DUAL VARIABLE DOMAIN IMMUNOGLOBULINS AND USES THEREOF
Reference to Related Applications
This application is a non-provisional application claiming priority to U.S. Provisional
Application Serial No. 6 1/358,23 1, filed June 24, 2010, the contents of which are hereby
incorporated by reference.
Field of the Invention
The present invention relates to multivalent and multispecific binding proteins that bind
Tumor Necrosis Factor (TNF) and Tumor Necrosis Factor - Related Weak Inducer of Apoptosis
(TWEAK), 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 capable of binding 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 A.C. Cuello (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 a so be produced by chemical conjugation of two different
mAbs (see Staerz, U.D., et al. (1985) Nature 3 14(6012): 628-3 1). 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 full IgG molecule.
A wide variety of other recombinant bispecific antibody formats have been developed
(see Kriangkum, J., et al. (2001) Biomol. Eng. 18(2): 31-40). Amongst them tandem single-chain
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 Gracie, 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., et al. (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 another 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
J. Krauss (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.
Bispecifjc diabodies (Db) utilize the diabody format for expression. Diabodies are
produced from scFv fragments by reducing the length of the linker connecting the V and VL
domain to approximately 5 residues (see Peipp, M. and T. Valerius (2002) Biochem. Soc. Trans.
30(4): 507-1 1). 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 approach was demonstrated for a bispecific diabody directed against
HER2 and CD3. A large knob was introduced in the VH domain by exchanging Val3 with Phe
and Leu45 with T p 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 only
slightly decrease as a result of these mutations. However, a reduction in antigen-binding activity
was observed for several 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. n 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 G. Winter (1997) Cancer
Immunol. Immunother. 45(3-4): 128-30; Wu, A.M., et al. (1996) Immunotechnology 2(1): p. 2 1-
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 G. Winter (1 97)
Cancer Immunol. Immunother. 45(3-4): 128-30; Wu, A.M., et al. (1996) Immunotechnol. 2(1):
21-36; Pluckthun, A. and P. Pack (1997) Immunotechnol. 3(2): 83-105; Ridgway, J.B., et al.
(1996) Protein Engin. 9(7): 6 17-21). 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 construct comprising two Fab repeats in the heavy chain of an IgG and
capable of binding four antigen molecules has been described (see PCT Publication No. WO
0177342A1, and Miller, K., et al. (2003) J. Immunol. 170(9): 4854-61).
Tumor Necrosis Factor - Related Weak Inducer of Apoptosis (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, F l4, is
expressed in CNS by endothelial cells, reactive astrocytes and neurons. TWEAK and Fn l4
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.
There is a need in the art for improved multivalent binding proteins capable of binding
TNF and TWEAK. U.S. Patent No. 7,612,1 8 1 provides a novel family of binding proteins
capable of binding two or more antigens with high affinity, which are called dual variable domain
immunoglobulins (DVD-Ig™). The present invention provides further novel binding proteins
capable of binding TNF and TWEAK.
Summary of the Invention
This invention pertains to multivalent binding proteins capable of binding TNF and
TWEAK. In an embodiment, the present invention provides a novel family of binding proteins
capable of binding TNF and TWEAK 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 VDl
and VD2 in the binding protein are heavy chain variable domains. In another embodiment, the
heavy chain variable domain is a murine heavy chain variable domain, a human heavy chain
variable domain, a CDR grafted heavy chain variable domain, or a humanized heavy chain
variable domain. In yet another, embodiment VDl and VD2 are capable of binding the same
antigen (e.g., TNF or TWEAK). In another embodiment VDl and VD2 are capable of binding
different antigens (e.g., TNF and TWEAK). In still another embodiment, C is a heavy chain
constant domain. For example, X is a linker with the proviso that XI is not CHI . For example,
XI is a linker comprising 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) (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: 8); 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); or GHEAAAVMQVQYPAS (SEQ ID NO: 26). 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 VDl 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.
In an embodiment, VDl and VD2 in the binding protein are light chain variable domains.
In an embodiment, the light chain variable domain is a murine light chain variable domain, a
human light chain variable domain, a CDR grafted light chain variable domain, or a humanized
light chain variable domain. In one embodiment VDl and VD2 are capable of binding the same
antigen (e.g., TNF or TWEAK). In another embodiment VDl and VD2 are capable of binding
different antigens (e.g., TNF and TWEAK). In an embodiment, C is a light chain constant
domain. In another embodiment, XI is a linker with the proviso that XI is not CL1 . In an
embodiment, X is a linker comprising 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); ADAAP (SEQ IDNO: 6); RADAAPTVS (SEQ
ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G4S) (SEQ ID NO: 9)
SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVS1FPP
(SEQ ID NO: ); 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: 2 1); ASTKGPSVFPLAP (SEQ ID NO: 22),
GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24);
GPAKELTPLKEAKVS (SEQ ID NO: 25); or GHEAAAVMQVQYPAS (SEQ ID NO: 26). 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. n
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 VDl-(Xl)n-VD2-C-(X2)n, wherein VDl 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, 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 VDl is a first heavy chain variable domain, VD2 is a second heavy chain variable
domain, C is a heavy chain constant domain, is a linker with the proviso that it is not CHI,
and X2 is an Fc region; and said second polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n,
wherein VDl is a first light chain variable domain, VD2 is a second light chain variable domain,
C is a light chain constant domain, X is a linker with the proviso that it is not CHI, 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 VDl-(Xl)n-VD2-C-(X2)n, respectively wherein VDl 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 the second two
polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n respectively, wherein VDl is a first light
chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant
domain, is a linker with the proviso that it is not CHI, 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 are capable of binding one
or more targets (e.g., TNF and/or TWEAK). Accordingly, in some embodiments, the binding
proteins of the invention comprise at least two variable domain sequences (e.g., VDl and VD2)
capable of binding at least two different targets. In some embodiments, VDl and VD2 are
independently chosen. Therefore, in some embodiments, VDl and VD2 comprise the same SEQ
D NO and, in other embodiments, VDl and VD2 comprise different SEQ ID NOS. In another
embodiment, the binding protein is capable of modulating a biological function of TNF and/or
TWEAK. In another embodiment, the binding protein is capable of neutralizing TNF and/or
TWEAK.
In an embodiment, the binding protein capable of binding TNF and TWEAK (seq.
1) comprises a DVD heavy chain amino acid sequence comprising SEQ ID NO. 38 or SEQ ID
NO. 40; and a DVD light chain amino acid sequence comprising SEQ ID NO. 39 or SEQ ID NO.
41. In an embodiment, the binding protein capable of binding TNF and TWEAK (seq. 1)
comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 38 and a DVD light chain
amino acid sequence of SEQ ID NO: 3 . In another embodiment, the binding protein capable of
binding TNF and TWEAK (seq. 1) has a reverse orientation and comprises a DVD heavy chain
amino acid sequence of SEQ ID NO. 40 and a DVD light chain amino acid sequence of SEQ ID
NO: 41.
In a second embodiment, the binding protein capable of binding TNF and TWEAK (seq.
1) comprises a DVD heavy chain amino acid sequence comprising SEQ D NO. 42 or SEQ ID
NO. 44; and a DVD light chain amino acid sequence comprising SEQ ID NO. 43 or SEQ ID NO.
45. In an embodiment, the binding protein capable of binding TNF and TWEAK (seq. 1)
comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 42 and a DVD light chain
amino acid sequence of SEQ ID NO: 43. In another embodiment, the binding protein capable of
binding TNF and TWEAK (seq. 1) has a reverse orientation and comprises a DVD heavy chain
amino acid sequence of SEQ ID NO. 44 and a DVD light chain amino acid sequence of SEQ ID
NO: 45.
In a third embodiment, the binding protein capable of binding TNF and TWEAK (seq.
1) comprises a DVD heavy chain amino acid sequence comprising SEQ ID NO. 46 or SEQ ID
NO. 48; and a DVD light chain amino acid sequence comprising SEQ ID NO. 47 or SEQ ID NO.
49. In an embodiment, the binding protein capable of binding TNF and TWEAK (seq. 1)
comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 46 and a DVD light chain
amino acid sequence of SEQ ID NO: 47. In another embodiment, the binding protein capable of
binding TNF and TWEAK (seq. 1) has a reverse orientation and comprises a DVD heavy chain
amino acid sequence of SEQ ID NO. 48 and a DVD light chain amino acid sequence of SEQ ID
NO: 49.
In a fourth embodiment, the binding protein capable of binding TNF and TWEAK (seq.
1) comprises a DVD heavy chain amino acid sequence comprising SEQ ID NO. 50 or SEQ ID
NO. 52; and a DVD light chain amino acid sequence comprising SEQ ID NO. 51 or SEQ ID NO.
53. In an embodiment, the binding protein capable of binding TNF and TWEAK (seq. 1)
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 TNF and TWEAK (seq. 1) 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 an embodiment, the binding protein capable of binding TNF and TWEAK (seq.
2) comprises a DVD heavy chain amino acid sequence comprising SEQ ID NO. 54 or SEQ ID
NO. 56; and a DVD light chain amino acid sequence comprising SEQ ID NO. 55 or SEQ ID NO.
57. In an embodiment, the binding protein capable of binding TNF and TWEAK (seq. 2)
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 TNF and TWEAK (seq. 2) 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 a second embodiment, the binding protein capable of binding TNF and TWEAK (seq.
2) comprises a DVD heavy chain amino acid sequence comprising SEQ ID NO. 58 or SEQ ID
NO. 60; and a DVD light chain amino acid sequence comprising SEQ ID NO. 59 or SEQ ID NO.
61. In an embodiment, the binding protein capable of binding TNF and TWEAK (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 TNF and TWEAK (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: 6 1.
In a third embodiment, the binding protein capable of binding TNF and TWEAK (seq.
2) comprises a DVD heavy chain amino acid sequence comprising SEQ ID NO. 62 or SEQ ID
NO. 64; and a DVD light chain amino acid sequence comprising SEQ ID NO. 63 or SEQ ID NO.
65. In an embodiment, the binding protein capable of binding TNF and TWEAK (seq. 2)
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 TNF and TWEAK (seq. 2) 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 a fourth embodiment, the binding protein capable of binding TNF and TWEAK (seq.
2) comprises a DVD heavy chain amino acid sequence comprising SEQ ID NO. 66 or SEQ ID
NO. 68; and a DVD light chain amino acid sequence comprising SEQ ID NO. 67 or SEQ ID NO.
69. In an embodiment, the binding protein capable of binding TNF and TWEAK (seq. 2)
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 TNF and TWEAK (seq. 2) 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 invention provides a binding protein comprising a
polypeptide chain, wherein said polypeptide chain comprises V l -( l)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; C is a heavy chain constant domain;
(Xl)n is a linker with the proviso that it is not CHI, wherein said (Xl)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-(X 1)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; C is a light chain constant domain;
(Xl)n is a linker with the proviso that it is not CHI, 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 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; C is a heavy chain constant domain;
(Xl)n is a linker with the proviso that it is not CHI, wherein said (Xl)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 VDl-(Xl )n-VD2-C-(X2)n, wherein VD 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; C is a light chain constant domain; (Xl)n is a linker with the
proviso that it is not CHI, wherein said ( )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 a native sequence Fc region or a variant
sequence Fc region. In still another embodiment, the Fc region is an Fc region from an IgGl ,
IgG2, IgG3, IgG4, gA, gM, IgE, or IgD.
In another embodiment the binding protein of the invention is a DVD-Ig capable of
binding two antigens comprising four polypeptide chains, wherein, first and third polypeptide
chains comprise 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;
C is a heavy chain constant domain; (Xl)n is a linker with the proviso that it is not CHI, wherein
said ( n is either present or absent; and (X2)n is an Fc region, wherein said (X2)n is either
present or absent; and wherein second and fourth polypeptide chains comprise VDl-(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; C is a light chain constant domain;
(Xl)n is a linker with the proviso that it is not CHI , 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.
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 capable of binding TNF and/or TWEAK comprising the steps of a) obtaining a
first parent antibody or antigen binding portion thereof, capable of binding a first antigen (e.g.,
TNF and/or TWEAK); b) obtaining a second parent antibody or antigen binding portion thereof,
capable of binding a second antigen (e.g., TNF and/or TWEAK); c) constructing first and third
polypeptide chains comprising VDl-(Xl)n-VD2-C-(X2)n, wherein, VDl 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; (Xl)n is a linker with the proviso
that it is not CHI , wherein said (Xl)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 VDl-(X l)n-VD2-C-(X2)n, wherein, VDl is a first light chain variable domain
obtained from said first parent antibody or antigen binding portion thereof (e.g., anti-TNF and/or
anti-TWEAK); VD2 is a second light chain variable domain obtained from said second parent
antibody or antigen binding thereof (e.g., anti-TNF and/or anti-TWEAK); C is a light chain
constant domain; (Xl)n is a linker with the proviso that it is not CHI, 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; 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 is
generated.
In still another embodiment, the invention provides a method of generating a Dual
Variable Domain Immunoglobulin capable of binding TNF and/or TWEAK with desired
properties comprising the steps of a) obtaining a first parent antibody or antigen binding portion
thereof, capable of binding a first antigen (e.g., TNF and/or TWEAK) and possessing at least one
desired property exhibited by the Dual Variable Domain Immunoglobulin; b) obtaining a second
parent antibody or antigen binding portion thereof, capable of binding a second antigen (e.g.,
TNF and/or TWEAK) and possessing at least one desired property exhibited by the Dual
Variable Domain Immunoglobulin; c) constructing first and third polypeptide chains comprising
VDl -(Xl)n-VD2-C-(X2)n, wherein; VDl 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; ( l )n is a linker with the proviso that it is not CHI , wherein
said ( )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 VDl -(X l)nVD2-
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; (Xl)n is a linker with the proviso that it is not CHI, 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; 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
(e.g., TNF and/or TWEAK) with desired properties is generated.
In one embodiment, the VD1 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 VD1 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 a human antibody, CDR
grafted antibody, or humanized antibody. In an embodiment, the antigen binding portions are 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 CH 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, or 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 one or more antibody parameters. In another embodiment,
the antibody parameters are antigen specificity, affinity to antigen, potency, biological function,
epitope recognition, stability, solubility, production efficiency, immunogenicity,
pharmacokinetics, bioavailability, tissue cross reactivity, or 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 which the
multivalent antibody is capable of binding to. 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 improvements)
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 of: at least about M ; at least about lC l at least about 10 M s"
at least about K M V ; or at least about lO' lv V 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 10 MV and 103 ; between M and 104M s ; between 1
and 10 M~V ; or between 1 and 10 M s 1, as measured by surface plasmon resonance.
In another embodiment the binding protein has an off rate constant (Koff) for one or
more targets of: at most about 10V at most about l V ;at most about V1;or at most about
OV, 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 C s to 10 V ; of V1to 10
V1;or of 10V to l OV1, as measured by surface plasmon resonance.
In another embodiment the binding protein has a dissociation constant (¾) to one or
more targets of: at most about 10 7 M; at most about 10~8 M; at most about 0 M; at most about
10 0 M; at most about 10 M; at most about 10 12 M; or at most 10 . In an embodiment, the
binding protein of the invention has a dissociation constant ( ) to its targets of 10 M to 10 8 M;
of 10 8 M to 10 M; of 10 9 M to 10 10 M; of 10 0 to 10 M; of 10 M to 10 12 M; or of 10 2 to
M 10 I M.
In another embodiment, the binding protein described herein is a conjugate further
comprising an agent. n yet another embodiment, the agent is an immunoadhesion molecule, an
imaging agent, a therapeutic agent, or a cytotoxic agent. In an embodiment, the imaging agent is a
radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a
magnetic label, or biotin. In another embodiment, the radiolabel is: H 1 C S, Y, Tc, in,
1 1, 1 I, Lu, Ho, or Sm. In yet another embodiment, the therapeutic or cytotoxic agent is
an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an antiangiogenic
agent, an anti-mitotic agent, an anthracycline, toxin, or 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 pcDNA, pTT (Durocher et al. (2002)
Nucleic Acids Res.30(2), pTT3 (pTT with additional multiple cloning site, pEFBOS (Mizushima,
S. and S. Nagata, S. (1990) Nucleic Acids Res. 18(1 7), pBV, pJV, pcDNA3.1 TOPO, pEF6
TOPO, or pBJ. In an embodiment, the vector is a vector disclosed in US Patent Publication No.
20090239259.
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 a protist cell, animal cell, plant cell, or fungal cell. In yet another embodiment, the host cell
is a mammalian cell including, but not limited to, CHO, COS; NSO, 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 and 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, in some
embodiments the polymeric carrier is: poly (acrylic acid), poly (cyanoacrylates), poly (amino
acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-coglycolic
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, or
blends and copolymers thereof. For example, in some embodiments the ingredient is albumin,
sucrose, trehalose, lactitol, gelatin, hydroxypropyl- b- cyclodextrin, methoxypolyethylene glycol,
or 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: a therapeutic agent, an imaging agent, a cytotoxic
agent, an angiogenesis inhibitor (including but not limited to an anti-VEGF antibody or a VEGFtrap),
a kinase inhibitor (including but not limited to a KDR and a TIE-2 inhibitor), a costimulation
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, or 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, capable of being bound by the binding protein
disclosed herein is 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
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, 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 Syndrome,
Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied
immunodeficiency (common variable hypogarnmaglobulinaemia), 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, cholestasis, 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) 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, 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, 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
mflammation 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 ateriosclerotic disease, Diffuse Levvy 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, Hallerrorden-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 infection/HIV
neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitity 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, ischemiareperfusion
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 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- hodgkins 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,
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, 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, 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, IPF/UIP, 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), 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, usual interstitial pneumonia (UIP),
vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet
macular degeneration, or wound healing.
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).
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.
In an embodiment, the antibodies of the invention or antigen-binding portions thereof, are
used to treat cancer or in the prevention 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, concurrent, or after the administration of a second agent, as discussed herein. In a
particular embodiment the second agent is 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- 1b 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, 1L-8, IL-12, IL-13, 1L-15, 1L-1 6, IL-1 8, IL-23, EMAR- , GM-CSF, FGF,
and PDGF, antibodies of CD2, CD3, CD4, CD8, CD-I , 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, p3 8, MAP kinase inhibitors, IL- 1b converting en2yme inhibitors, TNFconverting
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-1 1, IL-1 3, or TGF .
In a particular embodiment the pharmaceutical compositions disclosed herein are
administered to the patient by 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 administration.
One aspect of the invention provides at least one anti-idiotype antibody to at least one
binding protein of the present invention. The anti-idiotype 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 complementarily 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 a Dual Variable Domain (DVD)-Ig construct
according to the invention and shows a strategy for generating a DVD-Ig from two parent
antibodies;
Figure IB, is a schematic representation of constructs DVDl-Ig, DVD2-Ig, and two
chimeric mono-specific antibodies from hybridoma clones 3D12.E3and 13F5.G5.
Detailed Description of the Invention
This invention pertains to multivalent and/or multispecific binding proteins capable of
binding TNF and/or TWEAK. 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 TNF and/or TWEAK related 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
monomeric or polymeric. Use of "polypeptide" herein is intended to encompass polypeptide and
fragments and variants (including fragments of variants) thereof, unless otherwise contradicted
by context. 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 receptor;
induction of cell proliferation, inhibiting cell growth, inductions of other cytokines, induction of
apoptosis, and enzymatic activity. Biological activity also includes activity of an g 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, CHI , 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 amino-terminus to carboxy-terminus in the following order: F 1, CDR1, FR2, CDR2, FR3,
CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, gA 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
therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on
the therapeutic objectives. Certain human IgG isotypes, particularly IgGl and IgG3, mediate
ADCC and CDC via binding to FcyRs and complement Cl , 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: 4 15-20; 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 (Dall'Acqua (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. 5 1: 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 g molecule may have certain advantage in tissue
penetration due to its smaller size than 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 IgGl 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 specifically
bind 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 CHI 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 CHI
domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody,
(v) a dAb fragment (Ward et al. (1989) Nature 34 1: 544-546, PCT Publication No. WO 90/0 144
Al ), 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, RJ. et al. (1994) Structure 2: 1121-1 123). Such
antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody
Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41 354-5). In addition
single chain antibodies also include "linear antibodies" comprising a pair of tandem Fv segments
(VH-CHl-VH-CHl) 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.
5,64 1,870).
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 capable of binding 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., capable of binding 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 A.C. Cuello (1983) Nature 305(5934):
537-40), by chemical conjugation of two different monoclonal antibodies (see Staerz, U.D., et al.
1 85) Nature 314(6012): 628-3 1), or by knob-into-hole or similar approaches which introduces
mutations in the Fc region (see Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90( 14):
6444-8. 18), 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. O 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 it binds to.
A "functional antigen binding site" of a binding protein is one that is capable of binding 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-1 1, IL-12, IL-13, IL-15,
IL-1 8, IL-2 1, IL-22, IL-23, IL-33; a tumor necrosis factor such as TNF-alpha or TNF-beta; 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 : 1 121-1 123). 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(G 4S) (SEQ ID NO: 9)
SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVS1FPP
(SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFEFPP(SEQ IDNO: 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: 1 ); AKTTAPSVYPLAP (SEQ
ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ IDNO: 22),
GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24);
GPAKELTPLKEAKVS (SEQ ID NO: 25); and GHEAAAVMQVQYPAS (SEQ ID NO: 26).
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 II C, below), antibodies isolated from a recombinant, combinatorial human
antibody library (Hoogenboom, H.R. ( 1 97) TIB Tech. 5: 62-70; Azzazy H., and W.E.
Highsmith (2002) Clin. Biochem. 35: 425-445; Gavilondo J.V., and J.W. Larrick (2002)
BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 2 1:371-
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 L.L. Green (2002) Curr. Opin. Biotech. 13: 593-597; Little M. et al. (2000)
Immunol. Today 2 : 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)
BidlTechnology 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. ( 94)
Proc at Acad. Sci. USA 9 1: 3809-3813; Schier et al. ( 1995) Gene 169: 147- 155; Yelton et al.
(1995) J . Immunol. 155: 1994-2004; Jackson et al. (1995) J. Immunol. 154(7): 33 10-9; 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 as described in
US Patent No. 6,91 4,1 28.
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 CHI,
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 CDRl, 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 capable of binding 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: 90 1-917 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 subportions
were designated as LI, L2 and L3 or HI, H2 and H3 where the "L" and the "H"
designates the light chain and the heavy chains 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
CDRl 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 sub- regions, 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 capable of specific binding 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. 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
macrornolecules. Antibodies are said to "bind to the same epitope" if the antibodies crosscompete
(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. 1:19-26; Jonsson, U., et
al. (199 1) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-13 1; and
Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.
The term " ", 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 below:
Antibody ("Ab") + Antigen ("Ag")®Ab-Ag.
The term "K0 ", as used herein, is intended to refer to the off rate constant for
dissociation, or "dissociation rate constant", of a binding protein (e.g., an antibody) from the,
e.g., antibody/antigen complex as is known in the art. 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.

We claim:
1. A binding protein capable of binding at least one target, the binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises VDI-(Xl)n-VD2-C-(X2)n, wherein
VDl 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;
n is 0 or 1; and
wherein the binding protein binds TNF and TWEAK, wherein the heavy chain variable domain binding
TNF comprises at least one CDR from SEQ ID NO: 28 and/or wherein the heavy chain variable domain
binding TWEAK comprises at least one CDR from SEQ ID NO: 30 or 32.
2. The binding protein according to claim 1, wherein the VDl and/or VD2 heavy chain variable domains
comprise three CDRs from SEQ ID NO: 28,30, or 32.
3. A binding protein capable of binding at least one target, the binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl 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;
nisOor l;and
wherein the binding protein binds TNF and TWEAK, wherein the heavy chain variable domain binding
TNF comprises at least one CDR from SEQ ID NO: 29 and/or wherein the heavy chain variable domain
binding TWEAK comprises at least one CDR from SEQ ID NO: 31 or 33.
4. The binding protein according to claim 3, wherein the VDl and/or VD2 light chain variable domains
comprise three CDRs from SEQ ID NO: 29,31, or 33.
AMENDED SHEET (ARTICLE 19)
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5. The binding protein according to claim 1 or 3, wherein (Xl)n on the heavy and/or light chain is 0
and/or (X2)n on the heavy and/or light chain is (X2)0.
6. A binding protein capable of binding at least one target, the binding protein comprising first and second
polypeptide chains, wherein said first polypeptide chain comprises a first VDl-(Xl)n-VD2-C-(X2)n,
wherein
VDl 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 first linker with the proviso that it is not CHI;
X2 is an Fc region; n is 0 or 1; and
wherein said second polypeptide chain comprises a second VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domam;
XI is a second linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
n is 0 or 1; and
wherein the binding protein binds TNF and TWEAK, wherein the heavy chain variable domain
binding TNF comprises at least one CDR fi-om SEQ K) NO: 28; and/or the light chain variable
domain binding TNF comprises at least one CDR fi-om SEQ ID NO: 29; and/or the heavy chain
variable domain binding TWEAK comprises at least one CDR fi-om SEQ ID NO: 30 or 32; and/or
the light chain variable domain binding TWEAK comprises at least one CDR fi-om SEQ ID NO:
31 or 33.
7. The binding protein according to claim 6, wherein the VDl and/or VD2 heavy chain variable domains
comprise three CDRs fi-om SEQ ID NO: 28,30, or 32, respectively; and/or wherein the VDl and/or VD2
light chain variable domains comprise three CDRs fi-om SEQ ID NO: 29,31, or 33, respectively.
8. The binding protein according to claim 1, 3, or 6, wherein XI is any one of SEQ ID NOs: 1-27, or a
G/S based sequence.
AMENDED SHEET (ARTICLE 19)
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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 1,6, 24, 89,91, or 92, wherein the Fc region is a variant
sequence Fc region.
11. The binding protein according to claim 1,6, 24, 89, 91, or 92, wherein the Fc region from an IgGl,
IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
12. The binding protein according to claim 6, wherein said VDl of the first polypeptide chain and said
VDl of the second polypeptide chain are from a same first and second parent antibody, respectively, or
binding portion thereof.
13. The binding protein according to claim 6, wherein said VDl of the first polypeptide chain and said
VDl of the second polypeptide chain are fi"om a different first and second parent antibody, respectively,
or 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 from a same first and second parent antibody, respectively, or
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 from a different first and second parent antibody, respectively,
or binding portion thereof.
16. The binding protein according to any one of claims 12-15, wherein said first and said second parent
antibodies bind different epitopes on said target.
17. The binding protein according to any one of claims 12-15, wherein said first parent antibody or
binding portion thereof, binds a first target with a potency different fi-om the potency with which said
second parent antibody or binding portion thereof, binds a second target.
18. The binding protein according to any one of claims 12-15, wherein said first parent antibody or
binding portion thereof, binds a first target with an affinity different fi-om the affinity with which said
second parent antibody or binding portion thereof, binds a second target.
19. The binding protein according to any one of claims 1,3, 6,24, and 89-92, wherein said first parent
antibody or binding portion thereof, and said second parent antibody or binding portion thereof, are a
human antibody, a CDR grafted antibody, or a humanized antibody.
20-23. (Canceled)
AMENDED SHEET (ARTICLE 19)
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24. A binding protein capable of binding at least one target, the binding protein comprising four
polypeptide chains, wherein two polypeptide chains comprise VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl 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 first linker with the proviso that it is not CHI;
X2 is an Fc region;
n isOor 1; and
wherein two polypeptide chains comprise VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl 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 second linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
nisOor 1; and
wherein the binding protein binds TNF and TWEAK, wherein the heavy chain variable domain
binding TNF comprises at least one CDR from SEQ ID NO: 28, and/or the light chain variable
domain binding TNF comprises at least one CDR from SEQ ID NO: 29; and/or wherein the
heavy chain variable domain binding TWEAK comprises at least one CDR from SEQ ID NO: 30
or 32, and/or the light chain variable domain binding TWEAK comprises at least one CDR from
SEQ ID NO: 3 l or 33.
25. The binding protein according to claim 24, wherein the VDl and/or VD2 heavy chain variable
domains comprise three CDRs from SEQ ID NO: 28,30, or 32, respectively; and/or wherein the VDl
and/or VD2 light chain variable domains comprise three CDRs from SEQ ID NO: 29,31, or 33,
respectively.
26. The binding protein according to claim 1,3,6, or 24, wherein said binding protein has an on rate
constant (Km) to said at least one target of: at least about lO^M's''; at least about lO^NT's''; at least about
10*M''s''; at least about 10'M"'S''; or at least about 10*M"'s'', as measured by surface plasmon resonance.
AMENDED SHEET (ARTICLE 19)
JP*. wo 2011/163478 PCTAJS2011/041633
27. The binding protein according to claim 1, 3, 6, or 24, wherein said binding jjrotein has an off rate
constant (Koff) to said at least one target of: at most about 10"^s''; at most about lO'^s''; at most about
lO'^s''; or at most about 10'*s'', as measured by surface plasmon resonance.
28. The binding protein according to claim 1, 3,6, or 24, wherein said binding protein has a dissociation
constant (KD) to said at least one target of: at most about 10'' M; at most about 10"* M; at most about 10''
M; at most about 10''° M; at most about 10'" M; at most about 10'"^ M; or at most 10''^ M, as measured
by surface plasmon resonance.
29. A binding protein conjugate comprising a binding protein according to any one of claims 1, 3,6,24,
and S9-92, said binding protein conjugate further comprising an immunoadhension molecule, an imaging
agent, a therapeutic agent, or a cytotoxic agent.
30. The binding protein conjugate according to claim 29, wherein said imaging agent is a radiolabel, an
enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin.
31. The binding protein conjugate according to claim 30, wherein said radiolabel is 'H, "C, " S , ""Y, "TC,
•"ln,'"l,"'l.'"Lu,'**Ho,or'»Sm.
32. The binding protein conjugate according to claim 29, wherein said therapeutic or cytotoxic agent is an
anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent,
an anti-mitotic agent, an anthracycline, toxin, or an apoptotic agent.
33. The binding protein according to any one of claims 1,3, 6,24, and 89-92, wherein said binding
protein is a crystallized binding protein.
34-36. (Canceled)
37. An isolated nucleic acid encoding a binding protein according to any one of claims 1,3,6,24, and 89-
92.
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, pA^,
pcDNA3.1 TOPO, pEF6 TOPO. pHybE, 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.
42. The host cell according to claim 41, wherein said prokaiyotic cell is Escherichia coli.
43. The host cell according to claim 40, wherein said host cell is a eukaryotic cell.
AMENDED SHEET (ARTICLE 19)
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44. The host cell according to claim 43, wherein said eukaryotic cell is a protist cell, an animal cell, a
plant cell, or a fungal cell.
45. The host cell according to claim 44, wherein said animal cell is a mammalian cell, an avian cell, or an
insect cell.
46. The host cell according to claim 45, wherein said mammalian cell is a CHO cell.
47. The host cell according to claim 45, wherein said mammalian cell is a COS cell.
48. The host cell according to claim 43, wherein said fungal cell is a yeast cell.
49. The host cell according to claim 48, wherein said yeast cell is Saccharomyces cerevisiae.
50. The host cell according to claim 45, wherein said insect cell is an Sf9 cell.
51. A method of jM'oducing a binding protein, comprising culturing a host cell according to any one of
claims 40-50 in culture medium under conditions sufficient to produce the binding protein.
52-54. (Canceled)
55. A protein produced according to the method of claim 51.
56. A pharmaceutical composition comprising the binding protein according to any one of claims 1,3, 6,
24, and 89-92, and a pharmaceutically acceptable carrier.
57. The pharmaceutical composition according to claim 56 further comprising at least one additional
therapeutic agent.
58. The pharmaceutical composition according to 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. Use of the binding protein according to claims any one of 1, 3,9,24, and 89-92, for treating a subject
for a disease or a disorder by administering to the subject the binding protein such that treatment is
achieved.
AMENDED SHEET (ARTICLE 19)
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60. The use according to 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, inflammatoiy 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, 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 H, Schmidt's syndrome, adult (acute)
respiratoiy distress syndrome, alopecia, alopecia areata, seronegative arthropathy, 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 Syndrome, Acquired Immunodeficiency Related Diseases, hepatitis B, hepatitis C,
common varied immunodeficiency (common variable hypogammaglobulinaemiaX 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, Sjdgren'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
as 191
AMENDED SHEET (ARTICLE 19)
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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, Sjdrgren's
syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia,
autoimmune thjo-oid 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, depression, schizophrenia,
Th2 Type and Thl Type mediated diseases, acute and chronic pain, cancer, lung cancer, breast cancer,
stomach cancer, bladder cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal
cancer, hematopoietic malignancies, leukemia, lymphoma^ abetalipoproteinemia, acrocyanosis, acute and
chronic parasitic or infectious processes, acute leukemia, acute lymphoblastk; 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
aneutyisms, 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, bums,
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
AMENDED SHEET (ARTICLE 19)
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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, ftmctional 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, Hallerrorden-
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 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 iheumatoid 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 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- hodgkins 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, reperfiision injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, senile
dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft
AMENDED SHEET (ARTICLE 19)
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rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific anythmias,
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, Tcell
or FAB ALL, telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants,
trauma/hemorrhage, type III hypersensitivity reactions, type FV 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, vitalassociated
hemaphagocytic syndrome, Wernicke- Korsakoff syndrome, Wilson's disease, xenograft
rejection of any organ or tissue, acute coronary ^ndromes, acute idiopathic polyneuritis, acute
inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult Still's disease, anaphylaxis,
anti-phospholipid antibody syndrome, aplastic anemia, atopic eczema, atopk: 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, childhood onset
psychiatric disorder, dacryocystitis, dermatomyositis, diabetic retinopathy, disk herniation, disk prolaps,
drug induced immune hemolytic anemia, endometriosis, endophthalmitis, episcleritis, erythema
multif<»ine, 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, inflammatoiy kidney disease, IPF/UIP,
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 artety, disease (PAD), phlebitis, polyarteritis nodosa (or
periarteritis nodosa), polychondritis, polymyalgia rheumatica, poliosis, polyarticular JRA, polyendocrine
deficiency syndrome, polymyositis, post-pump syndrome, primary Parkinsonism, 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,
^ 2.00
AMENDED SHEET (ARTICLE 19)
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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,
usual interstitial pneumonia (UIP), vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome
(VKH syndrome), wet macular degeneration, or wound healing.
61. The use 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 binding protein capable of binding at least one target comprising the steps
of
a) obtaining a first parent antibody or binding portion thereof;
b) obtaining a second parent antibody or binding portion thereof;
c) constructing the polypeptide chain or chains according to any one of claims 1, 3, 9,24,
and 89-92; and
d) expressing the polypeptide chain or chains such that a binding protein capable of binding
at least one target is generated.
63. The method according to claim 62, wherein the VDI and/or VD2 heavy chain variable domains
comprise three CDRs from SEQ ID NO: 28, 30, or 32, respectively; and/or wherein the VDI and/or VD2
light chain variable domains comprise SEQ ID NO: 29,31, or 33, respectively.
64. The method according to claim 62, wherein said first parent antibody or binding portion thereof, and
said second parent antibody or binding portion thereof, are a human antibody, a CDR grafted antibody, or
a humanized antibody.
65-67. (Canceled)
68. The method according to claim 62, wherein the Fc region, if present, is a variant sequence Fc region.
69. The method according to claim 62, wherein the Fc region, if present, is from an IgGl, IgG2, lgG3,
IgG4,IgA,IgM,IgE,orIgD.
70-73. (Canceled)
tti20l
AMENDED SHEET (ARTICLE 19)
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74. The method according to claim 62, wherein said first parent antibody or binding portion thereof, binds
a first target with a different affinity than the affinity with which said second parent antibody or binding
portion thereof, binds a second target.
75. The method according to claim 62, wherein said first parent antibody or binding portion thereof, binds
a first target with a different potency than the potency with which said second parent antibody or binding
portion thereof, binds a second target.
76. A method of determining the presence of at least one target 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 binding protein comprises the binding protein according to any one of
claims 1,3,6,24, and 89-92.
77. The method according to claim 76, further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding protein
binds to an epitope on the target 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 target or fragment thereof that is not bound by the
binding protein to form a second complex; and
(iii) detecting the presence of the target 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 target or fragment
thereof is directly correlated with the signal generated by the detectable label.
78. The method according to claim 76, further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding protein
binds to an epitope on the target or fhigment 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 target or fragment thereof for binding to the binding protein so as to form a second
complex; and
AMENDED SHEET (ARTICLE 19)
C WO 2011/163478 PCT/US2011/041633
(iii) detecting the presence of the target 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 target or fragment
thereof is indirectly correlated with the signal generated by the detectable label.
79. The method according to any one of claims 77-78, wherein the test sample is from a patient and the
method further comprises diagnosing, prognosticating, or assessing the efficiency of
therapeutic/prophylactic 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 therapeutic/prophylactic
treatment of the patient as needed to improve efficacy.
80. The method according to any one of claims 77-78, wherein the method is adapted for use in an
automated system or a semi-automated system.
81. The method according to any one of claims 77-78, wherein the method determines the presence of
more than one antigen in the sample.
82. A method of determining the amount or concentration of an target 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 target 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 target or fragment thereof,
and wherein the at least one binding protein comprises the binding protein according to claim 1,
3,6,24, and 89-92.
83. The method of claim 82, further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding protein
binds to an epitope on the target or fragment thereof so as to form a first complex;
AMENDED SHEET (ARTICLE 19)
jm, WO 2011/163478 PCTAJS2011/041633
(ii) contacting the complex with the at least one detectable label, wherein the detectable label
binds to an epitope on the target 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 target 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 target or fragment thereof is directly proportional to the signal generated by the
detectable label.
84. The method according to claim 82, further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding protein
binds to an epitope on the target 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 target 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 target 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
target or fragment thereof is indirectly proportional to the signal generated by the detectable label.
85. The method according to any one of claims 82-84, wherein the test sample is from a patient and the
method frirther comprises diagnosing, prognosticating, or assessing the efficiency of
therapeutic/prophylactic 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 therapeutic/prophylactic
treatment of the patient as needed to improve efficacy.
86. The method according to any one of claims 83-85, wherein the method is adapted for use in an
automated system or a semi-automated system.
87. The method according to any one of claims 83-84, wherein the method determines the amount or
concentration of more than one antigen in the sample.
88. A kit for assaying a test sample for the presence, amount, or concentration of an target or fragment
thereof,
att2oM
AMENDED SHEET (ARTICLE 19)
C wo 2011/163478 PCT/US2011/041633
said kit comprising (a) instructions for assaying the test sample for the target n or fragment thereof and (b)
at least one binding protein comprising the binding protein according to claim 1, 3,6, 24, and 89-92.
89. A binding protein capable of binding at least one target, die binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl 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;
nisOor l;and
wherein the bind protein binds TNF and TWEAK; and
further wherein the binding protein binds:
(a) TNF, and has an off rate constant (Koff) of at most about 8.20 x lO"* s"' and/or a
dissociation constant (KD) of at most about 6.20 x 10'^' M, as determined by surface
plasmon resonance; and/or
(b) TWEAK, and has an off rate constant (Kofi) of less than 1.0 x 10"* s'' and/or a
dissociation constant (KD) of at most about 8.3 x 10''^ M, as determined by surface
plasmon resonance.
90. A binding protein capable of binding at least one target, the binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl 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;
nisOor l;and
wherein the bind protein binds TNF and TWEAK; and
fiuther wherein the binding protein binds:
aM205
AMENDED SHEET (ARTICLE 19)
^ wo 2011/163478 PCT/US2011/041633
(a) TNF, and has an off rate constant (Kosr) of at most about 8.20 x 10"^ s"' and/or a
dissociation constant (KQ) of at most about 6.20 x 10"" M, as determined by surface
plasmon resonance; and/or
(b) TWEAK, and has an off rate constant (Koff) of less than 1.0 x IC* s'' and/or a
dissociation constant (KD) of at most about 8.3 x 10"^^ M, as determined by surface
plasmon resonance.
91. A binding protein capable of binding at least one target, the binding protein comprising 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;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a luiker with the proviso that it is not CHI;
X2 is an Fc region;n is 0 or 1; and
wherein said second polypeptide chain comprises a second VDl-(Xl)n-VD2-C-{X2)n, wherein
VDl 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;
nisOor l,and
wherein the bind protein binds TNF and TWEAK; and
further wherein the binding protein binds:
(a) TNF, and has an off rate constant (Kos) of at most about 8.20 x 10"^ s'' and/or a
dissociation constant (KD) of at most about 6.20 x 10"" M, as determined by surface
plasmon resonance; and/or
SB 206
AMENDED SHEET (ARTICLE 19)
C WO 2011/163478 PCT/US2011/041633
(b) TWEAK, and has an off rate constant (Koff) of less than 1.0 x 10"* s'' and/or a
dissociation constant (KD) of at most about 8.3 x 10''^ M, as determined by surface
plasmon resonance.
92. A binding protein capable of binding at least one target, the binding protein comprising four
polypeptide chains, wherein two polypeptide chains comprise VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl 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;
nisOor l;and
wherein two polypeptide chains comprise VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl 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 widi the proviso that it is not CHI;
X2 does not comprise an Fc region;
nisOorl;and
wherein the bind protein binds TNF and TWEAK; and
further wherein the binding protein binds:
(a) TNF, and has an off rate constant (Koff) of at most about 8.20 x 10"^ s'* and/or a
dissociation constant (Ko) of at most about 6.20 x 10'" M, as determined by surface
plasmon resonance; and/or
(b) TWEAK, and has an off rate constant (Koir) of less than 1.0 x lO"* s'' and/or a
dissociation constant (Kp) of at most about 8.3 x 10''^ M, as determined by surface
plasmon resonance.
93. The binding protein according to claim 1 or 89, wherein the binding protein binds:
TBS ZCl
AMENDED SHEET (ARTICLE 19)
^ wo 2011/163478 PCT/US2011/041633
TNF and TWEAK, and wherein the heavy chain variable domain binding TNF comprises tiiree CDRs
from SEQ ID NO: 28; and/or wherein the heavy chain variable domain binding TWEAK comprises three
CDRs from SEQ ID NO: 30 or 32, respectively.
94. The binding protein according to claim 3 or 90, wherein the binding protein binds:
TNF and TWEAK, and wherein the light chain variable domain binding TNF comprises three CDRs from
SEQ ID NO: 29; and/or wherein the light chain variable domain binding TWEAK comprises three CDRs
from SEQ ID NO: 31 or 33, respectively.
95. The binding protein according to claim 6, 24,91 or 92, wherein the binding protein binds:
TNF and TWEAK, and wherein the heavy chain variable domain binding TNF comprises three CDRs
from SEQ ID NO: 28 and the light chain variable domain binding TNF comprises three CDRs from SEQ
ID NO: 29; and/or wherein the heavy chain variable domain binding TWEAK comprises three CDRs
from SEQ ID NO: 30 or 32, respectively, and the light chain variable domain binding TWEAK comprises
three CDRs from SEQ ID NO: 31 or 33, respectively.
96. The binding protein according to claim 24,91, or 92, wherein the binding protein is:
DVDl 127 (comprising SEQ ID NOs:38 and 39); DVDl 128 (comprising SEQ ID NOs:40 and 41);
DVDl 129 (comprising SEQ ID NOs:42 and 43); DVDl 130 (comprising SEQ ID NOs:44 and 45);
DVDl 131 (comprising SEQ IDNOs:46 and 47); DVDl 132 (comprising SEQ ID NOs:50 and 51);
DVDl 133 (comprising SEQ ID NOs:48 and 49); DVDl 134 (comprising SEQ ID NOs:52 and 53);
DVDl 135 (comprismg SEQ ID NOs:54 and 55); DVDl 136 (comprising SEQ ID NOs:56 and 57);
DVDl 137 (comprising SEQ ID NOs:58 and 59); DVDl 138 (comprising SEQ ID NOs:60 and 61);
DVDl 139 (comprising SEQ ID NOs:62 and 63); DVDl 140 (comprising SEQ ID NOs:66 and 67);
DVDl 141 (comprising SEQ ID NOs:64 and 65); or DVDl 142 (comprising SEQ ID NOs:68 and 69).
97. The binding protein according to any one of claims 89-92, wherein the VDl and/or VD2 heavy chain
variable domains, if present, comprise three CDRs from SEQ ID NO: 28,30, or 32, respectively; and/or
wherein the VDl and/or VD2 light chain variable domains, if present, comprise three CDRs from SEQ ID
NO: 29,31, or 33, respectively.
98. The binding protein according to any one of claims 1,3,6,24, and 89-92, wherein the VDl and/or
VD2 heavy chain variable domains, if present, comprise SEQ ID NO: 28, 30, or 32, respectively; and/or
wherein the VDl and/or VD2 light chain variable domains, if present, comprise SEQ ID NO: 29,31, or
33, respectively.
VB 2.0&
AMENDED SHEET (ARTICLE 19)
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99. The binding protein according to any one of claims 1, 3, 6,24, and 89-92, wherein (Xl)n between the
first and second light chain variable domain is not CL.
100. The method according to claim 62, wherein the VDl and/or VD2 heavy chain variable domains, if
present, comprise SEQ ID NO: 28,30, or 32, respectively; and/or wherein the VDl and/or VD2 light
chain variable domains, if present, comprise SEQ ID NO: 29,31, or 33, respectively.
101. The method according to claim 62, wherein (Xl}n between the first and second light chain variable
domain is not CL.
102. The binding protein according to any one of claims 1,3,6,24, and 89-92, wherein the binding
protein neutralizes TNF with an ECjo of at most about 1.951 nM, as measured by a murine TNF
neutralization assay.
103. The binding protein according to any one of claims 1,3, 6,24, and 89-92, wherein the binding
protein neutralizes TWEAK with an EC50 of at most about 4.336 nM, as measured by a human TWEAK
neutralization assay.
104. The binding protein according to any one of claims 1,3,6,24, and 89-92, wherein the VDl and
VD2 heavy chain variable domains, if present, comprise three CDRs from SEQ ID NO: 28,30, or 32,
respectively; and wherein the VDl and VD2 light chain variable domains, if present, comprise three
CDRs from SEQ ID NO: 29,31, or 33, respectively.
105. The binding protein according to any one of claims 1,3,6,24, and 89-92, wherein the VDl and
VD2 heavy chain variable domains, if present, comprise SEQ ID NO: 28,30, or 32, respectively; and
wherein the VDl and VD2 light chain variable domains, if present, comprise SEQ ID NO: 29,31, or 33,
respectively.
106. The method according to claim 62, wherein the VDl and VD2 heavy chain variable domains, if
present, comprise SEQ ID NO: 28,30, or 32, respectively; and wherein the VDl and VD2 light chain
variable domains, if present, comprise SEQ ID NO: 29,31, or 33, respectively.