DUAL VARIABLE DOMAIN IMMUNOGLOBULINS AND USES THEREOF
Cross Reference to Related Applications
This application is a non-provisional application claiming priority to U.S. Provisional
Application Serial No. 6 1/409,35 1, filed November 2, 2010, the entire content of which is hereby
incorporated by reference.
Field
Multivalent and multispecific binding proteins, methods of making, and their uses in the,
diagnosis, prevention and/or treatment of acute and chronic inflammatory diseases, cancer, and
other diseases are provided.
Background
Engineered proteins, such as multispecific antibodies that bind 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 and
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 g species are generated, of which only one is the functional bispecific antibody. The
presence of mis-paired by-products, and significantly reduced production yields, means
sophisticated purification procedures are required.
Bispecific antibodies can also be produced by chemical conjugation of two different
mAbs (see Staerz et al. ( 1985) Nature 314(601 2):628-3 1) . This approach does not yield
homogeneous preparation. Other approaches have used chemical conjugation of two different
mAbs or smal ler antibody fragments (see Brennan et al. ( 1985) Science 229(4708):8 1-3).
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 sign ificant 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 et al. (1987) J . Immunol. 39(7):2367-75). Bu this method
results in Fab'2 fragments, not full IgG molecule.
A wide variety of other recombinant bispecific antibody formats have been developed
(see riangk et ai. (200 ) B o oi. Eng. 1 (2): -40). Amongst e tandem single-cha in 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 et al. (2003) Nat. Med. 9( l ):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 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 et al. (200 1) 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 et al. (1999) J . Clin. Invest. 104( 10): 393-401 ) . 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 et al. (2000) J , Immunol. 164( 2):6495-502; Ito et
al . (2003) J . Immunol. J70(9):4 802-9; Ka i et al. (2002) J. Neuroimmunol. 5( -2): 134-40)
using either a very short A!a3 linker or lo g glycine/serine-rich linkers. a recent study, phage
display of a tandem scFv repertoire containing randomized middle linkers with a length of 3 or 6
residues was employed to enrich for those molecules that are produced in soluble and active form
in bacteria. This approach resulted in the isolation of a tandem scFv molecule with a 6 amino acid
residue linker (see Arndt and Krauss (2003) Methods Mol. Biol. 207:305-2 ) . 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
molecu les, which can be expressed i bacteria in an active for .
Bispecific diabodies (Db) utilize the diahody format for expression. Diabodies are
produced from scFv fragments by reducing the length of the linker connecting the VH and VL
domain to approximately 5 residues (see Peipp and Valerius (2002) Biochem. Soc. Trans.
30(4):507-i 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 et al. ( 995) Proc. Natl. Acad. Sci. USA 92( 5):702 -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 et al. (1993) Proc. Natl. Acad. Sci. USA 90( 14):6444-8. 18). This was
demonstrated for a bispecific diabody directed against HER2 and CD3 . A large knob was
introduced in the VH domain by exchanging Val37 with Phe and Leu45 with Trp and a
complementary hole was produced in the VL domain by mutating Phe98 to Met and Tyr87 to
Ala, either in the anti- HER2 or the anti-CD3 variable domains. By using this approach the
production of bispecific diabodies could be increased from 72% by the parental diabody to over
90% by the knob-into-hole diabody. Importantly, production yields 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 wel l as
undesirable pharmacokinetics.
Single-chain diabodies (scDb) represent an alternative strategy for improving the
formation of bispecific diabody-like molecules (see Holliger and Winter ( 1997) Cancer Immunol.
Immunother. 45(3-4): 128-30; Wu et al. (1996) Immunotechnology 2( 1):2 -36). Bispecific singlechain
diabodies are produced by connecting the two diabody-forming polypeptide chains with an
additional middle linker with a length of approximately 1 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 and Winter (1997) Cancer Immunol. Immunother. 45(3-4): 128-30;
Wu et al. ( 1996) Immunotechnol. 2(l ):2 1-36; Pluckthun and Pack ( 1997) Immunotechnol.
3(2):83- 105; Ridgway et al. (1996) Protein Engin. 9(7):6 17-2 1) . 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 et al. (2004) J. Biol. Chem. 2?9(4):2856-65). n addition, multivalent
antibody constructs comprising two Fab repeats in the heavy chain of an gG and that bind four
antigen molecules have been described (see PCT Publication No. WO 0177342, and Miller et al
(2003) J . Immunol. 170(9):4854-6 1) .
There is a need in the art for improved multivalent binding proteins that bind two or more
antigens. U.S. Patent No. 7,6 12,181 provides a novel family of binding proteins that bind two or
more antigens with high affinity, and which are called dual variable domain immunoglobulins
(DVD-Ig™). Novel binding proteins that bind two or more antigens are provided.
Summary
Multivalent binding proteins that bind two or more antigens are provided. A novel family
of binding proteins that bind two or more antigens with high affinity are also provided.
In one embodiment, a dual variable domain (DVD) binding protein comprising a
polypeptide chain, wherein the polypeptide chain comprises VDl -(X l )n-VD2-C-(X2)n, wherein
VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X
represents an amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1 is provided.
In an embodiment the VD1 and VD2 in the binding protein are heavy chain variable domains. In
another embodiment, the heavy chain variable domain is 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 VD1 and VD2 bind the same antigen.
In another embodiment VD and VD2 bind different antigens. In siiil another embodiment, C is a
heavy chain constant domain. For example, XI is a linker with the proviso that XI is not CHI .
For example, XI is 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„S) (SEQ ID NO: 9) SAKTTPKLEEGEFSEARV (SEQ D NO:
0); ADAAP (SEQ ID NO: 1 ); ADAAPTVS1FPP (SEQ D NO: 2); TVAAP (SEQ D NO: 13);
TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 5); QPKAAPSVTLFPP (SEQ ID
NO: 16); AKTTPP (SEQ ID NO: 7); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID
NO: 9); AKTTAPSVYPLAP (SEQ D NO: 20); ASTKGP (SEQ ID NO: 21);
ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23);
GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); and
GHEAAAVMQVQYPAS (SEQ ID NO: 26); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO:
27); or ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28). n an embod iment, X2 is an
Fc region. In another embodiment, X2 is a variant Fc region.
In an embodiment, the DVD-binding proteins disclosed herein comprises a polypeptide
chain, wherein the polypeptide chain comprises VDl -(X l )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, X is a linker with the proviso that it is not CH , and X2 is an Fc region.
In an embodiment, VD1 and VD2 in the binding protein are light chain variable domains.
In an embodiment, the light chain variable domain is 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 VD1 and VD2 bind the same antigen. In another
embodiment VD1 and VD2 bind different antigens in an embodiment, C is a light chain constant
domain. In another embodiment, XI is a linker with the proviso that XI is not CL. In an
embodiment, X is AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ
ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ
ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS
(SEQ ID NO: 8); RADAAAA(G4S)4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO:
10); ADAAP (SEQ ID NO: 1); ADAAPTVSIFPP (SEQ ID NO: 2); TVAAP (SEQ ID NO: 3);
TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID
NO: 16); AKTTPP (SEQ ID NO: 7); AKTTPPSVTPLAP (SEQ ID NO: 8); AKTTAP (SEQ ID
NO: 9); 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); and
GHEAAAVMQVQYPAS (SEQ ID NO: 26); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO:
27); or ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28). In an embodiment, the DVDbinding
protein does not comprise X2.
In an embodiment, both the variable heavy and variable light chain comprise the same
linker in another embodiment, the variable heavy and variable light chain comprise different
linkers. In another embodiment, both the variable heavy and variable light chain comprise a short
(about 6 amino acids) linker. In another embodiment, both the variable heavy and variable light
chain comprise a long (greater than 6 amino acids) linker. In another embodiment, the variable
heavy chain comprises a short linker and the variable light chain comprises a long linker. In
another embodiment, the variable heavy chain comprises a long linker and the variable light chain
comprises a short linker.
In an embodiment, the DVD-binding proteins disclosed herein comprises a polypeptide
chain, wherein said polypeptide chain comprises VDl-(X l )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 (CL), XI is a linker with the proviso that it is not CL, and X2 does not comprise
an Fc region.
In another embodiment, a DVD-binding protein comprising two polypeptide chains,
wherein said first polypeptide chain comprises VDl -(X l )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, X is a first linker, and X2 is an Fc region; and said second polypeptide chain
comprises VDl -(X l )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,
and X2 does not comprise an Fc region is provided. In some embodiments, the first and second
XI are the same. In other embodiments, the first and second XI are different. In some
embodiments the first X is not a C I domain. In some embodiments the second XI is not a CL
domain.
In a particular embodiment, the binding protein is a DVD binding protein comprising
four polypeptide chains wherein the first two polypeptide chains comprises VD -(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 first linker, and X2 is an Fc
region; and the second two polypeptide chain comprises VD -(X l )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, XI is a second linker, and X2 does not comprise an Fc region.
Such a DVD-binding protein has four antigen binding sites. In some embodiments, the first and
second are the same. In other embodiments, the first and second XI are different. In some
embodiments the first s not a C domain in some embodiments the second is not a CL
domain.
In another embodiment, the DVD-binding proteins disclosed herein bind one or more
targets. In an embodiment, the DVD Ig comprises at least two of the VH and/or VL regions listed
in Table 2, in any orientation. n some embodiments, VD1 and VD2 are independently chosen.
Therefore, in some embodiments, VD1 and VD2 comprise the same SEQ ID NO and, in other
embodiments, VD1 and VD2 comprise different SEQ ID NOS.
In an embodiment, the target is a cytokine, cell surface protein, enzyme, or receptor. In
another embodiment, the DVD-binding protein is capable of modulating a biological function of
one or more targets. In another embodiment, the DVD-binding protein is capable of neutralizing
one or more targets. In another embodiment, the cytokines are lymphokines, monokines,
polypeptide hormones, receptors, or tumor markers. For example, the DVD-binding proteins are
capable of binding two or more of the following: Tumor Necrosis Factor (TNF), Prostaglandin
E2 (PGE2), Vascular Endothelial Growth Factor (VEGF), Delta-Like Ligand 4 (DLL4) (see also
Table 2). In an embodiment, the DVD-binding proteins comprise CDR grafted VH and VL. In
another embodiment, the DVD-binding proteins comprise CDR grafted VH and VL and further
mutations to identify optimal frameworks for the DVD-binding proteins. In a specific
embodiment the DVD-binding proteins are capable of binding pairs of targets. In certain
embodiments, the pair of targets is TNF (seq. 1) and PGE2 (AB00 ); TNF (seq. 1) and PGE2
(AB003); TNF (seq. 1) and PGE2 (AB004); TNF (seq. 1) and PGE2 (AB0 11); TNF (seq. 1) and
PGE2 (AB01 4); TNF (seq. 1) and PGE2 (AB0 5); TNF (seq. 1) and PGE2 (AB0 16); TNF (seq.
1) and PGE2 (AB033); TNF (seq. 1) and PGE2 (ABO 17); TNF (seq. 1) and PGE2 (ABO 18); TNF
(seq. 1) and PGE2 (AB022); TNF (seq. 1) and PGE2 (AB023); TNF (seq. 1) and PGE2 (AB026);
TNF (seq. 1) and PGE2 (AB029); TNF (seq. 1) and PGE2 (AB050); TNF (seq. 1) and PGE2
(AB054); TNF (seq. 1) and PGE2 (AB043); TNF (seq. 1) and PGE2 (AB046); TNF (seq. 1) and
PGE2 (AB052); TNF (seq. 1) and PGE2 (AB060); TNF (seq. 2) and PGE2 (seq. 1); PGE2 (seq.
2) and TNF (seq. 3); VEGF (seq. 2) and DLL4 (seq. 1); DLL4 (seq. 2) and VEGF (seq. 3); VEGF
(seq. 2) and DLL4 (seq. 3); DLL4 (seq. 4) and VEGF (seq. 3); TNF (seq. 4) and PGE2 (seq. 3);
TNF (seq. 5) and PGE2 (seq. 4); PGE2 (seq. 5) and TNF (seq. ); VEGF (seq. 4) and DLL4 (seq.
5); DLL4 (seq. 6) and VEGF (seq. 5); VEGF (seq. 4) and DLL4 (seq. 7); DLL4 (seq. 8) and
VEGF (seq. 5); TNF (seq. 1) and PGE2 (seq. 6); PGE2 (seq. 4) and TNF (seq. 6); VEGF (seq. 5)
and DLL4 (seq. 9); DLL4 (seq. 5) and VEGF (seq. 6); VEGF (seq. 5) and DLL4 (seq. 10); DLL4
(seq. 7) and VEGF (seq. 6); TNF (seq. 6) and PGE2 (seq. 4); PGE2 (seq. 6) and TNF (seq. 1);
VEGF (seq. 6) and DLL4 (seq. 5); DLL4 (seq. 9) and VEGF (seq. 5); VEGF (seq. 6) and DLL4
(seq. 7); DLL4 (seq. 10) and VEGF (seq. 5); VEGF (seq. 1) and DLL4 (seq. 1); VEGF (seq. 1)
and DLL4 (seq. 2); or DLL4 (seq. 13) and VEGF (seq. 7).
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABOO )
comprises heavy chain amino acid sequences of SEQ D NO. 138 and SEQ ID NO. 140; and light
chain amino acid sequences of SEQ D NO. 39 and SEQ ID NO. 4 ] . In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB001 ) comprises a heavy chain amino
acid sequence of SEQ ID NO. 138 and a light chain amino acid sequence of SEQ ID NO: 139. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB00 1 has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 140 and a
light chain amino acid sequence of SEQ ID NO: 14 1.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB003)
comprises heavy chain amino acid sequences of SEQ ID NO. 142 and SEQ ID NO. 144; and light
chain amino acid sequences of SEQ ID NO. 1 3 and SEQ D NO. 145. n an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB003) comprises a heavy chain amino
acid sequence of SEQ ID NO. 142 and a light chain amino acid sequence of SEQ ID NO: 143 . In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB003) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 144 and a
light chain amino acid sequence of SEQ ID NO: 145.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB004)
comprises heavy chain amino acid sequences of SEQ ID NO. 146 and SEQ ID NO. 148; and light
chain amino acid sequences of SEQ ID NO. 1 7 and SEQ D NO. 149. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB004) comprises a heavy chain amino
acid sequence of SEQ ID NO. 146 and a light chain amino acid sequence of SEQ ID NO: 147. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB004) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 48 and a
light chain amino acid sequence of SEQ ID NO: 149.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB0 11)
comprises heavy chain amino acid sequences of SEQ ID NO. 150 and SEQ ID NO. 52; and light
chain amino acid sequences of SEQ ID NO. and SEQ ID NO. 53 . In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB0 11) comprises a heavy chain amino
acid sequence of SEQ ID NO. 0 and a light chain amino acid sequence of SEQ ID NO: 5 . n
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB0 1) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 52 and a
ght chain amino acid sequence of SEQ D NO: 3 .
In an embodiment, the DVD-binding protein that binds TNF (seq. 3) and PGE2 (ABO 14)
comprises heavy chain amino acid sequences of SEQ ID NO. 54 and SEQ ID NO. 56; and light
chain amino acid sequences of SEQ ID NO. 5 and SEQ 3D NO 7. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 14) comprises a heavy chain amino
acid sequence of SEQ ID NO. 54 and a light chain amino acid sequence of SEQ ID NO: 155. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 14) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 156 and a
light chain amino acid sequence of SEQ ID NO: 157.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 15)
comprises heavy chain amino acid sequences of SEQ ID NO. 158 and SEQ ID NO. 160; and light
chain amino acid sequences of SEQ ID NO. 9 and SEQ ID NO. 161. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 15) comprises a heavy chain amino
acid sequence of SEQ ID NO. 58 and a light chain amino acid sequence of SEQ ID NO: 159. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB0 15) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 160 and a
light chain amino acid sequence of SEQ ID NO: 161.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB0 16)
comprises heavy chain amino acid sequences of SEQ ID NO. 1 2 and SEQ ID NO. 164; and light
chain amino acid sequences of SEQ ID NO. 163 and SEQ ID NO. 165. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 16) comprises a heavy chain amino
acid sequence of SEQ ID NO. 162 and a light chain amino acid sequence of SEQ ID NO: 1 , In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 16) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 64 and a
light chain amino acid sequence of SEQ ID NO: 165.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB033)
comprises heavy chain amino acid sequences of SEQ ID NO. 166 and SEQ ID NO. 168; and light
chain amino acid sequences of SEQ ID NO. 167 and SEQ ID NO. 169. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB033) comprises a heavy chain amino
acid sequence of SEQ ID NO. 166 and a light chain amino acid sequence of SEQ ID NO: 167. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB033) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 68 and a
light chain amino acid sequence of SEQ ID NO: 69.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 17)
comprises heavy chain amino acid sequences of SEQ ID NO. 170 and SEQ ID NO. 72; and light
chain amino acid sequences of SEQ ID NO. 17 1 and SEQ ID NO. 173 . In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB0 1 ) comprises a heavy chain amino
acid sequence of SEQ ID NO. 170 and a light chain amino acid sequence of SEQ ID NO: ! . In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 17) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 172 and a
light chain amino acid sequence of SEQ ID NO: 173.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB0 18)
comprises heavy chain amino acid sequences of SEQ ID NO 4 and SEQ D NO. 176; and light
chain amino acid sequences of SEQ ID NO. !75 and SEQ ID NO. 177. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 18) comprises a heavy chain amino
acid sequence of SEQ ID NO. 1 4 and a light chain amino acid sequence of SEQ ID NO: 1 5. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (ABO 18) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 176 and a
light chain amino acid sequence of SEQ ID NO: 1 7.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB022)
comprises heavy chain amino acid sequences of SEQ ID NO. 1 8 and SEQ ID NO. 180; and light
chain amino acid sequences of SEQ ID NO. 179 and SEQ ID NO. 181. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB022) comprises a heavy chain amino
acid sequence of SEQ ID NO. 78 and a light chain amino acid sequence of SEQ ID NO: 79.
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB022) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 180 and a
light chain amino acid sequence of SEQ ID NO: 81.
In an embodiment, the DVD-binding protein thai binds TNF (seq. 1) PGE2 (AB023)
comprises heavy chain amino acid sequences of SEQ ID NO. 2 and SEQ D NO. 184; and light
chain amino acid sequences of SEQ ID NO. 83 and SEQ ID NO. 185. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB023) comprises a heavy chain amino
acid sequence of SEQ ID NO. 182 and a light chain amino acid sequence of SEQ ID NO: 1 3 . In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB023) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 184 and a
light chain amino acid sequence of SEQ ID NO: 185.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB026)
comprises heavy chain amino acid sequences of SEQ ID NO. 86 and SEQ ID NO. 188; and light
chain amino acid sequences of SEQ) D NO. 7 and SEQ ID NO. 89. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB026) comprises a heavy chain amino
acid sequence of SEQ ID NO. 186 and a light chain amino acid sequence of SEQ D NO: 87. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB026) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 188 and a
light chain amino acid sequence of SEQ ID NO: 189.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB029)
comprises heavy chain amino acid sequences of SEQ ID NO. 190 and SEQ ID NO. 92; and light
chain amino acid sequences of SEQ ID NO. 1 1 and SEQ ID NO. 3. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB029) comprises a heavy chain amino
acid sequence of SEQ ID NO. 190 and a light chain amino acid sequence of SEQ ID NO: 191. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB029) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 192 and a
light chain amino acid sequence of SEQ ID NO: 193.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB050)
comprises heavy chain amino acid sequences of SEQ ID NO. 194 and SEQ ID NO. 96; and light
chain amino acid sequences of SEQ ID NO. 95 and SEQ ID NO. 97. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB050) comprises a heavy chain amino
acid sequence of SEQ ID NO. 194 and a light chain amino acid sequence of SEQ ID NO: 195. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB050) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 196 and a
light chain amino acid sequence of SEQ ID NO: 97.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB054)
comprises heavy chain amino acid sequences of SEQ ID NO. 198 and SEQ ID NO. 200; and light
chain amino acid sequences of SEQ ID NO. 199 and SEQ ID NO. 201. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB054) comprises a heavy chain amino
acid sequence of SEQ ID NO. 1 8 and a light chain amino acid sequence of SEQ ID NO: 199. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB054) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 200 and a
light chain amino acid sequence of SEQ ID NO: 201.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB043)
comprises heavy chain amino acid sequences of SEQ ID NO. 202 and SEQ ID NO. 204; and light
chain amino acid sequences of SEQ ID NO. 203 and SEQ ID NO. 205 . n an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB043) comprises a heavy chain amino
acid sequence of SEQ ID NO. 202 and a light chain amino acid sequence of SEQ ID NO: 203 . In
another embodiment, the DVD-binding protein that binds TNF (seq. 3) and PGE2 (AB043) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ D NO. 204 and a
light chain amino acid sequence of SEQ ID NO: 205 .
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB046)
comprises heavy chain amino acid sequences of SEQ ID NO. 206 and SEQ ID NO. 208; and light
chain amino acid sequences of SEQ ID NO. 207 and SEQ JD NO. 209. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB046) comprises a heavy chain amino
acid sequence of SEQ ID NO. 206 and a light chain amino acid sequence of SEQ ID NO: 207. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB046) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 208 and a
light chain amino acid sequence of SEQ ID NO: 209.
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB052)
comprises heavy chain amino acid sequences of SEQ ID NO. 2 10 and SEQ ID NO. 2 12; and light
chain amino acid sequences of SEQ ID NO. 211 and SEQ ID NO. 2 13. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB052) comprises a heavy chain amino
acid sequence of SEQ ID NO. 2 10 and a light chain amino acid sequence of SEQ ID NO: 2 11. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB052) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 212 and a
light chain amino acid sequence of SEQ ID NO: 2 13.
In an embodiment, the DVD-binding protein that binds TNF (seq. ) and PGE2 (AB060)
comprises heavy chain amino acid sequences of SEQ ID NO. 2 14 and SEQ ID NO. 2 16; and light
chain amino acid sequences of SEQ ID NO. 2 15 and SEQ ID NO. 217. In an embodiment, the
DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB060) comprises a heavy chain amino
acid sequence of SEQ ID NO. 2 4 and a light chain amino acid sequence of SEQ ID NO: 2 5. In
another embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (AB060) has a
reverse orientation and comprises a heavy chain amino acid sequence of SEQ ID NO. 2 16 and a
light chain amino acid sequence of SEQ ID NO: 217. In an embodiment, the DVD-binding
protein that binds TNF (seq. 2) and PGE2 (seq. 1) comprises the heavy chain amino acid
sequence of SEQ ID NO. 218 and the light chain amino acid sequence of SEQ ID NO. 21 .
In an embodiment, the DVD-binding protein that binds PGE2 (seq. 2) and TNF (seq. 3)
comprises the heavy chain amino acid sequence of SEQ ID NO. 220 and the light chain amino
acid sequence of SEQ ID NO. 22 1.
In an embodiment, the DVD-binding protein that binds VEGF (seq. 2) and DLL4 (seq. 1)
comprises the heavy chain amino acid sequence of SEQ ID NO. 222 and the light chain amino
acid sequence of SEQ ID NO. 223 .
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 2) and VEGF (seq. 3)
comprises the heavy chain amino acid sequence of SEQ ID NO. 224 and the light chain amino
acid sequence of SEQ ID NO. 225.
n an embodiment the DVD-binding protein that binds VEGF (seq. 2) and DLL4 (seq. 3)
comprises the heavy chain amino acid sequence of SEQ ID NO. 226 and the light chain amino
acid sequence of SEQ ID NO. 227.
n an embodiment, the DVD-binding protein tha binds DLL4 (seq 4) and VEGF (seq. 3)
comprises the heavy chain amino acid sequence of SEQ ID NO. 228 and the light chain amino
acid sequence of SEQ ID NO. 229.
In an embodiment, the DVD-binding protein that binds TNF (seq. 4) and PGE2 (seq. 3)
comprises the heavy chain amino acid sequence of SEQ ID NO. 230 and the light chain amino
acid sequence of SEQ ID NO. 23 .
In an embodiment, the DVD-binding protein that binds TNF (seq. 5) and PGE2 (seq. 4)
comprises the heavy chain amino acid sequence of SEQ ID NO. 232 and the light chain amino
acid sequence of SEQ ID NO. 233 .
n an embodiment, the DVD-bind ing protein that binds PGE2 (seq. 5) and TNF (seq. 1)
comprises the heavy chain amino acid sequence of SEQ ID NO. 234 and the light chain amino
acid sequence of SEQ ID NO. 23 .
In an embodiment, the DVD-binding protein that binds VEGF (seq. 4) and DLL4 (seq. 5)
comprises the heavy chain amino acid sequence of SEQ ID NO. 236 and the light chain amino
acid sequence of SEQ ID NO. 237.
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 6) and VEGF (seq. 5)
comprises the heavy chain amino acid sequence of SEQ ID NO. 238 and the light chain amino
acid sequence of SEQ ID NO. 239.
In an embodiment, the DVD-binding protein that binds VEGF (seq. 4) and DLL4 (seq. 7)
comprises the heavy chain amino acid sequence of SEQ ID NO. 240 and the light chain amino
acid sequence of SEQ ID NO. 24 1.
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 8) and VEGF (seq. 5)
comprises the heavy chain amino acid sequence of SEQ ID NO. 242 and the light chain amino
acid sequence of SEQ D NO. 243 .
In an embodiment, the DVD-binding protein that binds TNF (seq. 1) and PGE2 (seq. 6)
comprises the heavy chain amino acid sequence of SEQ ID NO. 244 and the light chain amino
acid sequence of SEQ ID NO. 245.
In an embodiment, the DVD-binding protein that binds PGE2 (seq. 4) and TNF (seq. 6)
comprises the heavy chain amino acid sequence of SEQ ID NO. 246 and the light chain amino
acid sequence of SEQ ID NO. 247.
In an embodiment, the DVD-binding protein that binds VEGF (seq. 5) and DLL4 (seq. 9)
comprises the heavy chain amino acid sequence of SEQ ID NO. 248 and the light chain amino
acid sequence of SEQ ID NO. 249.
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 5) and VEGF (seq. 6)
comprises the heavy chain amino acid sequence of SEQ ID NO. 250 and the light chain amino
acid sequence of SEQ ID NO. 251.
In an embodiment, the DVD-binding protein that binds VEGF (seq. 5) and DLL4 (seq.
10) comprises the heavy chain amino acid sequence of SEQ ID NO. 252 and the light chain
amino acid sequence of SEQ ID NO. 253 .
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 7) and VEGF (seq. 6)
comprises the heavy chain amino ac d sequence of SEQ D NO. 254 and the light chain amino
acid sequence of SEQ ID NO. 255 .
In an embodiment, the DVD-binding protein that binds TNF (seq. 6) and PGE2 (seq. 4)
comprises the heavy chain amino acid sequence of SEQ JD NO. 256 and the light chain amino
acid sequence of SEQ ID NO. 257.
In an embodiment, the DVD-binding protein that binds PGE2 (seq. 6) and TNF (seq 1)
comprises the heavy chain amino acid sequence of SEQ ID NO. 258 and the light chain amino
acid sequence of SEQ ID NO. 259.
In an embodiment, the DVD-binding protein that binds VEGF (seq. 6) and DL 4 (seq. 5)
comprises the heavy chain a i o acid sequence of SEQ NO. 260 and the light chain amino
acid sequence of SEQ ID NO. 26 .
!4
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 9) and VEGF (seq. 5)
comprises the heavy chain amino acid sequence of SEQ ID NO. 262 and the light chain amino
acid sequence of SEQ ID NO. 263
In an embodiment, the DVD-binding protein that binds VEGF (seq. 6) and DLL4 (seq. 7)
comprises the heavy chain amino acid sequence of SEQ ID NO. 264 and the light chain amino
acid sequence of SEQ ID NO. 265 .
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 10) and VEGF (seq.
5) comprises the heavy chain amino acid sequence of SEQ ID NO. 266 and the light chain amino
acid sequence of SEQ ID NO. 267.
In an embodiment, the DVD-binding protein that binds VEGF (seq. 1) and DLL4 (seq.
11) comprises the heavy chain amino acid sequence of SEQ ID NO. 268 and the light chain
amino acid sequence of SEQ ID NO. 269.
In an embodiment, the DVD-binding protein that binds VEGF (seq. 1) and DLL4 (seq.
2) comprises the heavy chain amino acid sequence of SEQ ID NO. 270 and the light chain
amino acid sequence of SEQ ID NO. 27 1.
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 13) and VEGF (seq.
7) comprises the heavy chain amino acid sequence of SEQ ID NO. 272 and the light chain amino
acid sequence of SEQ ID NO. 273 .
In an embodiment, the DVD-binding protein that binds PGE2 and TNF comprises the
heavy chain amino acid sequence of SEQ ID NO. 304 and the light chain amino acid sequence of
SEQ ID NO. 305.
In an embodiment, the DVD-binding protein that binds VEGF and DLL4 (seq. 1)
comprises the heavy chain amino acid sequence of SEQ D NO. 306 and the light chain amino
acid sequence of SEQ ID NO. 307.
In an embodiment, the DVD-binding protein that binds DLL4 and VEGF (seq. 1)
comprises the heavy chain amino acid sequence of SEQ ID NO. 308 and the light chain amino
acid sequence of SEQ ID NO. 309
In a embod iment, the DVD-binding protein that binds VEGF and DLL-4 (seq. 2)
comprises the heavy chain amino acid sequence of SEQ ID NO. 3 and the ligh chain amino
acid sequence of SEQ D NO. .
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 2) and VEGF (seq. 1)
comprises the heavy chain amino acid sequence of SEQ ID NO. 312 and the light chain amino
acid sequence of SEQ ID NO. 3 13.
In an embodiment, the DVD-binding protein that binds TNF and PGE2 comprises the
heavy chain amino acid sequence of SEQ ID NO. 314 and the light chain amino acid sequence of
SEQ D NO. 3 15.
In an embodiment the DVD-binding protein that binds PGE2 and TNF comprises the
heavy chain amino acid sequence of SEQ ID NO. 316 and the light chain amino acid sequence of
SEQ ID NO. 317.
In an embodiment, the DVD-binding protein that binds DLL4 (seq. 1) and VEGF (seq. 7)
comprises the heavy chain amino acid sequence of SEQ D NO. 318 and the light chain amino
acid sequence of SEQ ID NO. 319.
In another embodiment, a DVD-binding protein comprising a polypeptide chain, wherein
said polypeptide chain comprises VDl -( 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 is provided. In an embodiment, the Fc region is absent from the
DVD-binding protein.
In another embodiment, a DVD-binding protein comprising a polypeptide chain, wherein
said polypeptide chain comprises 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, which can be the same or different from the first parent antibody; C is a light
chain constant domain; (X )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 is provided. In an embodiment (X2)n is absent from the DVD-binding protein.
In another embodiment the DVD-binding protein comprises first and second polypeptide
chains, wherein said first polypeptide chain comprises a first VDl -(X l )n-VD2-C-(X2)n, wherein
VD is a first heavy chain variable domain obtained from a first parent antibody or antigen
binding portion thereof; VD2 is a second heavy chain variable domain obtained from a second
parent antibody or antigen binding portion thereof, which can be the same or different from the
first parent antibody; C is a heavy chain constant domain; ( l )n is a first linker^ 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 -(X )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, which can be the same or different
from the first parent antibody; C is a light chain constant domain; (XI )n is a second linker,
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 one embodiment the first and second XI are the
same. In another embodiment, the first and second XI are different. In an embodiment, the first
XI does not comprise a CHI domain. In another embodiment, the second XI does not comprise a
CL domain.
In another embodiment, the DVD-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. In another embodiment, the Fc region if present in the first
polypeptide is a variant sequence Fc region. In still another embodiment, the Fc region is from an
IgG l , IgG2, IgG3, IgG4, IgA, IgM, IgE, or an IgD.
In another embodiment the DVD-binding protein binds two antigens comprising four
polypeptide chains, wherein, first and third polypeptide chains comprise VDl -(X l )n-VD2-C-
(X2)n, wherein.VD l is a first heavy chain variable domain obtained from a first parent antibody
or antigen binding portion thereof; VD2 is a second heavy chain variable domain obtained from a
second parent antibody or antigen binding portion thereof, which can be the same or different
from the first parent antibody; C is a heavy chain constant domain; ( )n is a first linker, wherein
said (X )n is either present or absent; and (X2)n is an Fc region, wherein said (X2)n is either
present or absent; and wherein each of the second and fourth polypeptide chains comprise VD1-
(Xl )n~VD2-C-(X2)n, wherein VD1 is a first light chain variable domain obtained from a first
parent antibody or antigen binding portion thereof; VD2 is a second light chain variable domain
obtained from a second parent antibody or antigen binding portion thereof, which can be the
same or different from the first parent antibody; C is a light chain constant domain; (X )n is a
second linker, wherein said (X )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 some embodiments the first and second
XI linkers are the same. In other embodiments, the first and second XI linkers are different. In
one embodiment, the first XI linker is not a CH domain. In one embodiment, the second XI
linker is not a CL domain.
A method of making a DVD-Ig binding protein by preselecting the parent antibodies is
provided. In an embodiment, the method of making a Dual Variable Domain Immunoglobulin
that binds two antigens comprising the steps of a) obtaining a first parent antibody or antigen
binding portion thereof, that binds a first antigen; b) obtaining a second parent antibody or
antigen binding portion thereof, that binds a second antigen; c) constructing first and third
polypeptide chains, each of which comprises VDl -(X )n-VD2-C-(X2)n, wherein, VD1 is a first
heavy chain variable domain obtained from said first parent antibody or antigen binding portion
thereof; VD2 is a second heavy chain variable domain obtained from said second parent antibody
or antigen binding portion thereof, which can be the same or different from the first parent
antibody; C is a heavy chain constant domain; (Xl )n is a first linker, 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, each of which comprises VD -(X l )n-VD2-C-
(X2)n, wherein, VD1 is a first light chain variable domain obtained from said first parent
antibody or antigen binding portion thereof; VD2 is a second light chain variable domain
obtained from said second parent antibody or antigen binding thereof, which can be the same or
different from the first parent antibody; C is a light chain constant domain; (Xl )n is a second
linker, wherein said (XI)n is either present or absent; and (X2)n does not comprise an Fc region,
wherein said (X2)n is either present or absent: and e) expressing said first, second, third and
fourth polypeptide chains; such that a DVD-Ig molecule that binds said first and said second
antigen is generated. In some embodiments the first and second X linkers are the same. In other
embodiments, the first and second XI linkers are different. In one embodiment, the first XI
linker is not a CH domain. In one embodiment, the second XI linker is not a CL domain.
In still another embodiment, a method of generating a DVD-binding protein molecule
that binds two antigens with desired properties comprising the steps of a) obtaining a first parent
antibody or antigen binding portion thereof, that binds a first antigen and possessing at least one
desired property exhibited by the DVD-Ig molecule; b) obtaining a second parent antibody or
antigen binding portion thereof, tha binds a second antigen and possessing at least one desired
property exhibited by the DVD-Ig molecule; c) constructing first a d third polypeptide chains
comprising VD1 (Xl )n-VD2-C-(X2)n, wherein; VD1 is a first heavy chain variable domain
obtained from said first parent antibody or antigen binding portion thereof; VD2 is a second
heavy chain variable domain obtained from said second parent antibody or antigen binding
portion thereof, which can be the same or different from the first parent antibody; C is a heavy
B
chain constant domain; (Xl )n is a first linker, 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 )n-VD2- C-(X2)n, wherein; VD1 is a first light
chain variable d m obtained from said first parent antibody or antigen binding portion thereof;
VD2 is a second light chain variable domain obtained from said second parent antibody or
antigen binding portion thereof, which can be the same or different from the first parent antibody;
C is a light chain constant domain; (XI )n is a second linker, 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; e) expressing said first, second, third and fourth polypeptide chains; such that a Dual
Variable Domain bindig protein that binds said first and said second antigen with desired
properties is generated is provided. In some embodiments the first and second XI linkers are the
same. In other embodiments, the first and second XI linkers are different. In one embodiment, the
first XI linker is not a CHI domain. n one embodiment, the second XI linker is not a CL
domain.
In one embodiment, the VDI of the first and second polypeptide chains disclosed herein
are obtained from the same parent antibody or antigen binding portion thereof. In another
embodiment, the VDI of the first and second polypeptide chains disclosed herein are obtained
from different parent antibodies or antigen binding portions thereof. In another embodiment, the
VD2 of the first and second polypeptide chains disclosed herein are obtained from the same
parent antibody or antigen binding portion thereof. In another embodiment, the VD2 of the first
and second polypeptide chains disclosed herein are obtained from different parent antibodies or
antigen binding portions thereof.
In one embodiment the first parent antibody or antigen binding portion thereof, and the
second parent antibody or antigen binding portion thereof, are the same antibody. In another
embodiment the first parent antibody or antigen binding portion thereof, and the second parent
antibody or antigen binding portion thereof, are different antibodies.
In one embodiment the first parent antibody or antigen binding portion thereof, binds a
first antigen and the second parent antibody or antigen binding portion thereof, binds a second
antigen. In a particular embodiment, the first and second antigens are the same antigen. In another
embodiment, the parent antibodies bind different epitopes on the same antigen. In another
embodiment the first and second antigens are different antigens. In another embodiment, the first
parent antibody or antigen binding portion thereof, binds the first antigen with a potency different
from the potency with which the second parent antibody or antigen binding portion thereof, binds
the second antigen. In yet another embodiment, the first parent antibody or antigen binding
portion thereof, binds the first antigen with an affinity different from the affinity with which the
second parent antibody or antigen binding portion thereof, binds the second antigen.
In another embodiment the first parent antibody or antigen binding portion thereof, and
the second parent antibody or antigen binding portion thereof, are a human antibody, CDR
grafted antibody, or a 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 CHI 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 DVD-binding protein 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 DVD-binding protein is multivalent. In another embodiment, the DVD bindirsg
protein is multispecific. The multivalent and or multispecific DVD-binding proteins described
herein have desirable properties particularly from a therapeutic standpoint. For instance, the
multivalent and or multispecific DVD-binding protein ay ( 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; and/or (3) induce cell death and/or apoptosis of a cell
expressing an antigen to which the multivalent DVD-binding protein binds. The "parent
antibody" which provides at least one antigen binding specificity of the multivalent and or
multispecific DVD-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 deathinducing,
and/or apoptosis-inducing antibody, and the multivalent and or multispecific DVDbinding
protein as described herein may display improvement(s) in one or more of these
properties. Moreover, the parent antibody may lack any one or more of these properties, but may
be endowed with them when constructed as a multivalent DVD-binding protein as described
herein.
In another embodiment the DVD-binding protein has an on rate constant (Kon) to one or
more targets of: at least about 0 M_ 1s '; at least about 10 M 's '; at least about 10 M 's '; at least
about s 1 or at least about 10 M ' s ', as measured by surface plasmon resonance. In an
embodiment, the DVD-binding protein has an on rate constant (Kon) to one or more targets
between about 10 M 's ' and about 10 M V;between about 10 M ' s and about 10 M S 1 ;
between about M s 1 and about 10 M V;or between about 1and about 10 M ', as
measured by surface plasmon resonance.
In another embodiment the DVD-binding protein has an off rate constant (Koff) for one
or more targets of: at most about 10V;at most about 10V;at most about 10V;or at most
about 10V,as measured by surface plasmon resonance. In an embodiment, the DVD-binding
protein has an off rate constant (Koff) to one or more targets of about 10 s_ to about 10V1;of
about 0Vto about 10V;or of about 10 s_ to about 10V,as measured by surface plasmon
resonance.
In another embodiment the DVD-binding protein has a dissociation constant (K ) to one
or more targets of: at most about 0 7 M; at most about 10 8 M; at most about 10 9 M; at most
about 10 10 M; at most about 10 " M; at most about 10 '2 M; or at most about 10 M. In an
embodiment, the DVD-binding protein has a dissociation constant (KD) to its targets of from
about 10 M to about 10 8 M; of from about 10 8 M to about 1 9 M; of from about 1 9 M to
about 10 '°M; of from about 10 10 to about 10 " M; of from about 0 M to about 10 12 M; or of
from about 10 12 to about M 0 M.
In another embodiment, the DVD-binding proteins described herein are conj ugates
further comprising an agent. In certain embodiments, 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 C S Y, Tc, " 'in,
I, 131 I, L i, Ho, or i 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 DVD-binding protein described herein binds to a cellular
protein and an agent. In certain embodiments, the cellular protein and 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
2 Ή , i C, S, Y, Tc, 1'In, l 1 L , 1 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 anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, or an
apoptotic agent.
In another embodiment, the DVD-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 DVDbinding
protein has a greater half life in vivo than the soluble counterpart of said DVD-binding
protein. In still another embodiment, the crystallized DVD-binding protein retains biological
activity.
In another embodiment, the DVD-binding proteins described herein are glycosylated. For
example, the glycosylation is a human glycosylation pattern.
An isolated nucleic acid encoding any one of the DVD-binding proteins disclosed herein
is provided. A further embodiment provides a vector comprising the isolated nucleic acid
disclosed herein. In certain embodiments, the vector is pcDNA; pTT (Durocher t a . (2002)
Nucl. Acids Res.30:2; pTT3 (pTT with additional multiple cloning site; pEFBOS (Mizushima
and Nagata, (1990) Nucl. Acids Res. 18: 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; NS0, SP2, PER.C6 or a fungal cell
such as Saccharomyces cerevisiae; or an insect cell such as Sf9.
In an embodiment, two or ore DVD-binding proteins, 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 7,429/1 86.
A method of producing a DVD-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 DVD-binding protein is provided. In an embodiment, 50%-75% of the binding
protein produced by this method is a dual specific tetravalent binding protein. In a particu lar
embodiment, 75%-90% of the binding protein produced by this method is a dual specific
tetravalent binding protein. In a particular embodiment, 90¾~95% of the binding protein
produced is a dual specific tetravalent binding protein.
One embodiment provides a composition for the release of a DVD-binding protein
wherein the composition comprises a formulation that in turn comprises a crystallized DVDbinding
protein, as disclosed herein, and an ingredient, and at least one polymeric carrier. For
example, in certain embodiments, the polymeric carrier comprises one or more of: poly (acrylic
acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly
(esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly
(caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide,
poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone),
maleic anhydride- alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and
cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides,
glycaminoglycans, sulfated polysaccharides, or blends and copolymers thereof. For example, in
certain embodiments, the ingredient is albumin, sucrose, trehalose, lactitol, gelatin,
hydroxypropyl- b- cyciodextrin, methoxypoiyethylene 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.
A pharmaceutical composition comprising a DVD-binding protein, as disclosed herein,
and a pharmaceutically acceptable carrier is provided. In a further embodiment the
pharmaceutical composition comprises at least one additional therapeutic agent for treating a
disorder. For example, in certain embodiments, 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 VEGF-trap). a kinase inhibitor (including but not limited to a KDR and a
TIE-2 inhibitor), a co-stimulation molecule blocker (including but not limited to anti-B7. 1, anti-
B7.2, CTLA4-Ig, anti-CD20), an adhesion molecule blocker (including but not limited to an anti-
LFA-1 antibody, an anti-E/L selectin antibody, a small molecule inhibitor), an anti-cytokine
antibody or functional fragment thereof (including but not limited to an anti-IL- 18, an anti-TNF,
and an anti-IL-6/cytokine receptor antibody), methotrexate, cyclosporin, rapamycin, FK506, a
detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a
non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local
anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an
anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive,
a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an
antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an
epinephrine or analog, a cytokine, or a cytokine antagonist.
A method for treating a human subject suffering from a disorder in which the target, or
targets, capable of being bound by the DVD-bind ing protein disclosed herein is detrimental,
comprising administering to the human subject a DVD-binding protein disclosed herein such thai
the activity of the target, or targets in the human subject is inhibited and one of more symptoms is
alleviated or treatment is achieved is provided. For example, in certain embodiments, the disorder
is arthritis, osteoarthritis, j uvenile 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 chron ic immune disease associated with organ transplantation,
sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's
disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-
Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis,
septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic
diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea,
Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia,
malignancies, heart failure, myocardial infarction, Addison's disease, sporadic polyglandular
deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute)
respiratory distress syndrome, alopecia, alopecia areata, seronegative arthopathy, arthropathy,
Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis,
chlamydia, yersinia and salmonella associated arthropathy, spondyloarthopathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris,
pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs
positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic
encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease
Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common
varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy,
female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic
fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis,
connective tissue disease associated interstitial lung disease, mixed connective tissue disease
associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid
arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung
disease, dermatomyositis/polymyositis associated lung disease, Sjogren's disease associated lung
disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease,
haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation
fibrosis, bronchiolitis obliterans, chronic eosinophil ic pneumonia, lymphocytic infiltrative u g
disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1
autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis
(anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance
with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ
transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis,
primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the
kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension
secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic
sclerosis, Sjorgren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia,
idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous
autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism,
primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic
liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis, idiosyncratic liver
disease, Drug-Induced hepatitis.

We claim:
1. A binding protein that binds a pair of antigens, 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;
is a linker with the proviso that it is not CHI;
X2 is an Fc region;
(Xl)n is (Xl)0 or (Xl)l; and
(X2)n is (X2)0 or (X2)l
wherein the pair of antigens is TNF and PGE2 or VEGF and DLL4, and
wherein the VDl and VD2 independently comprise three CDRs from SEQ D NO: 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,
90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 16, 118, 120, 122, 124, 126, 128, 130,
132, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, or 302.
2. The binding protein according to claim 1, wherein VDl and VD2 independently comprise SEQ
ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,
78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 14, 116, 118, 120,
122, 124, 126, 128, 130, 132. 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, or 302.
3. A binding protein that binds a pair of antigens, 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 CL;
X2 does not comprise an Fc region;
(Xl)n is (X1)0 or (Xl)l; and
(X2)n is (X2)0 or (X2)l
wherein the pair of antigens is TNF and PGE2 or VEGF and DLL4, and
wherein the VDl and VD2 independently comprise three CDRs from SEQ ID NO: 31, 33, 35, 37,
39 41, 43, 45, 47, 49, 5 1 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 87, 89,
91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,
133, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, or 303.
4. The binding protein according to claim 3, wherein the VDl and VD2 independently comprise
SEQ ID NO: , 33, 35, 37, 39, 41, 43, 45, 47, 49, 5 ί 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,
75, 77, 79, 8 , 83, 85 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 13, 15, 117, 119,
121, 123, 125, 127, 129, 131, 133, 281, 283, 285, 287, 289, 291 , 293, 295, 297, 299, 301, or 303.
5. The binding protein according to claim 1 or 3, wherein (Xl)n is (X1)0 and/or (X2)n is (X2)0.
6. A binding protein that binds a pair of antigens, 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;
X2 is an Fc region:
(Xl)n is (XI )0 or (Xl)l; and
(X2)n is (X2)0 or (X2)l
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 second linker;
X2 does not comprise an Fc region;
(X )n is (Xl )0 or (Xl ) l ; and
(X2)n is (X2)0 or (X2)l ;
wherein the first and second I linker are the same or different;
wherein the first XI linker is not C I and/or the second X linker is not CL;
wherein the pair of antigens is TNF and PGE2 or VEGF and DLL4, and
wherein the heavy chainVD l and VD2 independently comprise three CDRs from SEQ ID
NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
74, 76, 78, 80, 2, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 280, 282, 284, 286, 288, 290, 292, 294, 296,
298, 300, or 302; and the light chain VD1 and VD2 independently comprise SEQ ID NO:
31, 33, 35, 37, 39, 4 1, 43, 45, 47, 49, 51 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
79, 81, 83, 85 87, 89, 9 1, 93, 95, 97, 99, 101, 103, 105, 107, 109, 1 1, 13, 1 5, 17, 119,
12 1, 123, 125, 127, 129, 1 , 133, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301 ,
or 303 .
7. The binding protein according to claim 6, wherein the VD1 and VD2 heavy chain variable
domains independently comprise SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,
106, 108, 10, 1 2, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 280, 282, 284, 286, 288,
290, 292, 294, 296, 298, 300, or 302; and the VD1 and VD2 light chain variable domains
independently comprise SEQ ID NO: 31, 33, 35, 37, 39, 4 1, 43, 45, 47, 49, 5 53, 55, 57, 59, 61,
63, 65, 67, 69, 7 1, 73, 75, 77, 79, 81, 83, 85 87, 89, 9 1, 93, 95, 97, 99, 10 1, 103, 105, 107, 109,
l , 113, 5, 117, 9, 12 1, 123, 125, 127, 129, 131, 133, 281, 283, 285, 287, 289, 29 1, 293,
295, 297, 299, 301, or 303 .
8. The binding protein according to claim 1, 3, or 6, wherein XI and/or X2 is at least one of SEQ
ID Os 1-28.
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, 3, or 6 wherein the region is a variant sequence
Fc region.
11. The binding protein according to claim 1, 3, or 6, wherein the Fc region is from an IgG l ,
IgG2, igG3, !g gA gM, IgE, or IgD.
1 . The binding protein according to claim 6, wherein said VDl of the first polypeptide chain and
said VDl of the second polypeptide chain are obtained from a same first and second parent
antibody, respectively, or antigen binding portion thereof.
13. The binding protein according to claim 6, wherein said VDl of the first polypeptide chain and
said VD of the second polypeptide chain are obtained from a different first and second parent
antibody, respectively, or antigen binding portion thereof.
4. The binding protein according to claim 6, wherein said VD2 of the first polypeptide chain and
said VD2 of the second polypeptide chain are obtained from a same first and second parent
antibody, respectively, or antigen binding portion thereof.
15. The binding protein according to claim 6, wherein said VD2 of the first polypeptide chain and
said VD2 of the second polypeptide chain are obtained from different first and second parent
antibody, respectively, or antigen binding portion thereof.
16. The binding protein according to claim 13 or 15, wherein said first and said second parent
antibodies bind different epitopes on said antigen.
17. The binding protein according to claim 13 or 15, wherein said first parent antibody or antigen
binding portion thereof, binds said first antigen with a potency different from the potency with
w ch said second parent antibody or antigen binding portion thereof, binds said second antigen.
18. The binding protein according to claim 13 or 15, wherein said first parent antibody or antigen
binding portion thereof, binds said first antigen with an affinity different from the affinity with
which said second parent antibody or antigen binding portion thereof, binds said second antigen.
. A binding protein that binds two antigens comprising four polypeptide chains, wherein two
polypeptide chains comprise VDl -( l )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;
I is a first linker;
X2 is an Fc region;
(Xl )n is (Xl ) l or (Xl )l ; and
(X2)n is (X2) l or (X2)l ;
wherein two polypeptide chains comprise VDl -(X l )n-VD2-C-(X2)n, wherein
VD1 is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a second linker;
X2 does not comprise an Fc region;
(X )n is (X1 )0 or (Xl ) l ; and
(X2)n is (X2)0 or (X2)l ;
wherein the first and second XI linker are the same or different;
wherein the first XI linker is not CHI and/or the second XI linker is not CL;
wherein the pair of antigens is TNF and PGE2 or VEGF and DLL4, and
wherein the heavy chainVD1 and VD2 independently comprise three CDRs from SEQ D
NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 1 4,
16, 8, 120, 122, 124, 26, 128, 130, 132, 280, 282, 284, 286, 288, 290, 292, 294, 296,
298, 300, or 302; and the light chain VD1 and VD2 independently comprise SEQ ID NO:
31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
79, 81, 83, 85 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 1 , 113, 15, 117, 1 9,
12 1, 123, 125, 127, 129, 131, 133, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301,
or 303 .
20. The binding protein of claim 19, wherein the VD1 and VD2 heavy chain variable domains
independently comprise SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,
62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 00, 102, 104, 106, 108,
10, 112, 4, 116, 118, 120, 122, 124, 126, 128, 130, 132, 280, 282, 284, 286, 288, 290, 292,
294, 296, 298, 300, or 302; and the VD1 and VD2 light chain variable domains independently
comprise SEQ ID NO: 31, 33, 35, 37, 39, 4 1, 43, 45, 47, 49, 51 53, 55, 57, 59, 6 1, 63, 65, 67, 69,
71, 73, 75, 77, 79, 81, 83, 85 87, 89, 9 1, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,
117, 119, 12 1, 123, 125, 127, 129, 131, 133, 281, 283, 285 , 287, 289, 29 1, 293, 295, 297, 299,
301 , or 303 .
2 1. The binding protein according to claim 1, 3, 6, or 19, wherein said binding protein has an on
rate constant (Kon) to said one or more targets of: at least about lO at least about
O - s at least about 104M V;at least about O'M or at least about 10 M 's , as
measured by surface plasmon resonance.
22. The binding protein according to claim 1, 3, 6, or 19, wherein said binding protein has an off
rate constant (Koff) to said one or more targets of: at most about 10V;at most about 10V;at
most about 10V ;or at most about 1 V, as measured by surface plasmon resonance.
23. The binding protein according to claim 1, 3, 6, or 19, wherein said binding protein has a
dissociation constant (KD) to said one or more targets of: at most about 10 7 M; at most about
10 8 M; at most about 10 M; at most about 10 i M; at most about 0 " M; at most about 0 12 M;
or at most 1 M.
24. A binding protein conjugate comprising a binding protein according to any one of claims l , 3,
6, or 19, said binding protein conjugate further comprising an immunoadhesion molecule, an
imaging agent, a therapeutic agent, or a cytotoxic agent.
25. The binding protein according to claim 3, 3, 6, or 19, wherein said binding protein is a
crystallized binding protein.
26. The binding protein according to claim 25, wherein said crystal is a carrier-free
pharmaceutical controlled release crystal.
27. The binding protein according to claim 25, wherein said binding protein has a greater half life
in vivo than the soluble counterpart of said binding protein.
28. An isolated nucleic acid encoding a binding protein amino acid sequence according to any
one of claims 1, 3, 6, or 19.
29. A vector comprising an isolated nucleic acid according to claim 28.
30. The vector according to claim 29, wherein said vector is pcDNA, pTT, pTT3, pEFBOS, pBV,
pjV, pcDNA3.1 TOPO, pEF6 TOPO, pBJ, or pHybE.
3 1. A host cell comprising a vector according to claim 30.
32. The host cell according to claim 3 , wherein said host cell is a prokaryotic cell.
33. The host cell according to claim 32, wherein said host cell is E.Coli.
34. The host cell according to claim 3 1, wherein said host cell is a eukaryotic cell.
35. The host cell according to claim 34, wherein said eukaryotic cell is a protist cell, animal cell,
plant cell, or fungal cell.
36. The host cell according to claim 35, wherein said animal cell is a mammalian cell, an avian
cell, or an insect cell.
37. The host cell according to claim 36, wherein said animal cell is a CHO cell.
38. The host cell according to claim 36, wherein said animal cell is COS.
39. The host cell according to claim 35, wherein said fungal ce is a yeast cell.
40. The host cell according to claim 39, wherein said yeast cell is Saccharomyces cerevisiae.
4 . The host cell according to claim 36, wherein said insect cell is an Sf9 cell.
42. A method of producing a binding protein, comprising culturing a host cell described in any
one of claims 3 -4 in culture medium under conditions sufficient to produce the binding protein
43. The method according to claim 42, wherein 50%-75% of the binding protein produced is a
dual specific tetrava!eint binding protein
44. The method according to claim 42, wherein 75%-90% of the binding protein produced is a
dual specific tetravalent binding protein.
45. The method according to claim 42, wherein 90%-95% of the binding protein produced is a
dual specific tetravalent binding protein.
46. A protein produced according to the method of claim 42.
47. A pharmaceutical composition comprising the binding protein of claim 1, 3, 6, or 1 , and a
pharmaceutically acceptable carrier.
48. The pharmaceutical composition of claim 47 further comprising at least one additional
therapeutic agent.
49. The pharmaceutical composition of claim 48, 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.
50. A method for treating a subject for a disease or a disorder by administering to the subject the
binding protein of claim 1, 3, 6, or such that treatment is achieved.
51. The method of claim 50, wherein said disorder is rheumatoid arthritis, osteoarthritis, juvenile
chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,
spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis,
inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic
diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant rejection,
acute or chronic immune disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, 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 hypogammaglobu ίinaemia),
dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibroiic
lung disease, cryptogenic fibrosing alveolitis, postinflammatory interstitial lung disease,
interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed
connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung
disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus
associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjogren's disease
associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung
disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis,
radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic
infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune
hepatitis, type- 1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2
autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated 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 thrombocytopaen ia, autoimmune thyroid disease,
hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atroph ic
autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis,
vitil igo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver
injury, cholestasis, idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholic
Steatobepatitis, allergy and asthma, group B streptococci (GBS) infection, mental disorders such
as depression and schizophrenia, Th2 Type and Th l Type mediated diseases, acute and chronic
pain, and cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and
rectal cancer and hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia,
Acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute
lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial
infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS
dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis,
allergic rhinitis, allograft rejection, alpha- 1-antitrypsin deficiency, amyotrophic lateral sclerosis,
anemia, angina pectoris, anterior horn cell degeneration, anti-cd3 therapy, antiphospholipid
syndrome, anti-receptor hypersensitivity reactions, aortic and peripheral aneuryisms, aortic
dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation
(sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft
rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma,
burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy,
cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical
degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapyassociated
disorders, chronic myelocytic leukemia (CML), chronic alcoholism, chronic
inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary
disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure,
conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob
disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders, Dementia
pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic
conditions, diabetes, diabetes mellitus, diabetic aterosclerotic disease, Diffuse Lewy body
disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's Syndrome in
middle age, drug- induced movement disorders induced by drugs which block CNS dopamine
receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis,
epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial
hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia,
functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, graft rejection
of any organ or tissue, gram negative sepsis, gra positive sepsis, granulomas due to intracellular
organisms, hairy cell leukemia, Hallervorden-Spatz disease, hashimoto's thyroiditis, hay fever,
heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic
syndrome/thromboiytic thrombocytopenic purpura hemorrhage, hepatitis A, His bundle
arryhthmias, HIV infection/HIV neu ropathy, Hodgkin's disease, hyperkinetic movement
disorders, hypersensitivity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic
movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's
disease, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, Asthenia, infantile spinal
muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation exposure,
iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile
rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant
rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver
transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis,
malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, migraine headache,
mitochondrial multisystem disorder, mixed connective tissue disease, monoclonal gammopathy,
multiple myeloma, multiple systems degenerations (Mencel Dejerine-Thomas Shy-Drager and
Machado-Joseph), myasthenia gravis, mycobacterium avium intracellulare, mycobacterium
tuberculosis, myelodyplastic syndrome, myocardial ischemic disorders, nasopharyngeal
carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases,
neurogenic I muscular atrophies , neutropenic fever, non-Hodgkin's lymphoma, occlusion of the
abdominal aorta and its branches, occulsive arterial disorders, okt3 therapy, orchitis/epidydimitis,
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 arteriosclerotic disease, peripheral vascular disorders, peritonitis, pernicious
anemia, Pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy,
organomegaly endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post
perfusion syndrome, post pump syndrome, post-MI cardiotoray syndrome, preeclampsia,
Progressive supranuclear Palsy, primary pulmonary hypertension, radiation therapy Raynaud's
phenomenon and disease, Raynoud's disease, Refsurn'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 fibril lation, viral and fungal infections, viral encephal itis/aseptic
meningitis, viral-associated hemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's
disease, xenograft rejection of any organ or tissue, acute coronary syndromes, acute idiopathic
polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult
Still's disease, anaphylaxis, anti-phospholipid antibody syndrome, aplastic anemia, atopic
eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disorder associated with
streptococcus infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune
lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian
failure, 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, sneddonwilkinson
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 ! allergic reaction,
type II diabetes, usual interstitial pneumonia (UIP), vernal conjunctivitis, viral retinitis, Vogt-
Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration, or wound heal ing,.
52. The method according to claim 50, wherein said administering to the subject is parenteral,
subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular, intracartilaginous, intracavitary, intracelial, intracerebeilar, 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.
53 . A method for generating the binding protein of claim 19, comprising
a) obtaining a first parent antibody or antigen binding portion thereof, that binds a first
antigen;
b) obtaining a second parent antibody or antigen binding portion thereof, that binds a
second antigen;
c) constructing first and third polypeptide chains comprising VDl-(X l )n-VD2-C-(X2)n,
wherein
VD1 is a first heavy chain variable domain obtained from said first parent
antibody or antigen binding portion thereof;
VD2 is a second heavy chain variable domain obtained from said second parent
antibody or antigen binding portion thereof;
C is a heavy chain constant domain;
XI is a first linker;
X2 is an F region:
(Xl ) is (Xl )0 or (Xl ) l ; and
(X2)n is (X2)0 or (X2)
d) constructing second and fourth polypeptide chains comprising VDl -(X l)n-VD2-C-
(X2)n, wherein
VD1 is a first light chain variable domain obtained from said first parent
antibody or antigen binding portion thereof;
VD2 is a second light chain variable domain obtained from said second parent
antibody or antigen binding thereof;
C is a light chain constant domain;
XI is a second linker;
X2 does not comprise an Fc region;
(Xl )n is (X1)0 or (Xl ) l ; and
(X2)0 is (X2)0 or (X2)l ; and
e) expressing said first, second, third and fourth polypeptide chains such that a binding
protein that binds said first and said second antigen is generated,
wherein the first and second XI linker are the same or different;
wherein the first XI linker is not CHI and/or the second XI linker is not CL;
wherein the pair of antigens is TNF and PGE2 or VEGF and DLL4, and
wherein the heavy chainVDl and VD2 independently comprise three CDRs from SEQ ID
NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 02, 104, 106, 108, 110, 112, 114,
1 6, 18, 120, 122, 124, 126, 28, 130, 32, 280, 282, 284, 286, 288, 290, 292, 294, 296,
298, 300, or 302; and the light chain VD1 and VD2 independently comprise SEQ ID NO:
31, 33, 35, 37, 39, 4 1, 43, 45, 47, 49, 51 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
79, 81, 83, 85 87, 89, 9 1, 93, 95. 97, 99, 101, 103, 105, 107, 109, 11, 113, 115, 7, 119,
2 1, 123, 125, 127, 129, 13 1, 133, 281, 283, 285, 287, 289, 29 1, 293, 295, 297, 299, 301,
or 303 .
54. The method of claim 53, wherein the VD1 and VD2 heavy chain variable domains
independently comprise S Q ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,
62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 08,
110, 112, 4, 6, 118, 120, 122, 124, 126, 128, 130, 32, 280, 282, 284, 286, 288, 290, 292,
294, 296, 298. 300, or 302; and the VD1 and VD2 light chain variable domains independently
comprise SEQ ID NO: 3 1, 33, 35, 37, 39, 4 1, 43, 45, 47, 49, 51 53, 55, 57, 59, 61, 63, 65, 67, 69,
71, 73, 75, 77, 79, 81, 83, 85 87, 89, 9 1, 93, 95, 97, 99, 101, 103, 105, 107, 109, 1, 1 3, 5,
117, 9, 12 1, 123, 125, 127, 129, 13 1, 133, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,
301, or 303 .
55. The method of claim 53, wherein the Fc region is s a variant sequence Fc region.
56. The method of claim 53, wherein the Fc region is from an gGl , lgG2, gG3, IgG4, gA, IgM,
IgE, or IgD.
57. The method of claim 53, wherein said first parent antibody or antigen binding portion thereof,
binds said first antigen with a different affinity than the affinity with which said second parent
antibody or antigen binding portion thereof, binds said second antigen.
58. The method of claim 53, wherein said first parent antibody or antigen binding portion thereof,
binds said first antigen with a different potency than the potency with which said second parent
antibody or antigen binding portion thereof, binds said second antigen.
59. A method of determining the presence of at least one antigen or fragment thereof in a test
sample by an immunoassay,
wherein the immunoassay comprises contacting the test sample with at least one binding
protein and at least one detectable label,
wherein the at least one binding protein comprises the binding protein of claim 1, 3, 6, or
19 .
60. The method of claim 59 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding
protein binds to an epitope on the antigen or fragment thereof so as to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein the detectable
label binds to the binding protein or an epitope on the antigen or fragment thereof that is not
bound by the binding protein to form a second complex; and
(iii) detecting the presence of the antigen or fragment thereof in the test sample based on
the signal generated by the detectable label in the second complex, wherein the presence of the
antigen or fragment thereof is directly correlated with the signal generated by the detectable
label.
61. The method of claim 59 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding
protein binds to an epitope on the antigen or fragment thereof so as to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein the detectable
label competes with the antigen or fragment thereof for binding to the binding protein so as to
form a second complex; and
(iii) detecting the presence of the antigen or fragment thereof in the test sample based on
the signal generated by the detectable label in the second complex, wherein the presence of the
antigen or fragment thereof is indirectly correlated with the signal generated by the detectable
label.
62 . The method according to any one of claims 59-6 1, 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.
63 . The method according to any one of claims 59-62, wherein the method is adapted for use in
an automated system or a semi-automated system.
64. The method according to any one of claims 59-63, wherein the method determines the
presence of more than one antigen in the sample.
65. A method of determining the amount or concentration of an antigen or fragment thereof in a
test sample by an immunoassay,
wherein the immunoassay (a) employs at least one binding protein and at least one
detectable label and (b) comprises comparing a signal generated by the detectable label with a
control or calibrator comprising the antigen or fragment thereof,
wherein the calibrator is optionally part of a series of calibrators in which each calibrator
differs from the other calibrators in the series by the concentration of the antigen or fragment
thereof,
and wherein the at least one binding protein comprises the binding protein of claim 1, 3,
6. or 9.
66. The method of claim 65 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding
protein binds to an epitope on the antigen or fragment thereof so as to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein the detectable
!abe! binds to an epitope on the antigen or fragment thereof that is not bound by the binding
protein to form a second complex; and
(iii) determining the amount or concentration of the antigen or fragment thereof in the
test sample based on the signal generated by the detectable label in the second complex, wherein
the amount or concentration of the antigen or fragment thereof is directly proportional to the
signal generated by the detectable label.
67. The method of claim 65 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding
protein binds to an epitope on the antigen or fragment thereof so as to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein the detectable
label competes with the antigen or fragment thereof for binding to the binding protein so as to
form a second complex; and
(iii) determining the amount or concentration of the antigen or fragment thereof in the
test sample based on the signal generated by the detectable label in the second complex, wherein
the presence of the antigen or fragment thereof is indirectly proportional to the signal generated
by the detectable label.
68. The method according to any one of claims 65-67, 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.
69. The method according to any one of claims 65-68, wherein the method is adapted for use in
an automated system or a semi-automated system.
70. The method according to any one of claims 65-69, wherein the method determines the amount
or concentration of more than one antigen in the sample.
7 . A kit for assaying a test sample for the presence, amount, or concentration of an antigen or
fragment thereof, said kit comprising
(a) instructions for assaying the test sample for the antigen or fragment thereof; and
(b) at least one binding protein comprising the binding protein of claim 1, 3, 6, or 19.