK cells and target cells were then aliquoted at a ratio of 9:1
onto 96-well V bottom plate by transferring 75 μL of NK cells and 75 μL of target cells to the
same well. Media was added instead of NK cells for wells that were used to determine
spontaneous calcein-AM release. 2% triton (Sigma-Aldrich, St. Louis, MO, Cat.#93443) was
added instead of NK cells to wells that were used to determine total lysis. All conditions were
plated in triplicate.
Cells were incubated for 2-2.5 hours at 37°C and then spun down at 1300 rpm for 5
minutes. 100 μL/well of supernatant was transferred to black cliniplates. Plates were read on a
2103 EnVision Multilabel Reader (Perkin Elmer, Waltham, MA)
The ADCC activity for the DVD-Igs is shown in Tables 7, 8, and 9.
Table 7: ADCC Activity Of NKG2P Containing DVD-Igs Using Unactivated NK CeU
Effectors. POHH2 Target Cells
All DVD-Igs containing VDs from NKG2D in either the N-terminal or C-terminal
position showed ADCC activity.
Table 8: ADCC Activity Of NKG2D Containing DVD-Igs Using Activated NK Cell
Effectors. A431 Target Cells
All DVD-Igs containing VDs from NKG2D in either the N-terminal or C-terminal
position showed ADCC activity.
Table 9: Adcc Activity Of NKG2D Containing DVD-Igs Using Activated NK Cell Effectors,
U87MGDE2-7 Target Cells
All DVD-Igs containing VDs from NKG2D in either the N-terminal or C-terminal
position showed ADCC activity.
Example 1.2.2.3.F: FcR Binding Method
Cells (FcRn: FcRnGPI-CHO, FcγRI: THP-1, FcγRIIa: K562, FcγRIIb: CHO-FcγRII-b-1)
were plated at 1x105 cells/well on a 96 well round bottom plate. Antibodies and DVD-Igs were
diluted 100 μg/ml in FACS buffer (1% FBS in PBS pH6.4 for FcRn samples, pH7.4 for the rest).
The supernatant was removed from the cells and 30 μL of diluted antibodies and DVD-Igs was
added to well. Cells were incubated with the antibodies at 4°C for 2 hours. Following incubation,
the cells were washed three times with 150 uL FACS buffer (pH 6.4 for FcRn samples, pH 7.4 for
the rest). The cells were resuspended in 50 uL FACS buffer (pH 6.4 for FcRn samples, pH 7.4
for the rest) with 1:125 diluted R-PE conjugated anti-human IgG F(Ab')2 (Jackson
ImmunoResearch, West Grove, PA, Cat.# 109-116-170) and incubated at 4°C for 40 minutes.
Cells were washed three times, and finally resuspended in 100 μL FACS buffer (pH 6.4 for FcRn
samples, pH 7.4 for the rest). Samples were run on a FACSCalibur machine (Becton Dickinson,
San Jose, CA). FACSCalibur settings for FL2 were adjusted such that a non-antibody-treated
control sample had a GMFI of 3. Experimental samples were run subsequently. Flow Jo software
(Treestar, Inc, Ashland, OR) was used to analyze the data and determine R-PE GMFI on live cells
as designated by a forward and side scatter gate.
The Fc Receptor binding for the DVD-Igs is shown in Table 10.
Table 10: Fc Receptor Binding for DVD-Igs
All DVD-Igs containing VDs from NKG2D in either the N-terminal or C-terminal
position showed Fc Recepor binding.
Example 1.4: Generation of a DVD-Ig
DVD-Ig molecules that bind two antigens are constructed using two parent monoclonal
antibodies, one against human antigen A, and the other against human antigen B, selected as
described herein.
Example 1.4.1: Generation Of A DVD-Ig Having Two Linker Lengths
A constant region containing μl Fc with mutations at 234, and 235 to eliminate
ADCC/CDC effector functions is used. Four different anti-A/B DVD-Ig constructs are generated:
2 with short linker (SL) and 2 with long linker (LL), each in two different domain orientations:
VA-VB-C and VB-VA-C (see Table 11). The linker sequences, derived from the N-terminal
sequence of human C1/Ck or CH1 domain, are as follows:
For DVDAB constructs:
light chain (if anti-A has λ):Short linker: QPKAAP (SEQ ID NO: 15); Long linker:
QPKAAPSVTLFPP (SEQ ID NO: 16)
light chain (if anti-A has K):Short linker: TVAAP (SEQ ID NO: 13); Long linker:
TVAAPSVFIFPP (SEQ ID NO: 14)
heavy chain (yl): Short linker: ASTKGP (SEQ ID NO: 21); Long linker:
ASTKGPSVFPLAP (SEQ ID NO: 22)
For DVDBA constructs:
light chain (if anti-B has X):Short linker: QPKAAP (SEQ ID NO: 15); Long linker:
QPKAAPSVTLFPP (SEQ ID NO: 16)
light chain (if anti-B has k):Short linker: TVAAP (SEQ ID NO: 13); Long linker:
TVAAPSVFIFPP (SEQ ID NO: 14)
heavy chain (yl): Short linker: ASTKGP (SEQ ID NO: 21); Long linker:
ASTKGPSVFPLAP (SEQ ID NO: 22)
Heavy and light chain constructs are subcloned into the pBOS expression vector, and
expressed in COS cells, followed by purification by Protein A chromatography. The purified
materials are subjected to SDS-PAGE and SEC analysis.
Table 11 describes the heavy chain and light chain constructs used to express each anti-
A/B DVD-Ig protein.
Table 11: Anti-A/B DVD-Ig Constructs
Example 1.4.2: Molecular cloning of DNA constructs for DVDABSL and DVDABLL:
To generate heavy chain constructs DVDABHC-LL and DVDABHC-SL, VH domain of
A antibody is PCR amplified using specific primers (3' primers contain short/long linker
sequence for SL/LL constructs, respectively); meanwhile VH domain of B antibody is amplified
using specific primers (5' primers contains short/long linker sequence for SL/LL constructs,
respectively). Both PCR reactions are performed according to standard PCR techniques and
procedures. The two PCR products are gel-purified, and used together as overlapping template
for the subsequent overlapping PCR reaction. The overlapping PCR products are subcloned into
Srf I and Sal I double digested pBOS-hCγl,z non-a mammalian expression vector (Abbott) by
using standard homologous recombination approach.
To generate light chain constructs DVDABLC-LL and DVDABLC-SL, VL domain of A
antibody is PCR amplified using specific primers (3' primers contain short/long linker sequence
for SL/LL constructs, respectively); meanwhile VL domain of B antibody is amplified using
specific primers (5' primers contains short/long linker sequence for SL/LL constructs,
respectively). Both PCR reactions are performed according to standard PCR techniques and
procedures. The two PCR products are gel-purified, and used together as overlapping template
for the subsequent overlapping PCR reaction using standard PCR conditions. The overlapping
PCR products are subcloned into Srf I and Not I double digested pBOS-hCk mammalian
expression vector (Abbott) by using standard homologous recombination approach. Similar
approach has been used to generate DVDBASL and DVDBALL as described below:
Example 1.4.3: Molecular cloning of DNA constructs for DVDBASL and DVDBALL
To generate heavy chain constructs DVDBAHC-LL and DVDBAHC-SL, VH domain of
antibody B is PCR amplified using specific primers (3' primers contain short/long linker sequence
for SL/LL constructs, respectively); meanwhile VH domain of antibody A is amplified using
specific primers (5' primers contains short/long linker sequence for SL/LL constructs,
respectively). Both PCR reactions are performed according to standard PCR techniques and
procedures. The two PCR products are gel-purified, and used together as overlapping template
for the subsequent overlapping PCR reaction using standard PCR conditions. The overlapping
PCR products are subcloned into Srf I and Sal I double digested pBOS-hCγl,z non-a mammalian
expression vector (Abbott) by using standard homologous recombination approach.
To generate light chain constructs DVDBALC-LL and DVDBALC-SL, VL domain of
antibody B is PCR amplified using specific primers (3' primers contain short/long linker sequence
for SL/LL constructs, respectively); meanwhile VL domain of antibody A is amplified using
specific primers (5' primers contains short/long linker sequence for SL/LL constructs,
respectively). Both PCR reactions are performed according to standard PCR techniques and
procedures. The two PCR products are gel-purified, and used together as overlapping template
for the subsequent overlapping PCR reaction using standard PCR conditions. The overlapping
PCR products are subcloned into Srf I and Not I double digested pBOS-hCk mammalian
expression vector (Abbott) by using standard homologous recombination approach.
Example 1.4.4: Construction and Expression of Additional DVP-Ig
Example 1.4.4.1: Preparation of DVD-Ig vector constructs
Parent antibody amino acid sequences for specific antibodies, which recognize specific
antigens or epitopes thereof, for incorporation into a DVD-Ig can be obtained by preparation of
hybridomas as described above or can be obtained by sequencing known antibody proteins or
nucleic acids. In addition, known sequences can be obtained from the literature. The sequences
can be used to synthesize nucleic acids using standard DNA synthesis or amplification
technologies and assembling the desired antibody fragments into expression vectors, using
standard recombinant DNA technology, for expression in cells.
For example, nucleic acid codons were determined from amino acids sequences and
oligonucleotide DNA was synthesized by Blue Heron Biotechnology, Inc.
(www.blueheronbio.com) Bothell, WA USA. The oligonucleotides were assembled into 300-
2,000 base pair double-stranded DNA fragments, cloned into a plasmid vector and sequenceverified.
Cloned fragments were assembled using an enzymatic process to yield the complete
gene and subcloned into an expression vector. (See 7,306,914; 7,297,541; 7,279,159; 7,150,969;
20080115243; 20080102475; 20080081379; 20080075690; 20080063780; 20080050506;
20080038777; 20080022422; 20070289033; 20070287170; 20070254338; 20070243194;
20070225227; 20070207171; 20070150976; 20070135620; 20070128190; 20070104722;
20070092484; 20070037196; 20070028321; 20060172404; 20060162026; 20060153791;
20030215458; 20030157643).
A group of pHybE vectors (US Patent Application Serial No. 61/021,282) were used for
parental antibody and DVD-Ig cloning. V1, derived from pJP183; pHybE-hCgl,z,non-a V2, was
used for cloning of antibody and DVD heavy chains with a wildtype constant region. V2, derived
from pJP191; pHybE-hCk V2, was used for cloning of antibody and DVD light chains with a
kappa constant region. V3, derived from pJP192; pHybE-hC1 V2, was used for cloning of
antibody and DVDs light chains with a lambda constant region. V4, built with a lambda signal
peptide and a kappa constant region, was used for cloning of DVD light chains with a lambdakappa
hybrid V domain. V5, built with a kappa signal peptide and a lambda constant region, was
used for cloning of DVD light chains with a kappa-lambda hybrid V domain. V7, derived from
pJP183; pHybE-hCg1,z,non-a V2, was used for cloning of antibody and DVD heavy chains with
a (234,235 AA) mutant constant region.
Example 1.4.4.2: Transfection And Expression In 293 Cells
The DVD-Ig vector constructs are tranfected into 293 cells for production of DVD-Ig
protein. The 293 transient transfection procedure used is a modification of the methods published
in Durocher et al. (2002) Nucleic Acids Res. 30(2):E9 and Pham et al. (2005) Biotech.
Bioengineering 90(3):332-44. Reagents that were used in the transfection included:
• HEK 293-6E cells (human embryonic kidney cell line stably expressing EBNA1;
obtained from National Research Council Canada) cultured in disposable Erlenmeyer
flasks in a humidified incubator set at 130 rpm, 37°C and 5% C02.
• Culture medium: FreeStyle 293 Expression Medium (Invitrogen 12338-018) plus 25
Hg/mL Geneticin (G418) (Invitrogen 10131-027) and 0.1% Pluronic F-68 (Invitrogen
24040-032).
• Transfection medium: FreeStyle 293 Expression Medium plus 10 mM HEPES
(Invitrogen 15630-080).
• Polyethylenimine (PEI) stock: 1 mg/mL sterile stock solution, pH 7.0, prepared with
linear 25kDa PEI (Polysciences) and stored at less than -15°C.
• Tryptone Feed Medium: 5% w/v sterile stock of Tryptone Nl (Organotechnie, 19554) in
FreeStyle 293 Expression Medium.
Cell preparation for transfection: Approximately 2-4 hours prior to transfection, HEK 293-6E
cells are harvested by centrifugation and resuspended in culture medium at a cell density of
approximately 1 million viable cells per mL. For each transfection, 40 mL of the cell suspension
is transferred into a disposable 250-mL Erlenmeyer flask and incubated for 2 - 4 hours.
Transfection: The transfection medium and PEI stock are prewarmed to room temperature (RT).
For each transfection, 25μg of plasmid DNA and 50μg of polyethylenimine (PEI) are combined in
5 mL of transfection medium and incubated for 15 - 20 minutes at RT to allow the DNA:PEI
complexes to form. For the BR3-Ig transfections, 25μg of BR3-Ig plasmid is used per
transfection. Each 5-mL DNA:PEI complex mixture is added to a 40-mL culture prepared
previously and returned to the humidified incubator set at 130 rpm, 37°C and 5% CO2. After 20-
28 hours, 5 mL of Tryptone Feed Medium is added to each transfection and the cultures are
continued for six days.
The expression profile for the DVD-Igs is shown in Table 12.
Table 12: Transient HEK293 Expression Yields of NKG2D Containing Antibodies and
DVD-Igs
All DVD-Igs expressed well in 293 cells. DVD-Igs could be easily purified over a protein
A column. In most cases >5 mg/L purified DVD-Ig could be obtained easily from supernatants of
293 cells.
Example 1.4.5: Characterization and lead selection of A/B DVD-Igs
The binding affinities of anti-A/B DVD-Igs are analyzed on Biacore against both protein
A and protein B. The tetravalent property of the DVD-Ig is examined by multiple binding studies
on Biacore. Meanwhile, the neutralization potency of the DVD-Igs for protein A and protein B
are assessed by bioassays, respectively, as described herein. The DVD-Ig molecules that best
retain the affinity and potency of the original parent mAbs are selected for in-depth
physicochemical and bio-analytical (rat PK) characterizations as described herein for each mAb.
Based on the collection of analyses, the final lead DVD-Ig is advanced into CHO stable cell line
development, and the CHO-derived material is employed in stability, pharmacokinetic and
efficacy studies in cynomolgus monkey, and preformulation activities.
Example 2: Generation and Characterization of Dual Variable Domain Immunoglobulins
(DVD-Ig)
Dual variable domain immunoglobulins (DVD-Ig) using parent antibodies with known
amino acid sequences were generated by synthesizing polynucleotide fragments encoding DVDIg
variable heavy and DVD-Ig variable light chain sequences and cloning the fragments into a
pHybC-D2 vector according to Example 1.4.4.1. The DVD-Ig contracts were cloned into and
expressed in 293 cells as described in Example 1,4.4.2. The DVD-Ig protein was purified
according to standard methods. Functional characteristics were determined according to the
methods described in Example 1.1.1 and 1.1.2 as indicated. DVD-Ig VH and VL chains for the
DVD-Igs of the invention are provided below.
Exmple 2.1: Generation of NKG2D and CD-20 DVD-Igs with Linker Set 1
Table 13
Example 2.2: Generation of NKG2D and CD-19 (seq. 1) PVP-Igs with Linker Set 1
Example 2.3: Generation of NKG2D and EGFR (sea. 2) DVD-Igs with Linker Set 1
Example 2.4: Generation of NKG2D and EGFR (sea. 1) DVD-Igs with Linker Set 1
Example 2.5: Generation of NKG2D and HER 2 (seq. 1) DVD-Igs with Linker Set 1
Example 2.6: Generation of NKG2D and IGF1R (sea. 1) DVD-Igs with Linker Set 1
Example 2.7: Generation of NKG2D and EGFR (sea. 3) DVD-Igs with Linker Set 1
Example 2.7: Cloning Vector Sequences Used to Clone Parent Antibody and DVD-Ig
Sequences
Table 20

The present invention incorporates by reference in their entirety techniques well known in
the field of molecular biology and drug delivery. These techniques include, but are not limited to,
techniques described in the following publications:
Ausubel et al. (eds.), Current Protocols in Molecular Biology. John Wiley & Sons, NY
(1993);
Ausubel et al. (eds.), Short Protocols In Molecular Biology. John Wiley & Sons, NY (4th
edition, 1999) (ISBN 0-471-32938-X)
Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a
Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, New York, (1999);
Goodson, in Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984);
Hammerling et al., in: Monoclonal Antibodies and T-Cell Hvbridomas 563-681 (Elsevier,
N.Y., 1981;
Harlow et a l , Antibodies: A Laboratory Manual. (Cold Spring Harbor Laboratory Press,
2nd ed. 1988);
Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of
Health, Bethesda, Md. (1987) and (1991);
Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest. Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No. 91-3242;
Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York.
790 pp. (ISBN 3-540-41354-5);
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990);
Langer and Wise (eds.), Medical Applications of Controlled Release. CRC Press, Boca
Raton, FL (1974);
Lu and Weiner eds., Cloning and Expression Vectors for Gene Function Analysis (2001)
BioTechniques Press. Westborough, MA. 298 pp. (ISBN 1-881299-21-X);
Old, R.W. & S.B. Primrose, Principles of Gene Manipulation: An Introduction To
Genetic Engineering (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in
Microbiology; V.2:409 pp. (ISBN 0-632-01318-4);
Robinson, J.R. (ed.), Sustained and Controlled Release Drug Delivery Systems, Marcel
Dekker, Inc., NY (1978);
Ruan, Q., Skinner, J.P. and Tetin, S.Y. Using non-fluorescent FRET acceptors in protein
binding studies. Analyt. Biochemistry (2009), 393, 196-204;
Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold
Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6>;
Smolen and Ball (eds.), Controlled Drug Bioavailability, Drug Product Design and
Performance, John Wiley & Sons, NY (1984);
Winnacker, EX. From Genes To Clones: Introduction To Gene Technology (1987) VCH
Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).
Incorporation by Reference
The contents of all cited references (including literature references, patents, patent
applications, databases and websites) that maybe cited throughout this application are hereby
expressly incorporated by reference in their entirety for any purpose, as are the references cited
therein. The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of immunology, molecular biology and cell biology, which are well
known in the art.
Equivalents
The invention may be embodied in other specific forms without departing from the spirit
or essential characteristics thereof. The foregoing embodiments are therefore to be considered in
all respects illustrative rather than limiting of the invention described herein. Scope of the
invention is thus indicated by the appended claims rather than by the foregoing description, and
all changes that come within the meaning and range of equivalency of the claims are therefore
intended to be embraced herein.

We Claim:
1. A binding protein comprising a polypeptide chain, wherein said polypeptide
chain comprises VDI-(X1)n-VD2-C-(XZ)n, wherein;
VD1 is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 is an Fc region;
(X1)n is (X1)O or (X1)l; and
(X2)n is (X2)O or (X2)l
wherein the binding protein is capable of binding a pair of antigens, and wherein
the pair of antigens is: NKG2D and CD-20; NKG2D and CD-19; NKG2D and
EGFR; NKG2D and HER-2; or NKG2D and IGFIR.
2. The binding protein according to claim I, wherein VD1 and VD2
independently comprise SEQ ID NO: 30, 32, 34, 36, 38,40,42, or 44.
3. A binding protein comprising a polypeptide chain, wherein said polypeptide
chain comprises VDI-(X1)n-VD2-C-(X2)n, wherein;
VD1 is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region; and
nisOor1;
wherein the binding protein is capable of binding a pair of antigens, and wherein
the pair of antigens is: NKG2D and CD-20; NKG2D and CD-19; NKG2D and
EGFR; NKG2D and HER-2; or NKG2D and IGFIR.
4. The binding protein according to claim 3, wherein the VD1 and VD2 light
chain variable domains independently comprise SEQ ID NO: 29, 31, 33, 35, 37,
39,41,43, or 45.
5. The binding protein according to claim 1 or 3, wherein n is 0.
6. A binding protein comprising first and second polypeptide chains, wherein
said first polypeptide chain comprises a first VDI-(X1)n-VD2-C-(X2)n, wherein
VD1 is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI; and
X2 is an Fc region; and
wherein said second polypeptide chain comprises a second VDI-(X1)n-
VD2-C-(X2)n, wherein
VD1 is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region; and
wherein (X1)n is (X1)O or (X1)l and (X2)n is (X2)O or (X2)1, wherein the binding
protein is capable of binding a pair of antigens, and wherein the pair of antigens
is: NKG2D and CD-20; NKG2D and CD-19; NKG2D and EGFR; NKG2D and
HER-2; or NKG2D and IGFI R.
7. The binding protein according to claim 6, wherein the VD1 and VD2 heavy
chain variable domains individually comprise SEQ ID NO: 30, 32, 34, 36, 38, 40,
42, or 44, and wherein the VD1 and VD2 light chain variable domains individually
comprise SEQ ID NO: 31, 33,35, 37, 39,41,43, or 45.
8. The binding protein according to claim I, 3, or 6, wherein XI is any one of
SEQ ID NOS 1-29.
9. The binding protein according to claim 6, wherein the binding protein
comprises two first polypeptide chains and two second polypeptide chains.
10. The binding protein according to claim I, 3, or 6, wherein the Fc region is
a variant sequence Fc region.
11. The binding protein according to claim 1, 3, or 6 4-0, wherein the Fc region
is an Fc region from an IgG1, lgG2, lgG3, lgG4, IgA, IgM, IgE, or IgD.
12. The binding protein according to claim 6, wherein said VD1 of the first
polypeptide chain and said VD1 of the second polypeptide chain are obtained
from the same first and second parent antibody, respectively, or antigen binding
portion thereof.
13. The binding protein according to claim 6, wherein said VD1 of the first
polypeptide chain and said VD1 of the second polypeptide chain are obtained
from a different first and second parent antibody, respectively, or antigen binding
portion thereof.
14. The binding protein according to claim 6, wherein said VD2 of the first
polypeptide chain and said VD2 of the second polypeptide chain are obtained
from the same first and second parent antibody, respectively, or antigen binding
portion thereof.
15. The binding protein according to claim 6, wherein said VD2 of the first
polypeptide chain and said VD2 of the second polypeptide chain are obtained
from different first and second parent antibody, respectively, or antigen binding
portion thereof.
16. The binding protein according to any one of claims 13-15, wherein said
first and said second parent antibodies bind different epitopes on said antigen.
17. The binding protein according to any one of claims 13-15, wherein said
first parent antibody or antigen binding portion thereof, binds said first antigen
with a potency different from the potency with which said second parent antibody
or antigen binding portion thereof, binds said second antigen.
18. The binding protein according to any one of claims 13-1 5, wherein said
first parent antibody or antigen binding portion thereof, binds said first antigen
with an affinity different from the affinity with which said second parent antibody
or antigen binding portion thereof, binds said second antigen.
24. A binding protein capable of binding two antigens comprising four
polypeptide chains, wherein two polypeptide chains comprise VD?-(X1)n-VD2-C-
(X2)n1 wherein
VD1 is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI; and
X2 is an Fc region; and
wherein two polypeptide chains comprise VDI-(X1)n-VD2-C-(X2)n,
wherein
VD1 is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region; and
wherein (X1)n is (X1)O or (X1)l and (X2)n is (X2)O or (X2)l; wherein the VD1 and
VD2 heavy chain variable domains individually comprise SEQ ID NO: 30, 32, 34,
36, 38,40, 42, or 44, and wherein the VD1 and VD2 light chain variable domains
individually comprise SEQ ID NO: 31, 33, 35, 37, 39,41,43, or 45.
25. A binding protein capable of binding two antigens comprising four
polypeptide chains, wherein two polypeptide chains comprise VDI-(X1)n-VD2-C-
(X2)n, wherein
VD1 is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 is an Fc region;
(X1)n is (X1)O or (X1)l; and
(X2)n is (X2)O or (X2)l; and
wherein two polypeptide chains comprise VDI-(X1)n-VD2-C-(X2)n,
wherein
VDI is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region; and
(X1)n is (X1)O or (X1)l; and
(X2)n is (X2)O or (X2)l
wherein at least one of the antigens is: CD-20, CD-19, human epidermal growth
factor receptor 2 (HER-2), epidermal growth factor receptor (EGFR), insulin-like
growth factor receptor (IGFIR), or NKG2D.
26. The binding protein according to claim I, 3,6, 24, or 25, wherein said
binding protein has an on rate constant (Kon to said one or more tar ets 1 of: at 4 -1 ? least about IO*M-~S-'a; t least about 10~1u1-l;~ a-t least about 10 M s- ; at least
about IO~M-~S"; or at least about 1 0 ~ ~a~s m~eassu"re~d b y surface plasmon
resonance.
27. The binding protein according to claim I, 3, 6, 24, or 25, wherein said
binding protein has an off rate constant (Koff) to said one or more targets of: at
most about 10"s-'; at most about 10~s";a t most about IO-~S-'; or at most about
IO's", as measured by surface plasmon resonance.
28. The binding protein according to claim I, 3, 6, 24, or 25, wherein said
binding protein has a dissociation constant (KC,) to said one or more targets of: at
most about lo-' M; at most about lom8M ; at most about lo-' M; at most about 10'
'O M; at most about 10"' M; at most about 10-l2 M; or at most M.
29. A binding protein conjugate comprising a binding protein according to any
one of claims I, 3, 6, 24, or 25, said binding protein conjugate further comprising
an agent, wherein said agent is: an immunoadhesion molecule, an imaging
agent, a therapeutic agent, or a cytotoxic agent.
30. The binding protein conjugate according to claim 29, wherein said agent is
an imaging agent, and wherein said imaging agent is: a radiolabel, an enzyme, a
fluorescent label, a luminescent label, a bioluminescent label, a magnetic label,
m or biotin.
33. The binding protein according to claim I, 3, 6, 24, or 25, wherein said
binding protein is a crystallized binding protein.
37. An isolated nucleic acid encoding a binding protein amino acid sequence
according to any one of claims 1, 3, 6, 24, or 25.
38. A vector comprising an isolated nucleic acid according to claim 37.
39. The vector according to claim 38, wherein said vector is pcDNA, pTT,
pTT3, pEFBOS, pBV, pJV, pcDNA3.1 TOPO, pEF6 TOPO, or pBJ.
40. A host cell comprising a vector according to claim 38.
41. The host cell according to claim 40, wherein said host cell is a prokaryotic
cell.
43. The host cell according to claim 40, wherein said host cell is a eukaryotic
cell.
44. The host cell according to claim 43, wherein said eukaryotic cell is a protist
cell, an animal cell, a plant cell, a yeast cell, a mammalian cell, an avian cell, an
insect cell, or fungal cell.
51. A method of producing a binding protein, comprising culturing a host cell
described in any one of claims 40,41,43, or 44 in culture medium under
conditions sufficient to produce the binding protein
55. A protein produced according to the method of claim 51.
56. A pharmaceutical composition comprising the binding protein of any one of
claims 1, 3, 6, 24, 25, or 55, and a pharmaceutically acceptable carrier.
57. The pharmaceutical composition of claim 56 further comprising at least
one additional therapeutic agent.
58. The pharmaceutical composition of claim 57, wherein said additional
therapeutic agent is an imaging agent, a cytotoxic agent, an angiogenesis
inhibitor, a kinase inhibitor, a co-stimulation molecule blocker, an adhesion
molecule blocker, an anti-cytokine antibody or functional fragment thereof,
methotrexate, cyclosporin, rapamycin, FK506, a detectable label or reporter, a
TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid
anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local
anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a
corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an
immunoglobulin, an immunosuppressive, a growth hormone, a hormone
replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a
stimulant, an asthma medication, a beta agonist, an inhaled steroid, an
epinephrine or analog, a cytokine, or a cytokine antagonist.
59. A method for treating a subject for a disease or a disorder by administering
to the subject the binding protein of any one of claims I, 3, 6, 24, 25, or 55 such
that treatment is achieved.
60. The method of claim 59, wherein said disorder is rheumatoid arthritis,
osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic
arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus,
Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent
diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis
scleroderma, graft versus host disease, organ transplant rejection, acute or
chronic immune disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease,
rave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's
granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the
kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,
sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acute
transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's
disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart
failure, myocardial infarction, Addison's disease, sporadic polyglandular
deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult
(acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative
arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic
arthropathy, enteropathic synovitis, chlamydia, yersinia and salmonella
associated arthropathy, spondyloarthopathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus
vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune
haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious
anaemia, juvenile pernicious anaemia, myalgic encephalitis1Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing
hepatitis, cryptogenic autoimmune hepatitis, Acquired lmmunodeficiency
Syndrome, Acquired lmmunodeficiency Related Diseases, Hepatitis B, Hepatitis
C, common varied immunodeficiency (common variable
hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian
failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing
alveolitis, postinflammatory interstitial lung disease, interstitial pneumonitis,
connective tissue disease associated interstitial lung disease, mixed connective
tissue disease associated lung disease, systemic sclerosis associated interstitial
lung disease, rheumatoid arthritis associated interstitial lung disease, systemic
lupus erythematosus associated lung disease, dermatomyositis/polymyositis
associated lung disease, Sjogren's disease associated lung disease, ankylosing
spondylitis associated lung disease, vasculitic diffuse lung disease,
haemosiderosis associated lung disease, drug-induced interstitial lung disease,
fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic
pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung
disease, gouty arthritis, autoimmune hepatitis, type-I autoimmune hepatitis
(classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM
antibody hepatitis), autoimmune mediated hypoglycaemia, type B insulin
resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease
associated with organ transplantation, chronic immune disease associated with
organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis
type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal
disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, lyme
disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia,
pulmonary hypertension secondary to connective tissue disease, Goodpasture's
syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic
fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's
syndrome, Takayasu's diseasetarteritis, autoimmune thrombocytopaenia,
idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism,
goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic
autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary
vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis,
alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, Drug-Induced
hepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci
(GBS) infection, mental disorders such as depression and schizophrenia, Th2
Type and Thl Type mediated diseases, acute and chronic pain, and cancers
such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and
rectal cancer and hematopoietic malignancies (leukemia and lymphoma),
abetalipoproteinemia, Acrocyanosis, acute and chronic parasitic or infectious
processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute
renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex,
alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis,
allergic rhinitis, allograft rejection, alpha-I -antitrypsin deficiency, amyotrophic
lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anticd3
therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions,
aortic and peripheral aneuryisms, aortic dissection, arterial hypertension,
arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or
paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone grafl
rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's
lymphoma, burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors,
cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage
transplant rejection, cerebellar cortical degenerations, cerebellar disorders,
chaotic or multifocal atrial tachycardia, chemotherapy-associated disorders,
chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory
pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary
disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive
heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery
disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis,
cytokine therapy associated disorders, Dementia pugilistica, demyelinating
diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions,
diabetes, diabetes mellitus, diabetic aterosclerotic disease, Diffuse Lewy body
disease, dilated congestive cardiomyopathy, disorders of the basal ganglia,
Down's Syndrome in middle age, drug- induced movement disorders induced by
drugs which block CNS dopamine receptors, drug sensitivity, eczema,
encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus
infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial
hematophagocytic lymphohistiocytosis, fetal thymus implant rejection,
Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas
gangrene, gastric ulcer, graft rejection of any organ or tissue, gram negative
sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell
leukemia, Hallervorden-Spatz disease, hashimoto's thyroiditis, hay fever, heart
transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis A, His
bundle arryhthmias, HIV infection1HIV neuropathy, Hodgkin's disease,
hyperkinetic movement disorders, hypersensitivity reactions, hypersensitivity
pneumonitis, hypertension, hypokinetic movement disorders, hypothalamicpituitary-
adrenal axis evaluation, idiopathic Addison's disease, idiopathic
pulmonary fibrosis, antibody-mediated cytotoxicity, Asthenia, infantile spinal
muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation
exposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic
stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's
sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of
the corticospinal system, lipedema, liver transplant rejection, lymphedema,
malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma,
meningitis, meningococcemia, metaboliclidiopathic, migraine headache,
mitochondria1 multisystem disorder, mixed connective tissue disease, monoclonal
gammopathy, multiple myeloma, multiple systems degenerations (Mencel
Dejerine-Thomas Shy-Drager and Machado-Joseph), myasthenia gravis,
mycobacterium avium intracellulare, mycobacterium tuberculosis, myelodyplastic
syndrome, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal
chronic lung disease, nephritis, nephrosis, neurodegenerative diseases,
neurogenic I muscular atrophies , neutropenic fever, non-Hodgkin's lymphoma,
occlusion of the abdominal aorta and its branches, occulsive arterial disorders,
okt3 therapy, orchitis/epidydimitis, orchitislvasectomy reversal procedures,
organomegaly, osteoporosis, pancreas transplant rejection, pancreatic
carcinoma, paraneoplastic syndromelhypercalcemia of malignancy, parathyroid
transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial
disease, peripheral arteriosclerotic disease, peripheral vascular disorders,
peritonitis, pernicious anemia, Pneumocystis carinii pneumonia, pneumonia,
POEMS syndrome (polyneuropathy, organomegaly. endocrinopathy, monoclonal
gammopathy, and skin changes syndrome), post perfusion syndrome, post pump
syndrome, post-MI cardiotomy syndrome, preeclampsia, Progressive
supranuclear Palsy, primary pulmonary hypertension, radiation therapy,
Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease,
regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury,
restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, senile
dementia of Lewy body type, seronegative arthropathies, shock, sickle cell
anemia, skin allograft rejection, skin changes syndrome, small bowel transplant
rejection, solid tumors, specific arrythmias, spinal ataxia, spinocerebellar
degenerations, streptococcal myositis, structural lesions of the cerebellum,
Subacute sclerosing panencephalitis, Syncope, syphilis of the cardiovascular
system, systemic anaphalaxis, systemic inflammatory response syndrome,
systemic onset juvenile rheumatoid arthritis, T-cell or Fab ALL, Telangiectasia,
thromboangitis obliterans, thrombocytopenia, toxicity, transplants,
trauma/hemorrhage, type I11 hypersensitivity reactions, type IV hypersensitivity,
unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose
veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral
and fungal infections, viral encephalitislaseptic meningitis, viral-associated
hemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,
xenograft rejection of any organ or tissue, acute coronary syndromes, acute
idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy,
acute ischemia, adult Still's disease, anaphylaxis, anti-phospholipid antibody
syndrome, aplastic anemia, atopic eczema, atopic dermatitis, autoimmune
dermatitis, autoimmune disorder associated with streptococcus infection,
autoimmune enteropathy, autoimmune hearing loss, autoimmune
lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune
premature ovarian failure, blephariiis, bronchiectasis, bullous pemphigoid,
cardiovascular disease, catastrophic antiphospholipid syndrome, celiac disease,
cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinically isolated
syndrome (cis) with risk for multiple sclerosis, childhood onset psychiatric
disorder, dacryocystitis, dermatomyositis, diabetic retinopathy, disk herniation,
disk prolaps, drug-induced immune hemolytic anemia, endometriosis,
endophthalmitis, episcleritis, erythema multiforme, erythema multiforme major,
gestational pemphigoid, Guillain-Barre syndrome (GBS), hay fever, Hughes
syndrome, idiopathic Parkinson's disease, idiopathic interstitial pneumonia, IgE -
mediated allergy, immune hemolytic anemia, inclusion body myositis, infectious
ocular inflammatory disease, inflammatory demyelinating disease, inflammatory
heart disease, inflammatory kidney disease, IPFIUIP, iritis, keratitis,
keratoconjunctivitis sicca, Kussmaul disease or Kussmaul-Meier disease,
Landry's paralysis, Langerhan's cell histiocytosis, livedo reticularis, macular
degeneration, microscopic polyangiitis, morbus bechterev, motor neuron
disorders, mucous membrane pemphigoid, multiple organ failure,
myelodysplastic syndrome, myocarditis, nerve root disorders, neuropathy, non-A
non-B hepatitis, optic neuritis, osteolysis, ovarian cancer, pauciarticular JRA,
peripheral artery occlusive disease (PAOD), peripheral vascular disease (PVD),
peripheral artery, disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis
nodosa), polychondritis, polymyalgia rheumatica, poliosis, polyarticular JRA,
polyendocrine deficiency syndrome, polymyositis, post-pump syndrome, primary
Parkinsonism, prostate and rectal cancer and hematopoietic malignancies
(leukemia and lymphoma), prostatitis, pure red cell aplasia, primary adrenal
insufficiency, recurrent neuromyelitis optica, restenosis, rheumatic heart disease,
sapho (synovitis, acne, pustulosis, hyperostosis, and osteitis), scleroderrna,
secondary amyloidosis, shock lung, scleritis, sciatica, secondary adrenal
insufficiency, silicone associated connective tissue disease, sneddon-wilkinson
dermatosis, spondilitis ankylosans, Stevens-Johnson syndrome (SJS), systemic
inflammatory response syndrome, temporal arteritis, toxoplasmic retinitis, toxic
epidermal necrolysis, transverse myelitis, TRAPS (tumor necrosis factor receptor,
type I allergic reaction, type I1 diabetes, usual interstitial pneumonia (UIP),
vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH
syndrome), wet macular degeneration, or wound healing.
61. The method according to claim 60, wherein said administering to the
subject is parenteral, subcutaneous, intramuscular, intravenous, intrarticular,
intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary,
intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical,
intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac,
intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,
intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,
bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
62. A method for generating a Dual Variable Domain immunoglobulin capable
of binding two antigens comprising the steps of
a) obtaining a first parent antibody or antigen binding portion thereof,
capable of binding a first antigen;
b) obtaining a second parent antibody or antigen binding portion thereof,
capable of binding a second antigen;
c) constructing:
(i) a polypeptide chain comprising VDI-(X1)n-VD2-C-(X2)n, wherein;
VD1 is a first heavy chain variable domain obtained from said first
parent antibody or antigen binding portion thereof;
VD2 is a second heavy chain variable domain obtained from said
second parent antibody or antigen binding portion thereof;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 is an Fc region;
(X1)n is (X1)O or (XI)?; and
(X2)n is (X2)O or (X2)l;
wherein the binding protein is capable of binding a pair of antigens, and
wherein the pair of antigens is: NKG2D and CD-20; NKG2D and CD-19;
NKG2D and EGFR; NKG2D and HER-2; or NKGZD and IGFIR;
(ii) a polypeptide chain comprising VDI-(XI)n-VD2-C-(X2)n, wherein;
VD1 is a first light chain variable domain obtained from said first
parent antibody or antigen binding portion thereof;
VD2 is a second light chain variable domain obtained from said
second parent antibody or antigen binding portion thereof;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
(X1)n is (X1)O or (X1)l; and
(X2)n is (X2)O or (X2)l
wherein the binding protein is capable of binding a pair of antigens, and
wherein the pair of antigens is: NKG2D and CD-20; NKG2D and CD-19;
NKG2D and EGFR; NKG2D and HER-2; or NKG2D and IGFI R;
(iii) a first and second polypeptide chain, wherein said first polypeptide
chain comprises a first VDI-(X1)n-VD2-C-(XZ)n, wherein
VD1 is a first heavy chain variable domain obtained from said first
parent antibody or antigen binding portion thereof;
VD2 is a second heavy chain variable domain obtained from said
second parent antibody or antigen binding portion thereof;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI; and
X2 is an Fc region; and
wherein said second polypeptide chain comprises a second VDI-(X1)n-
VD2-C-(X2)n, wherein
VD1 is a first light chain variable domain obtained from said first
parent antibody or antigen binding portion thereof;
VD2 is a second light chain variable domain obtained from said
second parent antibody or antigen binding portion thereof;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
wherein (X1)n is (X1)O or (X1)l and (X2)n is (X2)O or (X2)l; wherein the
binding protein is capable of binding a pair of antigens, and wherein the
pair of antigens is: NKG2D and CD-20; NKG2D and CD-19; NKG2D
and EGFR; NKG2D and HER-2; or NKG2D and IGFIR;
(iv) four polypeptide chains, wherein two polypeptide chains comprise
VD1 -(XI )n-VD2-C-(X2)n, wherein
VD1 is a first heavy chain variable domain obtained from said first
parent antibody or antigen binding portion thereof;
VD2 is a second heavy chain variable domain obtained from said
second parent antibody or antigen binding portion thereof;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI; and
X2 is an Fc region; and
wherein two polypeptide chains comprise VDI-(X1)n-VD2-C-(X2)n,
wherein
VD1 is a first light chain variable domain obtained from said first
parent antibody or antigen binding portion thereof;
VD2 is a second light chain variable domain obtained from said
second parent antibody or antigen binding portion thereof;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
wherein (X1)n is (X1)O or (X1)l and (X2)n is (X2)O or (X2)l; wherein the
binding protein is capable of binding a pair of antigens, and wherein the
pair of antigens is: NKG2D and CD-20; NKG2D and CD-19; NKG2D
and EGFR; NKG2D and HER-2; or NKG2D and IGFI R;
(v) four polypeptide chains, wherein two polypeptide chains comprise
VDl -(Xl)n-VD2-C-(X2)n, wherein
VD1 is a first heavy chain variable domain obtained from said first
parent antibody or antigen binding portion thereof;
VD2 is a second heavy chain variable domain obtained from said
second parent antibody or antigen binding portion thereof;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 is an Fc region;
(X1)n is (X1)O or (X1)l and (X2)n is (X2)O or (X2)l; and
wherein two polypeptide chains comprise VDI-(X1)n-VD2-C-(X2)n,
wherein
VD1 is a first light chain variable domain obtained from said first
parent antibody or antigen binding portion thereof;
VD2 is a second light chain variable domain obtained from said
second parent antibody or antigen binding portion thereof;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region; and
(X1)n is (X1)O or (X1)l and (X2)n is (X2)O or (X2)l;
wherein the binding protein binds two antigens, and wherein at least one
of the antigens is: CD-20, CD-19, human epidermal growth factor
receptor 2 (HER-2), epidermal growth factor receptor (EGFR), insulin-like
growth factor receptor (IGFI R), or NKG2D;
d) expressing said polypeptide chains such that a Dual Variable Domain
immunoglobulin capable of binding said first and said second antigen is
generated.
63. The method of claim 62, wherein the VD1 and VD2 heavy chain variable
domains independently comprise SEQ ID NO: 30, 32, 34,36,38,40,42, or 44,
and wherein the VD1 and VD2 light chain variable domains independently
comprise SEQ ID NO: 31, 33, 35, 37, 39,41,43, or 45.
68. The method of claim 62, wherein the Fc region is a variant sequence Fc
region.
69. The method of claim 62, wherein the Fc region is an Fc region from an
IgG1, lgG2, lgG3, lgG4, IgA, IgM, IgE, or IgD.
74. The method of claim 62, wherein said first parent antibody or antigen
binding portion thereof, binds said first antigen with a different affinity and/or
potency than the affinity and/or potency with which said second parent antibody
or antigen binding portion thereof, binds said second antigen.
125. A method of determining the presence of at least one antigen or fragment
thereof in a test sample by an immunoassay,
wherein the immunoassay comprises contacting the test sample with at
least one binding protein and at least one detectable label,
wherein the at least one antigen or fragment thereof is NKG2D; CD-20;
CD-19; EGFR; HER-2; and IGFIR,
and wherein the at least one binding protein comprises the binding protein
of claim 1, 3,6, 24, 25, or 55.
126. The method of claim 125 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein
the binding protein binds to an epitope on the antigen or fragment thereof so as
to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein
the detectable label binds to the binding protein or an epitope on the antigen or
fragment thereof that is not bound by the binding protein to form a second
complex; and
(iii) detecting the presence of the antigen or fragment thereof in the test
sample based on the signal generated by the detectable label in the second
complex, wherein the presence of the antigen or fragment thereof is directly
correlated with the signal generated by the detectable label.
127. The method of claim 125 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein
the binding protein binds to an epitope on the antigen or fragment thereof so as
to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein
the detectable label competes with the antigen or fragment thereof for binding to
the binding protein so as to form a second complex; and
(iii) detecting the presence of the antigen or fragment thereof in the test
sample based on the signal generated by the detectable label in the second
complex, wherein the presence of the antigen or fragment thereof is indirectly
correlated with the signal generated by the detectable label.
128. The method according to any one of claims 125-127, wherein the test
sample is from a patient and the method further comprises diagnosing,
prognosticating, or assessing the efficiency of therapeuticlprophylactic treatment
of the patient, and
wherein if the method further comprises assessing the efficacy of
therapeuticlprophylactic treatment of the patient, the method optionally further
comprises modifying the therapeuticlprophylactic treatment of the patient as
needed to improve efficacy.
129. The method according to any one of claims 125-128, wherein the method is
adapted for use in an automated system or a semi-automated system.
130. The method according to any one of claims 125-129, wherein the method
determines the presence of more than one antigen in the sample.
131. A method of determining the amount or concentration of an antigen or
fragment thereof in a test sample by an immunoassay,
wherein the immunoassay (a) employs at least one binding protein and at
least one detectable label and (b) comprises comparing a signal generated by the
detectable label with a control or calibrator comprising the antigen or fragment
thereof,
wherein the calibrator is optionally part of a series of calibrators in which
each calibrator differs from the other calibrators in the series by the concentration
of the antigen or fragment thereof,
wherein the at least one antigen or fragment thereof is NKG2D; CD-20;
CD-19; EGFR; HER-2; and IGFIR,
and wherein the at least one binding protein comprises the binding protein
of claim 1, 3, 6, 24, 25, or 55.
132. The method of claim 131 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein
the binding protein binds to an epitope on the antigen or fragment thereof so as
to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein
the detectable label binds to an epitope on the antigen or fragment thereof that is
not bound by the binding protein to form a second complex; and
(iii) determining the amount or concentration of the antigen or fragment
thereof in the test sample based on the signal generated by the detectable label
in the second complex, wherein the amount or concentration of the antigen or
fragment thereof is directly proportional to the signal generated by the detectable
label.
133. The method of claim 131 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein
the binding protein binds to an epitope on the antigen or fragment thereof so as
to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein
the detectable label competes with the antigen or fragment thereof for binding to
the binding protein so as to form a second complex; and
(iii) determining the amount or concentration of the antigen or fragment
thereof in the test sample based on the signal generated by the detectable label
in the second complex, wherein the presence of the antigen or fragment thereof
is indirectly proportional to the signal generated by the detectable label.
134. The method according to any one of claims 131-133, wherein the test
sample is from a patient and the method further comprises diagnosing,
prognosticating, or assessing the efficiency of therapeuticlprophylactic treatment
of the patient, and
wherein if the method further comprises assessing the efficacy of
therapeutic/prophylactic treatment of the patient, the method optionally further
comprises modifying the therapeutidprophylactic treatment of the patient as
needed to improve efficacy.
135. The method according to any one of claims 131-134, wherein the method is
adapted for use in an automated system or a semi-automated system.
136. The method according to any one of claims 131-135, wherein the method
determines the amount or concentration of more than one antigen in the sample.
137. A kit for assaying a test sample for the presence, amount, or concentration
of an antigen or fragment thereof,
wherein the antigen or fragment thereof is NKGZD; CD-20; CD-19; EGFR;
HER-2; and IGFI R,
said kit comprising (a) instructions for assaying the test sample for the
antigen or fragment thereof and (b) at least one binding protein comprising the
binding protein of claim 1, 3,6,24, 25, or 55.