DUAL VARIABLE DOMAIN IMMUNO GLOBULINS AND USES
THERE OF
Cross Reference to Related Applications
This application is a non-provisional application claiming priority to U.S. Provisional
Application Serial No. 61/363,120, filed July 9, 2010, the contents of which are hereby
incorporated by reference.
Field
Multivalent and multispecific binding proteins, methods of making, and specifically to
their uses in the, diagnosis, prevention and/or treatment of acute and chronic inflammatory
diseases, cancer, and other diseases are provided.
Background
Engineered proteins, such as multispecific antibodies capable of binding two or more
antigens are known in the art. Such multispecific binding proteins can be generated using cell
fusion, chemical conjugation, or recombinant DNA techniques.
Bispecific antibodies have been produced using quadroma technology (see Milstein, C.
and A.C. Cuello (1983) Nature 305(5934):537-40) based on the somatic fusion of two different
hybridoma cell lines expressing murine monoclonal antibodies (mAbs) with the desired
specificities of the bispecific antibody. Because of the random pairing of two different
immunoglobulin (Ig) heavy and light chains within the resulting hybrid-hybridoma (or
quadroma) cell line, up to ten different Ig species are generated, of which only one is the
functional bispecific antibody. The presence of mis-paired by-products, and significantly reduced
production yields, means sophisticated purification procedures are required.
Bispecific antibodies can also be produced by chemical conjugation of two different
mAbs (see Staerz, U.D., et al. (1985) Nature 314(6012): 628-31). This approach does not yield
homogeneous preparation. Other approaches have used chemical conjugation of two different
mAbs or smaller antibody fragments (see Brennan, M., et al. (1985) Science 229(4708): 81-3).
Another method used to produce bispecific antibodies is the coupling of two parental
antibodies with a hetero-bifunctional crosslinker, but the resulting bispecific antibodies suffer
from significant molecular heterogeneity because reaction of the crosslinker with the parental
antibodies is not site-directed. To obtain more homogeneous preparations of bispecific antibodies
two different Fab fragments have been chemically crosslinked at their hinge cysteine residues in
a site-directed manner (see Glennie, M.J., et al. (1987) J. Immunol. 139(7): 2367-75). But this
method results in Fab'2 fragments, not full IgG molecule.
A wide variety of other recombinant bispecific antibody formats have been developed
(see Kriangkum, J., et al. (2001) Biomol. Eng. 18(2): 31-40). Amongst them tandem single-chain
Fv molecules and diabodies, and various derivatives thereof, are the most widely used. Routinely,
construction of these molecules starts from two single-chain Fv (scFv) fragments that recognize
different antigens (see Economides, A.N., et al. (2003) Nat. Med. 9(1): 47-52). Tandem scFv
molecules (taFv) represent a straightforward format simply connecting the two scFv molecules
with an additional peptide linker. The two scFv fragments present in these tandem scFv
molecules form separate folding entities. Various linkers can be used to connect the two scFv
fragments and linkers with a length of up to 63 residues (see Nakanishi, K., et al. (2001) Ann.
Rev. Immunol. 19: 423-74). Although the parental scFv fragments can normally be expressed in
soluble form in bacteria, it is, however, often observed that tandem scFv molecules form
insoluble aggregates in bacteria. Hence, refolding protocols or the use of mammalian expression
systems are routinely applied to produce soluble tandem scFv molecules. In a recent study, in
vivo expression by transgenic rabbits and cattle of a tandem scFv directed against CD28 and a
melanoma-associated proteoglycan was reported (see Gracie, J.A., et al. (1999) J. Clin. Invest.
104(10): 1393-401). In this construct, the two scFv molecules were connected by a CHI linker
and serum concentrations of up to 100 mg L of the bispecific antibody were found. Various
strategies including variations of the domain order or using middle linkers with varying length or
flexibility were employed to allow soluble expression in bacteria. A few studies have now
reported expression of soluble tandem scFv molecules in bacteria (see Leung, B.P., et al. (2000)
J . Immunol. 164(12): 6495-502; Ito, A., et al. (2003) J. Immunol. 170(9): 4802-9; Kami, A., et al.
(2002) J. Neuroimmunol. 125(1-2): 134-40) using either a very short Ala3 linker or long
glycine/serine-rich linkers. In another recent study, phage display of a tandem scFv repertoire
containing randomized middle linkers with a length of 3 or 6 residues was employed to enrich for
those molecules that are produced in soluble and active form in bacteria. This approach resulted
in the isolation of a tandem scFv molecule with a 6 amino acid residue linker (see Arndt, M. and
J . Krauss (2003) Methods Mol. Biol. 207: 305-21). It is unclear whether this linker sequence
represents a general solution to the soluble expression of tandem scFv molecules. Nevertheless,
this study demonstrated that phage display of tandem scFv molecules in combination with
directed mutagenesis is a powerful tool to enrich for these molecules, which can be expressed in
bacteria in an active form.
Bispecific diabodies (Db) utilize the diabody format for expression. Diabodies are
produced from scFv fragments by reducing the length of the linker connecting the VH and VL
domain to approximately 5 residues (see Peipp, M. and T. Valerius (2002) Biochem. Soc. Trans.
30(4): 507-11). This reduction of linker size facilitates dimerization of two polypeptide chains by
crossover pairing of the VH and VL domains. Bispecific diabodies are produced by expressing,
two polypeptide chains with, either the structure VHA-VLB and VHB-VLA (VH-VL
configuration), or VLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. A
large variety of different bispecific diabodies have been produced in the past and most of them
are expressed in soluble form in bacteria. However, a recent comparative study demonstrates that
the orientation of the variable domains can influence expression and formation of active binding
sites (see Mack, M. et al.(1995) Proc. Natl. Acad. Sci. U S A 92(15): 7021-5). Nevertheless,
soluble expression in bacteria represents an important advantage over tandem scFv molecules.
However, since two different polypeptide chains are expressed within a single cell inactive
homodimers can be produced together with active heterodimers. This necessitates the
implementation of additional purification steps in order to obtain homogenous preparations of
bispecific diabodies. One approach to force the generation of bispecific diabodies is the
production of knob-into-hole diabodies (see Holliger, P., T. Prospero, and G. Winter (1993) Proc.
Natl. Acad. Sci. U S A 90(14): 6444-8.18). This approach was demonstrated for a bispecific
diabody directed against HER2 and CD3. A large knob was introduced in the VH domain by
exchanging Val37 with Phe and Leu45 with Tip 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. In addition, such approach
requires mutational modification of the immunoglobulin sequence at the constant region, thus
creating non-native and non-natural form of the antibody sequence, which may result in increased
immunogenicity, poor in vivo stability, as well as undesirable pharmacokinetics.
Single-chain diabodies (scDb) represent an alternative strategy for improving the
formation of bispecific diabody-like molecules (see Holliger, P. and G. Winter (1997) Cancer
Immunol. Immunother. 45(3-4): 128-30; Wu, A.M., et al. (1996) Immunotechnology 2(1): p. 21-
36). Bispecific single-chain diabodies are produced by connecting the two diabody-forming
polypeptide chains with an additional middle linker with a length of approximately 15 amino acid
residues. Consequently, all molecules with a molecular weight corresponding to monomeric
single-chain diabodies (50-60 kDa) are bispecific. Several studies have demonstrated that
bispecific single chain diabodies are expressed in bacteria in soluble and active form with the
majority of purified molecules present as monomers (see Holliger, P. and G. Winter (1997)
Cancer Immunol. Immunother. 45(3-4): 128-30; Wu, A.M., et al. (1996) Immunotechnol. 2(1):
21-36; Pluckthun, A. and P. Pack (1997) Immunotechnol. 3(2): 83-105; Ridgway, J.B., et al.
(1996) Protein Engin. 9(7): 617-21). Thus, single-chain diabodies combine the advantages of
tandem scFvs (all monomers are bispecific) and diabodies (soluble expression in bacteria).
More recently diabodies have been fused to Fc to generate more Ig-like molecules,
named di-diabodies (see Lu, D., et al. (2004) J . Biol. Chem. 279(4): 2856-65). In addition,
multivalent antibody construct comprising two Fab repeats in the heavy chain of an IgG and
capable of binding four antigen molecules has been described (see WO 0177342A1, and Miller,
K., et al. (2003) J . Immunol. 170(9): 4854-61).
There is a need in the art for improved multivalent binding proteins capable of binding
two or more antigens. U.S. Patent Application Serial No. 11/507,050 provides a novel family of
binding proteins capable of binding two or more antigens with high affinity, which are called
dual variable domain immunoglobulins (DVD-Ig™). Further, novel binding proteins capable of
binding two or more antigens are provided.
Summary
Multivalent binding proteins capable of binding two or more antigens are provided. A
novel family of binding proteins capable of binding two or more antigens with high affinity are
provided.
In one embodiment, a binding protein comprising a polypeptide chain, wherein the
polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein VDl is a first variable domain,
VD2 is a second variable domain, C is a constant domain, XI represents an amino acid or
polypeptide, X2 represents an Fc region and n is 0 or 1 is provided. In an embodiment the VDl
and VD2 in the binding protein are heavy chain variable domains. In another embodiment, the
heavy chain variable domain is a murine heavy chain variable domain, a human heavy chain
variable domain, a CDR grafted heavy chain variable domain, or a humanized heavy chain
variable domain. In yet another, embodiment VDl and VD2 are capable of binding the same
antigen. In another embodiment VDl and VD2 are capable of binding different antigens. In still
another embodiment, C is a heavy chain constant domain. For example, XI is a linker with the
proviso that XI is not CHI. For example, XI is a linker comprising AKTTPKLEEGEFSEAR
(SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3);
SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6);
RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G4S)4 (SEQ
ID NO: 9) SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 1);
ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO:
14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO:
17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP
(SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22),
GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24);
GPAKELTPLKEAKVS (SEQ ID NO: 25); or GHEAAAVMQVQYPAS (SEQ ID NO: 26). In an
embodiment, X2 is an Fc region. In another embodiment, X2 is a variant Fc region.
In an embodiment the binding protein disclosed herein comprises a polypeptide chain,
wherein the polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein VD1 is a first
heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain
constant domain, XI is a linker with the proviso that it is not CHI, and X2 is an Fc region.
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 are capable of binding the same
antigen. In another embodiment VD1 and VD2 are capable of binding different antigens. In an
embodiment, C is a light chain constant domain. In another embodiment, X is a linker with the
proviso that XI is not CL1 . In an embodiment, XI comprises 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: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO:
14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO:
17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP
(SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22),
GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24);
GPAKELTPLKEAKVS (SEQ ID NO: 25); or GHEAAAVMQVQYPAS (SEQ ID NO: 26). In an
embodiment, the binding protein does not comprise X2.
In an embodiment, both the variable heavy and variable light chain comprise the same
linker. In another embodiment, the variable heavy and variable light chain comprise different
linkers. In another embodiment, both the variable heavy and variable light chain comprise a short
(about 6 amino acids) linker. In another embodiment, both the variable heavy and variable light
chain comprise a long (greater than 6 amino acids) linker. In another embodiment, the variable
heavy chain comprises a short linker and the variable light chain comprises a long linker. In
another embodiment, the variable heavy chain comprises a long linker and the variable light chain
comprises a short linker.
In an embodiment the binding protein disclosed herein comprises a polypeptide chain,
wherein said polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein VDl is a first light
chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant
domain, XI is a linker with the proviso that it is not CHI, and X2 does not comprise an Fc
region.
In another embodiment, a binding protein comprising two polypeptide chains, wherein
said first polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein VDl is a first heavy
chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant
domain, XI is a linker with the proviso that it is not CHI, and X2 is an Fc region; and said
second polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein VDl is a first light
chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant
domain, XI is a linker with the proviso that it is not CHI, and X2 does not comprise an Fc region
is provided. In a particular embodiment, the Dual Variable Domain (DVD) binding protein
comprises four polypeptide chains wherein the first two polypeptide chains comprises VD1-
(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, X is a linker with the
proviso that it is not CHI, and X2 is an Fc region; and the second two polypeptide chain
comprises VDl-(Xl)n-VD2-C-(X2)n respectively, wherein VDl is a first light chain variable
domain, VD2 is a second light chain variable domain, C is a light chain constant domain, XI is a
linker with the proviso that it is not CHI, and X2 does not comprise an Fc region. Such a Dual
Variable Domain (DVD) protein has four antigen binding sites.
In another embodiment the binding proteins disclosed herein are capable of binding one
or more targets. Accordingly, in some embodiments, the binding proteins comprise at least two
variable domain sequences (e.g., VDl and VD2) capable of binding at least two different targets.
In some embodiments, VDl and VD2 are independently chosen. Therefore, in some
embodiments, VDl and VD2 comprise the same SEQ ID NO and, in other embodiments, VDl
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 binding protein is capable of modulating a biological function of one or
more targets. In another embodiment, the binding protein is capable of neutralizing one or more
targets. In another embodiment, the binding protein is capable of binding the following
exemplary cytokines: lymphokines, monokines, polypeptide hormones, receptors, or tumor
markers. For example, in some embodiments, the DVD-Ig is capable of binding two or more of
the following: Nerve Growth Factor (NGF), Methotrexate (MTX); NKG2D; IGF1,2; RON;
ErbB3; CD-3; IGFR; HGF; VEGF; DLL4; PIGF; CD-20; EGFR; HER-2; CD-19; CD-80; CD-22;
CD-40; c-MET; and NRP1 (see also Table 2). In a specific embodiment the binding protein is
capable of binding the following exemplary pairs of targets: NGF and MTX; NGF and NKG2D;
NGF and IGF1,2; NGF and RON; NGF and ErbB3; NGF and CD-3; NGF and IGFR; NGF and
HGF; NGF and VEGF; NGF and DLL4; NGF and PIGF; NGF and CD-20; NGF and EGFR;
NGF and HER-2; NGF and CD-19; NGF and CD-80; NGF and CD-22; NGF and CD-40; NGF
and c-MET; or NGF and NRP1 (see Examples 2.1 to 2.60).
In an embodiment, the binding protein comprises VDl and VD2 heavy chain variable
domains comprising SEQ ID NO: 28, 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, or 90; and VDl and VD2 light chain variable
domains comprising SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,
63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91.
In another embodiment, the binding protein comprises a heavy chain and a light chain
sequence as shown in Tables 12-71.
In an embodiment, the binding protein capable of binding NGF (seq. 2) and MTX
comprises a heavy chain comprising SEQ ID NO: 96 or SEQ ID NO: 98; and a light chain amino
acid sequence comprising SEQ ID NO: 97 or SEQ ID NO: 99. In an embodiment, the binding
protein capable of binding NGF (seq. 2) and MTX comprises a heavy chain amino acid sequence
of SEQ ID NO: 96 and a light chain amino acid sequence of SEQ ID NO: 97. In another
embodiment, the binding protein capable of binding NGF (seq. 1) and IL-6R has a reverse
orientation and comprises a heavy chain amino acid sequence of SEQ ID NO: 98 and a light
chain amino acid sequence of SEQ ID NO: 99.
In another embodiment, a binding protein comprising a polypeptide chain, wherein said
polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein; VDl is a first heavy chain
variable domain 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
binding protein.
In another embodiment, a 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; C is a light chain constant domain; (Xl)n is a linker with the proviso that it is not
CHI, wherein said (Xl)n is either present or absent; and (X2)n does not comprise an Fc region,
wherein said (X2)n is either present or absent is provided. In an embodiment, (X2)n is absent
from the binding protein.
In another embodiment the binding protein comprises first and second polypeptide
chains, wherein said first polypeptide chain comprises a first VDl-(Xl)n-VD2-C-(X2)n, wherein
VD1 is a first heavy chain variable domain obtained from a first parent antibody or antigen
binding portion thereof; VD2 is a second heavy chain variable domain obtained from a second
parent antibody or antigen binding portion thereof; C is a heavy chain constant domain; (Xl)n is
a linker with the proviso that it is not CHI, wherein said (Xl)n is either present or absent; and
(X2)n is an Fc region, wherein said (X2)n is either present or absent; and wherein said second
polypeptide chain comprises a second VDl-(Xl)n-VD2-C-(X2)n, wherein VD1 is a first light
chain variable domain obtained from a first parent antibody or antigen binding portion thereof;
VD2 is a second light chain variable domain obtained from a second parent antibody or antigen
binding portion thereof; C is a light chain constant domain; (Xl)n is a linker with the proviso that
it is not CHI, wherein said (Xl)n is either present or absent; and (X2)n does not comprise an Fc
region, wherein said (X2)n is either present or absent. In another embodiment, the binding protein
comprises two first polypeptide chains and two second polypeptide chains. In yet another
embodiment, (X2)n is absent from the second polypeptide. In still another embodiment, the Fc
region, if present in the first polypeptide is a native sequence Fc region or a variant sequence Fc
region. In still another embodiment, the Fc region is an Fc region from an IgGl, IgG2, IgG3,
IgG4, IgA, IgM, IgE, or lgD.
In another embodiment the binding protein is a capable of binding two antigens
comprising four polypeptide chains, wherein, first and third polypeptide chains comprise VD1-
(Xl)n-VD2-C-(X2)n, wherein,VDl is a first heavy chain variable domain obtained from a first
parent antibody or antigen binding portion thereof; VD2 is a second heavy chain variable domain
obtained from a second parent antibody or antigen binding portion thereof; C is a heavy chain
constant domain; (Xl)n is a linker with the proviso that it is not CHI, wherein said (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 second and fourth polypeptide chains comprise VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first light chain variable domain obtained from a first parent antibody or antigen binding
portion thereof; VD2 is a second light chain variable domain obtained from a second parent
antibody or antigen binding portion thereof; C is a light chain constant domain; (Xl)n is a linker
with the proviso that it is not CHI , wherein said (Xl)n is either present or absent; and (X2)n does
not comprise an Fc region, wherein said (X2)n is either present or absent.
A method of making a binding protein by preselecting the parent antibodies is provided.

Aitorney Docket No.: 1016.49-304
NGF and HGF, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting HGF
comprises at least one CDR from SEQ ID NO: 50;
NGF and VEGF, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting VEGF
comprises at least one CDR from SEQ ID NO: 54, 78, 80, or 84;
NGF and DLL4;
NGFandPlGF;
NGF and CD-20, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting CD-20
comprises at least one CDR from SEQ ID NO: 28;
NGF and EGFR, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting EGFR
comprises at least one CDR from SEQ ID NO: 32, 62, or 76;
NGF and HER-2, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting HER-2
comprises at least one CDR from SEQ ID NO: 34;
NGF and CD-19, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting CD-19
comprises at least one CDR from SEQ ID NO: 38;
NGF and CD-80, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting CD-80
comprises at least one CDR from SEQ ID NO: 40;
NGF and CD-22, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting CD-22
comprises at least one CDR from SEQ ID NO: 42;
NGF and CD-40, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting CD-40
comprises at least one CDR from SEQ ID NO: 44;
NGF and c-MET; or
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NGF and NRPl, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting NRPl
comprises at least one CDR from SEQ ID NO: 58 or 66.
2. The binding protein according to claim 1, wherein the VDl and/or VD2 heavy chain variable domains
comprise three CDRs from SEQ ID NO: 28,30, 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, or 90, respectively.
3. A binding protein capable of binding at least one target, the binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
nisOor l;and
wherein the binding protein binds :
NGFandMTX;
NGFandNKG2D;
NGF and IGFl, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting IGFl
comprises at least one CDR from SEQ ID NO: 47;
NGF and IGF2, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting IGF2
comprises at least one CDR from SEQ ID NO: 47;
NGF and RON;
NGFandErbB3;
NGF and CD-3, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting CD-3
comprises at least one CDR from SEQ ID NO: 30 or 68;
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NGF and IGFR, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting IGFR
comprises at least one CDR from SEQ ID NO: 48;
NGF and HGF, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting HGF
comprises at least one CDR from SEQ ID NO: 50;
NGF and VEGF, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting VEGF
comprises at least one CDR from SEQ ID NO: 54,78, 80, or 84;
NGF and DLL4;
NGFandPlGF;
NGF and CD-20, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting CD-20
comprises at least one CDR from SEQ ID NO: 28;
NGF and EGFR, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR fixjm SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting EGFR
comprises at least one CDR from SEQ ID NO: 32, 62, or 76;
NGF and HER-2, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting HER-2
comprises at least one CDR from SEQ ID NO: 34;
NGF and CD-19, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting CD-19
comprises at least one CDR from SEQ ID NO: 38;
NGF and CD-80, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting CD-80
comprises at least one CDR from SEQ ID NO: 40;
NGF and CD-22, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting CD-22
comprises at least one CDR from SEQ ID NO: 42;
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^ ^
NGF and CD-40, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting CD-40
comprises at least one CDR from SEQ ID NO: 44;
NGF and c-MET; or
NGF and NRPl, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain targeting NRPl
comprises at least one CDR from SEQ ID NO: 58 or 66.
4. The binding protein according to claim 3, wherein the VDl and/or VD2 light chain variable domains
comprise three CDRs from SEQ ID NO: 29, 31,33, 35,37,39,41,43,45,47,49, 51, 53, 55, 57, 59,61,
63, 65, 67, 69,71,73, 75,77, 79, 81, 83, 85, 87, 89, or 91, respectively.
5. The binding protein according to claim 1 or 3, wherein (Xl)n on the heavy and/or light chain is 0
and/or (X2)n on the heavy and/or light chain is (X2)0.
6. A binding protein capable of binding at least one target, the binding protein comprising first and second
polypeptide chains, wherein said first polypeptide chain comprises a first VDl-(Xl)n-VD2-C-pC2)n,
wherein
VDl is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 is an Fc region; n is 0 or 1; and
wherein said second polypeptide chain comprises a second VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
n isOor 1, and
wherein the binding protein binds :
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NGF and MTX;
NGFandNKG2D;
NGF and IGFl, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting IGFl comprises at least one CDR from SEQ ID NO: 46, and/or wherein the light chain variable
domain targeting IGFl comprises at least one CDR from SEQ ID NO: 47;
NGF and IGF2, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting IGF2 comprises at least one CDR from SEQ ID NO: 46, and/or wherein the light chain variable
domain targeting IGF2 comprises at least one CDR fix)m SEQ ID NO: 47;
NGF and RON;
NGF and ErbB3;
NGF and CD-3, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR fix)m SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-3 comprises at least one CDR from SEQ ID NO: 30 or 68, and/or wherein the light chain
variable domain targeting CD-3 comprises at least one CDR from SEQ ID NO: 31 or 69;
NGF and IGFR, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting IGFR comprises at least one CDR from SEQ ID NO: 48, and/or wherein the light chain variable
domain targeting IGFR comprises at least one CDR from SEQ ID NO: 49;
NGF and HGF, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting HGF comprises at least one CDR from SEQ ID NO: 50, and/or wherein the light chain variable
domain targeting HGF comprises at least one CDR from SEQ ID NO; 51;
NGF and VEGF, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
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comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting VEGF comprises at least one CDR from SEQ ID NO: 54, 78, 80, or 84, and/or wherein the light
chain variable domain targeting VEGF comprises at least one CDR from SEQ ID NO: 55, 79, 81, or 85;
NGF and DLL4;
NGF and PIGF;
NGF and CD-20, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-20 comprises at least one CDR from SEQ ID NO: 28, and/or wherein the light chain
variable domain targeting CD-20 comprises at least one CDR from SEQ ID NO: 29;
NGF and EGFR, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting EGFR comprises at least one CDR from SEQ ID NO: 32, 62, or 76, and/or wherein the light
chain variable domain targeting EGFR comprises at least one CDR from SEQ ID NO: 33, 63, or 77;
NGF and HER-2, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting HER-2 comprises at least one CDR from SEQ ID NO: 34, and/or wherein the light chain
variable domain targeting HER-2 comprises at least one CDR from SEQ ID NO: 35;
NGF and CD-19, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-19 comprises at least one CDR from SEQ ID NO: 38, and/or wherein the light chain
variable domain targeting CD-19 comprises at least one CDR from SEQ ID NO: 39;
NGF and CD-80, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-80 comprises at least one CDR from SEQ ID NO: 40, and/or wherein the light chain
variable domain targeting CD-80 comprises at least one CDR from SEQ ID NO: 41;
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NGF and CD-22, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-22 comprises at least one CDR from SEQ ID NO: 42, and/or wherein the light chain
variable domain targeting CD-22 comprises at least one CDR from SEQ ID NO: 43;
NGF and CD-40, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-40 comprises at least one CDR from SEQ ID NO: 44; and/or wherein the light chain
variable domain targeting CD-40 comprises at least one CDR from SEQ ID NO: 45;
NGF and c-MET; or
NGF and NRPl, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting NRPl comprises at least one CDR from SEQ ID NO: 58 or 66, and/or wherein the heavy chain
variable domain targeting NRPl comprises at least one CDR from SEQ ID NO: 59 or 67.
7. The binding protein according to claim 6, wherein the VDl and/or VD2 heavy chain variable domains
comprise three CDRs from SEQ ID NO: 28, 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, or 90, respectively; and/or wherein the VDl and/or
VD2 light chain variable domains comprise three CDRs from SEQ ID NO: 29,31,33,35,37,39,41,43,
45,47,49, 51, 53, 55, 57, 59, 61,63, 65,67, 69, 71,73,75, 77,79, 81, 83, 85, 87, 89, or 91, respectively.
8. The binding protein according to claim 1, 3,6,24, or 91-94, wherein XI is at least one of SEQ ID
NOs; 1-27, or a G/S based sequence.
9. The binding protein according to claim 6, wherein the binding protein comprises two first polypeptide
chains and two second polypeptide chains.
10. The binding protein according to claim 1, 6,24,91,93, or 94, wherein the Fc region is a variant
sequence Fc region.
11. The binding protein according to claim 1, 6,24,91,93, or 94, wherein the Fc region is from an IgG 1,
IgG2, IgG3, IgG4, IgA. IgM, IgE, or IgD.
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12. The binding protein according to claim 6, wherein said VDl of the first polypeptide chain and said
VDl of the second polypeptide chain are from a same first and second parent antibody, respectively, or
binding portion thereof.
13. The binding protein according to claim 6, wherein said VDl of the first polypeptide chain and said
VDl of the second polypeptide chain are from a different first and second parent antibody, respectively,
or binding portion thereof.
14. The binding protein according to claim 6, wherein said VD2 of the first polypeptide chain and said
VD2 of the second polypeptide chain are from a same first and second parent antibody, respectively, or
binding portion thereof.
15. The binding protein according to claim 6, wherein said VD2 of the first polypeptide chain and said
VD2 of the second polypeptide chain are from a different first and second parent antibody, respectively,
or binding portion thereof.
16. The binding protein according to claims 13-15, wherein said first and said second parent antibodies
bind different epitopes on said target.
17. The binding protein according to claims 13-15, wherein said first parent antibody or binding portion
thereof, binds a first target with a potency different from the potency with which said second parent
antibody or binding portion thereof, binds a second target.
18. The binding protein according to claims 13-15, wherein said first parent antibody or binding portion
thereof, binds a first target with an affinity different from the affinity with which said second parent
antibody or binding portion thereof, binds a second target.
19. The binding protein according to claims 13-15, wherein said first parent antibody or binding portion
thereof, and said second parent antibody or binding portion thereof, are a human antibody, a CDR grafted
antibody, or a humanized antibody.
20-23. (Canceled)
24. A binding protein capable of binding at least one target, wherein the binding protein comprising four
polypeptide chains, wherein two polypeptide chains comprise VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI;
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I X2 is an Fc region; n is 0 or 1; and
wherein two polypeptide chains comprise VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
n isOor l;and
wherein the binding protein binds:
NGF and MTX;
NGF and NKG2D;
NGF and IGFl, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting IGFl comprises at least one CDR from SEQ ID NO: 46, and/or wherein the light chain variable
domain targeting IGFl comprises at least one CDR from SEQ ID NO: 47;
NGF and IGF2, wherein tiie heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting IGF2 comprises at least one CDR from SEQ ID NO: 46, and/or wherein the light chain variable
domain targeting IGF2 comprises at least one CDR from SEQ ID NO: 47;
NGF and RON;
NGF and ErbB3;
NGF and CD-3, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-3 comprises at least one CDR from SEQ ID NO: 30 or 68, and/or wherein the light chain
variable domain targeting CD-3 comprises at least one CDR from SEQ ID NO: 31 or 69;
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NGF and IGFR, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting IGFR comprises at least one CDR from SEQ ID NO: 48, and/or wherein the light chain variable
domain targeting IGFR comprises at least one CDR from SEQ ID NO: 49;
NGF and HGF, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting HGF comprises at least one CDR from SEQ ID NO: 50, and/or wherein the light chain variable
domain targeting HGF comprises at least one CDR from SEQ ID NO: 51;
NGF and VEGF, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting VEGF comprises at least one CDR from SEQ ID NO: 54, 78, 80, or 84, and/or wherein the light
chain variable domain targeting VEGF comprises at least one CDR from SEQ ID NO: 55,79, 81, or 85;
NGF and DLL4;
NGF and PIGF;
NGF and CD-20, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-20 comprises at least one CDR from SEQ ID NO: 28, and/or wherein the light chain
variable domain targeting CD-20 comprises at least one CDR from SEQ ID NO: 29;
NGF and EGFR, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting EGFR comprises at least one CDR from SEQ ID NO: 32, 62, or 76, and/or wherein the light
chain variable domain targeting EGFR comprises at least one CDR from SEQ ID NO: 33, 63, or 77;
NGF and HER-2, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting HER-2 comprises at least one CDR from SEQ ID NO: 34, and/or wherein the light chain
variable domain targeting HER-2 comprises at least one CDR from SEQ ID NO: 35;
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NGF and CD-19, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-19 comprises at least one CDR from SEQ ID NO: 38, and/or wherein the light chain
variable domain targeting CD-19 comprises at least one CDR from SEQ ID NO: 39;
NGF and CD-80, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-80 comprises at least one CDR from SEQ ED NO: 40, and/or wherein the light chain
variable domain targeting CD-80 comprises at least one CDR from SEQ ID NO: 41;
NGF and CD-22, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-22 comprises at least one CDR from SEQ ID NO: 42, and/or wherein the light chain
variable domain targeting CD-22 comprises at least one CDR from SEQ ID NO: 43;
NGF and CD-40, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting CD-40 comprises at least one CDR from SEQ ID NO: 44; and/or wherein the light chain
variable domain targeting CD-40 comprises at least one CDR from SEQ ID NO: 45;
NGF and c-MET; or
NGF and NRPl, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86, and/or wherein the light chain variable domain targeting NGF
comprises at least one CDR from SEQ ID NO: 61 or 87; and/or wherein the heavy chain variable domain
targeting NRPl comprises at least one CDR from SEQ ID NO: 58 or 66, and/or wherein the heavy chain
variable domain targeting NRPl comprises at least one CDR from SEQ ID NO: 59 or 67 .
25. The binding protein according to claim 24, wherein the VDl and/or VD2 heavy chain variable
domains comprise three CDRs from SEQ ID NO: 28, 30, 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, or 90, respectively; and/or wherein the VDl
and/or VD2 light chain variable domains comprise three CDRs from SEQ ID NO:. 29, 31, 33,35, 37, 39,
41,43,45,47,49, 51, 53, 55, 57, 59, 61,63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91,
respectively.
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26. The binding protein according to claim 1,3, 6,24, or 91-94, wherein said binding protein has an on
rate constant (Kon) to said at least one target of: at least about 10^M''s''; at least about IO^M'S"'; at least
about 10*M"'s''; at least about 10'M''S''; or at least about 10*M'S'', as measured by surface plasmon
resonance.
27. The binding protein according to claim 1,3,6,24, or 91-94, wherein said binding protein has an off
rate constant (KOH) to said at least one target of: at most about lO'^s"'; at most about lO'^s''; at most about
lO'^s''; or at most about lO'^s"*, as measured by surface plasmon resonance.
28. The binding protein according to claim 1,3, 6,24, or 91-94, wherein said binding protein has a
dissociation constant (KD) to said at least one target of: at most about 10"'M; at most about lO'^M; at most
about lO'^M; at most about 10"'°M; at most about 10'"M; at most about 10''^M; or at most 10'"M, as
measured by surface plasmon resonance.
29. A binding protein conjugate comprising a binding protein according to claims 1,3, 6,24, or 91-94,
said binding protein conjugate further comprising an immunoadhesion molecule, an imaging agent, a
therapeutic agent, or a cytotoxic agent.
30. The binding protein conjugate according to claim 29, wherein said imaging agent is a radiolabel, an
enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin.
31. (Canceled)
32. The binding protein conjugate according to claim 29, wherein said therapeutic or cytotoxic agent is:
an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic
agent, an anti-mitotic agent, an anthracycline, toxin, or an apoptotic agent.
33. The binding protein according to claim 1,3, 6,24, or 91-94, wherein said binding protein is a
crystallized binding protein.
34-36. (Canceled)
37. An isolated nucleic acid encoding a binding protein amino acid sequence according to claims 1,3, 6,
24, or 91-94.
38. A vector comprising an isolated nucleic acid according to claim 37.
39. The vector according to claim 38, wherein said vector is pcDNA, pTT, pTT3, pEFBOS, pBV, pJV,
pcDNA3.1 TOPO, pEF6 TOPO, pHybE, or pBJ.
40. A host cell comprising a vector according to claim 38.
41. The host cell according to claim 40, wherein said host cell is a prokaryotic cell.
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42. The host cell according to claim 41, wherein said prokaryotic cell is Escherichia coli.
43. The host cell according to claim 40, wherein said host cell is a eukaryotic cell.
44. The host cell according to claim 43, wherein said eukaryotic cell is a protist cell, an animal cell, a
plant cell, or a fungal cell.
45. The host cell according to claim 44, wherein said animal cell is a mammalian cell, an avian cell, or an
insect cell.
46. The host cell according to claim 45, wherein said manunalian cell is a CHO cell.
47. The host cell according to claim 45, wherein said mammalian cell is a COS cell.
48. The host cell according to claim 44, wherein said fimgal cell is a yeast cell.
49. The host cell according to claim 48, wherein said yeast cell is Saccharomyces cerevisiae.
50. The host cell according to claim 45, wherein said insect cell is an Sf9 cell.
51. A method of producing a binding protein, comprising culturing a host cell according to of claims 40-
50 in culture medium under conditions sufficient to produce the binding protein.
52-54. (Canceled)
55. A protein produced according to the method of claim 51.
56. A pharmaceutical composition comprising the binding protein of claims 1,3, 6,24, or 91-94, and a
pharmaceutically acceptable carrier.
57. The pharmaceutical composition of claim 56 further comprising at least one additional therapeutic
agent.
58. The pharmaceutical composition of claim 57, wherein said additional therapeutic agent is: an imaging
agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a co-stimulation molecule blocker,
an adhesion molecule blocker, an anti-cytokine antibody or functional fragment thereof, methotrexate,
cyclosporin, rapamycin, FK506, a detectable label or reporter, a TNF antagonist, an antirheumatic, a
muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a
sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod,
an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a
growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an
antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or
analog, a cytokine, or a cytokine antagonist.
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59. Use of the binding protein according to claims 1,3, 6, 24, or 91-94, for treating a subject for a disease
or a disorder by administering to the subject the binding protein such that treatment is achieved.
60. The use according to claim 59, wherein said disorder is rheumatoid arthritis, osteoarthritis, juvenile
chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy,
systemic lupus erythematosus, Crohn's disease, ulcerative colitis, 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, cadiexia, 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 tj^je I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute)
respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's
disease, psoriatic arthropathy, ulcerative oolitic 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 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, Sj&gren'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
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hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune
disease associated with organ transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2,
idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic
vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS,
sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension
secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis
nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's
syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia,
autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's
disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary
vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver
injury, cholestasis, idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis,
allergy and asthma, group B streptococci (GBS) infection, mental disorders, depression, schizophrenia,
Th2 Type and TTil Type mediated diseases, acute and chronic pain, cancer, lung cancer, breast cancer,
stomach cancer, bladder cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal
cancer, hematopoietic malignancies, leukemia, lymphoma, abetalipoproteinemia, acrocyanosis, acute and
chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute
myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure,
adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic
conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-1- antitrypsin
deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti
cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aordic and peripheral
aneuryisms, acotic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial
fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft
rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, bums,
cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass
inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar
disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic myelocytic
leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia
(CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal
carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery
disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated
disorders, dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis,
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dermatologic conditions, diabetes, diabetes mellitus, diabetic ateriosclerotic disease, diffuse Lewy body
' disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's syndrome in middle
age, drug- induced movement disorders induced by drugs which block CNS dopamine receptors, drug
sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus
infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic
lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial
disorders, fungal sepsis, gas gangrene, gastric ulcer, graft rejection of any organ or tissue, gram negative
sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallerrorden-
Spatz disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis,
hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis
A, His bundle arryhthmias, HIV infection/HTV neuropathy, Hodgkin's disease, hyperkinetic movement
disorders, hypersensitity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement
disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic
pulmonary fibrosis, antibody mediated cytotoxicity, asthenia, infantile spinal muscular atrophy,
inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis,
ischemia- reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular
atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of the
corticospinal system, lipedema, liver transplant rejection, lymphederma, malaria, malignamt Lymphoma,
malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic,
migraine headache, mitochondrial multi.system disorder, mixed connective tissue disease, monoclonal
gammopathy, multiple myeloma, multiple systems degenerations (Mencel Dejerine- Thomas Shy-Drager
and Machado-Joseph), myasthenia gravis, mycobacterium avium intracellulare, mycobacterium
tuberculosis, myelodyplastic syndrome, myocardial ischemic disorders, nasopharyngeal carcinoma,
neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular
atrophies, neutropenic fever, non- hodgkins lymphoma, occlusion of the abdominal aorta and its
branches, occulsive arterial disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal
procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma,
paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic
inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherlosclerotic disease, peripheral
vascular disorders, peritonitis, pernicious anemia, Pneumocystis carinii pneumonia, pneumonia, POEMS
syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes
syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia,
progressive supranucleo palsy, primary pulmonary hypertension, radiation therapy, Raynaud's
phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia,
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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, Tcell
or FAB ALL, telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants,
trauma/hemorrhage, type ni hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia,
urosepsis, urticaria, valvular heart diseases, varicose veins, .vasculitis, venous diseases, venous
thrombosis, ventricular fibrillation, viral and fungal infections, vital encephalitis/aseptic meningitis, vitalassociated
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 disordo- 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 honiation, disk prolaps,
drug induced immune hemolytic anemia, endometriosis, endophthalmitis, episcleritis, erythema
multiforme, erythema multiforme major, gestational pemphigoid, Guillain-Barr6 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, prostatitis, pure red cell
aplasia, primary adrenal insufficiency, recurrent neuromyelitis optica, restenosis, rheiunatic heart disease,
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sapho (synovitis, acne, pustulosis, hyperostosis, and osteitis), scleroderma, secondary amyloidosis, shock
lung, scleritis, sciatica, secondary adrenal insufficiency, silicone associated connective tissue disease,
sneddon-wilkinson dermatosis, spondilitis ankylosans, Stevens-Johnson syndrome (SJS), systemic
inflammatory response syndrome, temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis,
transverse myelitis, TRAPS (tumor necrosis factor receptor, type 1 allergic reaction, type II diabetes,
usual interstitial pneumonia (UIP), vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome
(VKH syndrome), wet macular degeneration, or wound healing.
61. The use according to claim 60, wherein said administering to the subject is parenteral, subcutaneous,
intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous,
intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural,
intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, mtraspinal, intrasynovial, intrathoracic,
intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
62. A method for generating a binding protein capable of binding at least one target comprising the steps
of
a) obtaining a first parent antibody or binding portion thereof;
b) obtaining a second parent antibody or binding portion thereof;
c) constructing the polypeptide chain or chains according to claims 1,3, 6, 24; or 91-94; and
d) expressing the polypeptide chain or chains such that a binding protein capable of binding
at least one target is generated.
63. The method according to claim 62, wherein the VDl and/or VD2 heavy chain variable domains, if
present, comprise three CDRs from SEQ ID NO: 28,30, 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, or 90, respectively; and/or wherein the VDl
and/or VD2 light chain variable domains, if present, comprise three CDRs from SEQ ID NO: 29,31, 33,
35,37,39,41,43,45,47,49, 51, 53, 55, 57, 59, 61, 63,65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,
or 91, respectively.
64. The method according to claim 62, wherein said first parent antibody or binding portion thereof, and
said second parent antibody or binding portion thereof, are a human antibody, a CDR grafted antibody, or
a humanized antibody.
65-67. (Canceled)
68. The method according to claim 62, wherein the Fc region, if present, is a variant sequence Fc region.
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69. The method according to claim 62, wherein the Fc region, if present, is from an IgGl, IgG2, IgG3,
IgG4,IgA,IgM,IgE,orIgD.
70-73. (Canceled)
74. The method according to claim 62, wherein said first parent antibody or binding portion thereof, binds
a first target with a different affinity than the affinity with which said second parent antibody or binding
portion thereof, binds a second target.
75. The method according to claim 62, wherein said first parent antibody or binding portion thereof, binds
a first target with a different potency than the potency with which said second parent antibody or binding
portion thereof, binds a second target.
76. A method of determining the presence of at least one target or fragment thereof in a test sample by an
immunoassay,
wherein the immunoassay comprises contacting the test sample with at least one bmding protein
and at least one detectable label,
wherein the at least one binding protein comprises the binding protein according to claims 1, 3, 6,
24, or 91-94 .
77. The method according to claim 76 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding protein
binds to an epitope on the target or fragment thereof so as to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein the detectable label
binds to the binding protein or an epitope on the target or fi'agment thereof that is not bound by the
binding protein to form a second complex; and
(iii) detecting the presence of the target or fi'agment thereof in the test sample based on the signal
generated by the detectable label in the second complex, wherein the presence of the target or firagment
thereof is directly correlated with the signal generated by the detectable label.
78. The method according to claim 76 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding protein
binds to an epitope on the target or fragment thereof so as to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein the detectable label
competes with the target or fi-agment thereof for binding to the binding protein so as to form a second
complex; and
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, (iii) detecting the presence of the target or fragment thereof in the test sample based on the signal
generated by the detectable label in the second complex, wherein the presence of the target or fragment
thereof is indirectly correlated with the signal generated by the detectable label.
79. The method according to -claim 77 or 78, wherein the test sample is from a patient and the method
further comprises diagnosing, prognosticating, or assessing the efficiency of therapeutic/prophylactic
treatment of the patient, and
wherein if the method further comprises assessing the efficacy of therapeutic/prophylactic
treatment of the patient, the method optionally further comprises modifying the therapeutic/prophylactic
treatment of the patient as needed to improve efficacy.
80. The method according to claim 77 or 78, wherein the method is adapted for use in an automated
system or a semi-automated system.
81. The method according to claim 77 or 78, wherein the method determines the presence of more than
one antigen in the sample.
82. A method of determining the amount or concentration of an target or fragment thereof in a test sample
by an immunoassay,
wherein the immunoassay (a) employs at least one bindmg protein and at least one detectable
label and (b) comprises comparing a signal generated by the detectable label with a control or calibrator
comprising the target or fragment thereof,
wherein the calibrator is optionally part of a series of calibrators in which each calibrator differs
from the other calibrators in the series by the concentration of the target or fragment thereof,
and wherein the at least one binding protein comprises the binding protein according to claim 1,
3,6,24, or 91-94.
83. The method according to claim 82 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding protein
binds to an epitope on the target or fragment thereof so as to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein the detectable label
binds to an epitope on the target or fragment thereof that is not bound by the binding protein to form a
second complex; and
(iii) determining the amount or concentration of the target or fragment thereof in the test sample
based on the signal generated by the detectable label in the second complex, wherein the amount or
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concentration of the target or fragment thereof is directly proportional to the signal generated by the
detectable label.
84. The method according to claim 82 further comprising:
(i) contacting the test sample with the at least one binding protein, wherein the binding protein
binds to an epitope on the target or fragment thereof so as to form a first complex;
(ii) contacting the complex with the at least one detectable label, wherein the detectable label
competes with the target or fragment thereof for binding to the binding protein so as to form a second
complex; and
(iii) determining the amount or concentration of the target or fragment thereof in the test sample
based on the signal generated by the detectable label in the second complex, wherein the presence of the
target or fragment thereof is indirectly proportional to the signal generated by the detectable label.
85. The method according to claim 83 or 84, 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.
86. The method according to claim 83 or 84, wherein the method is adapted for use in an automated
system or a semi-automated system.
87. The method according to claim 83 or 84, wherein the method determines the amoimt or concentration
of more than one antigen in the sample.
88. A kit for assaying a test sample for the presence, amount, or concentration of a target or fragment
thereof, said kit comprising (a) instructions for assaying the test sample for the target or fragment thereof
and (b) at least one binding protein comprising the binding protein according to claims 1,3,6,24, or 91-
94.
89. The binding protein according to claims 1,3,6, or 24, wherein the VDl and/or VD2 heavy chain
variable domains, if present, comprise SEQ ID NO: 28,30, 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, or 90, respectively; and/or wherein the
VDl and/or VD2 light chain variable domains, if present, comprise SEQ ID NO: 29, 31, 33, 35,37, 39,
41,43,45,47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89. or 91,
respectively.
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90. The method according to claim 62, wherein the VDl and/or VD2 heavy chain variable domains, if
present, comprise SEQ ID NO: 28,30,32,34,36, 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, or 90, respectively; and/or wherein the VDl and/or VD2 light
chain variable domains, if present, comprise SEQ ID NO: 29, 31,33,35,37, 39,41,43, 45,47,49, 51,
53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, or 91, respectively.
91. A binding protein capable of binding at least one target, the binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker wifli the proviso that it is not CHI;
X2 is an Fc region;
n is Cor 1; and
wherein the binding protein binds: NGF and MTX; NGF and NKG2D; NGF and IGF 1,2; NGF and RON;
NGF and ErbB3; NGF and CD-3; NGF and IGFR; NGF and HGF; NGF and VEGF; NGF and DLL4;
NGF and PIGF; NGF and CD-20; NGF and EGFR; NGF and HER-2; NGF and CD-19; NGF and CD-80;
NGF and CD-22; NGF and CD-40; NGF and c-MET; or NGF and NRPl; and
further wherein the binding protein binds:
(a) NGF, and has an off rate constant (Koff) of at most about 2.80 x 10"* s"' and/or a dissociation
constant (KD) of at most about 1.80 x 10'' M, as determined by surface plasmon resonance;
(b) EGFR, and has an off rate constant (K^a) of at most about 1.60 x 10'^ s'' and/or a dissociation
constant (KD) of at most about 1.10 x 10'' M, as determined by surface plasmon resonance;
(c) ErbB3, and has an off rate constant (Koff) of less than 1 x lO"* s"' and/or a dissociation constant
(KD) of less than 4.2 x 10"'^ M, as determined by surface plasmon resonance;
(d) VEGF, and has an off rate constant (Koff) of at most aboxrt 7.70 x 10"' s'* and/or a dissociation
constant (KD) of at most about 8.00 x 10'" M, as determined by surface plasmon resonance;
(e) DLL4, and has an off rate constant (Koff) of at most about 2.00 x 10"* s'' and/or a dissociation
constant (KD) of at most about 4.14 x 10''° M, as determined by surface plasmon resonance;
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(f) RON, and has an off rate constant (Koir) of at most about 6.70 x 10'^ s ' and/or a dissociation
constant (KD) of at most about 5.70 x 10'' M, as determined by surface plasmon resonance; and/or
(g) PIGF, and has an off rate constant (Koir) of at most about 2.80 x 10"^ s"' and/or a dissociation
constant (KD) of at most about 1.40 x 10'' M, as determined by surface plasmon resonance.
92. A binding protein capable of binding at least one target, the binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
nisOorl;and
wherein the binding protein binds: NGF and MTX; NGF and NKG2D; NGF and IGF1,2; NGF and RON;
NGF and ErbB3; NGF and CD-3; NGF and IGFR; NGF and HGF; NGF and VEGF; NGF and DLL4;
NGF and PIGF; NGF and CD-20; NGF and EGFR; NGF and HER-2; NGF and CD-19; NGF and CD-80;
NGF and CD-22; NGF and CD-40; NGF and c-MET; or NGF and NRPl; and
further wherein the binding protein binds:
(a) NGF, and has an off rate constant (K<,ff) of at most about 2.80 x 10"^ s'' and/or a dissociation
constant (KD) of at most about 1.80 x 10"' M, as determined by surface plasmon resonance;
(b) EGFR, and has an off rate constant (KOA) of at most about 1.60 x 10"^ s'' and/or a dissociation
constant (KD) of at most about 1.10 x 10''M, as determined by surface plasmon resonance;
(C) ErbB3, and has an off rate constant (Kow) of less than 1x10"* s"' and/or a dissociation constant
(KD) of less than 4.2 x 10"'^ M, as determined by surface plasmon resonance;
(d) VEGF, and has an off rate constant (KOB) of at most about 7.70 x 10'^ s"' and/or a dissociation
constant (KQ) of at most about 8.00 x 10"" M, as determined by surface plasmon resonance;
(e) DLL4, and has an off rate constant (KOA) of at most about 2.00 x 10"* s"' and/or a dissociation
constant (KD) of at most about 4.14 x 10"'° M, as determined by surface plasmon resonance;
(f) RON, and has an off rate constant (Koo) of at most about 6.70 x 10"^ s"' and/or a dissociation
constant (KD) of at most about 5.70 x 10"' M, as determined by surface plasmon resonance; and/or
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(g) PIGF, and has an off rate constant (Koff) of at most about 2.80 x 10"^ s"' and/or a dissociation
constant (KD) of at most about 1.40 x 10"' M, as determined by surface plasmon resonance.
93. A binding protein capable of binding at least one target, the binding protein comprising first and
second polypeptide chains, wherein said first polypeptide chain comprises a first VDl-(Xl)n-VD2-C-
(X2)n, wherein
VDl is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 is an Fc region;n is 0 or 1; and
wherein said second polypeptide chain comprises a second VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
n is 0 or 1, and
wherein the binding protein binds: NGF and MTX; NGF and NKG2D; NGF and IGF 1,2; NGF and RON;
NGF and ErbB3; NGF and CD-3; NGF and IGFR; NGF and HGF; NGF and VEGF; NGF and DLL4;
NGF and PIGF; NGF and CD-20; NGF and EGFR; NGF and HER-2; NGF and CD-19; NGF and CD-80;
NGF and CD-22; NGF and CD-40; NGF and c-MET; or NGF and NRPl; and
fiirther wherein the binding protein binds:
(a) NGF, and has an off rate constant (Koff) of at most about 2.80 x 10"* s'' and/or a dissociation
constant (KD) of at most about 1.80 x 10'' M, as determined by surface plasmon resonance;
(b) EGFR, and has an off rate constant (Koff) of at most about 1.60 x 10'^ s'' and/or a dissociation
constant (Kp) of at most about 1.10 x 10"'M, as determined by surface plasmon resonance;
(c) ErbB3, and has an off rate constant (Koff) of less than 1x10* s"' and/or a dissociation constant
(KD) of less than 4.2 x 10"'^ M, as determined by surface plasmon resonance;
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(d) VEGF, and has an off rate constant (KOA) of at most about 7.70 x 10'' s'' and/or a dissociation
constant (KD) of at most about 8.00 x 10'" M, as determined by surface plasmon resonance;
(e) DLL4, and has an off rate constant (Koff) of at most about 2.00 x 10"^ s"' and/or a dissociation
constant (Kp) of at most about 4.14 x 10""* M, as determined by surface plasmon resonance;
(f) RON, and has an off rate constant (Kos) of at most about 6.70 x 10"^ s"^ and/or a dissociation
constant (KD) of at most about 5.70 x 10'' M, as determined by surface plasmon resonance; and/or
(g) PIGF, and has an off rate constant (KOH) of at most about 2.80 x 10"^ s'' and/or a dissociation
constant (KD) of at most about 1.40 x 10"' M, as determined by surface plasmon resonance.
94. A binding protein capable of binding at least one target, the binding protein comprising four
polypeptide chains, wherein two polypeptide chains comprise VDl-(XI )n-VD2-C-(X2)n, wherein
VDl is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 is an Fc region;
nisOor 1; and
wherein two polypeptide chains comprise VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first light chain variable domain;
VD2 is a second light chain variable domain;
C is a light chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 does not comprise an Fc region;
nis Oor 1; and
wherein the binding protein binds: NGF and MTX; NGF and NKG2D; NGF and IGF 1,2; NGF and RON;
NGF and ErbB3; NGF and CD-3; NGF and IGFR; NGF and HGF; NGF and VEGF; NGF and DLL4;
NGF and PIGF; NGF and CD-20; NGF and EGFR; NGF and HER-2; NGF and CD-19; NGF and CD-80;
NGF and CD-22; CD-40; NGF and c-MET; or NGF and NRP1; and
further wherein the binding protein binds:
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We claim:
1. A binding protein capable of binding at least one target, the binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises VDl-(Xl)n-VD2-C-(X2)n, wherein
VDl is a first heavy chain variable domain;
VD2 is a second heavy chain variable domain;
C is a heavy chain constant domain;
XI is a linker with the proviso that it is not CHI;
X2 is an Fc region;
nisOor 1; and
wherein the binding protein binds :
NGFandMTX;
NGF and NKG2D;
NGF and IGFl, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting IGFl
comprises at least one CDR from SEQ ID NO: 46;
NGF and IGF2, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting IGF2
comprises at least one CDR from SEQ ID NO: 46;
NGF and RON;
NGF and ErbB3;
NGF and CD-3, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting CD-3
comprises at least one CDR from SEQ ID NO: 30 or 68;
NGF and IGFR, wherein the heavy chain variable domain targeting NGF comprises at least one
CDR from SEQ ID NO: 60 or 86; and/or wherein the heavy chain variable domain targeting IGFR
comprises at least one CDR from SEQ ID NO: 48;
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(a) NGF, and has an off rate constant (Koff) of at most about 2.80 x 10"^ s"' and/or a dissociation
constant (KD) of at most about 1.80 x 10'^ M, as determined by surface plasmon resonance;
(b) EGFR, and has an off rate constant (Koff) of at most about 1.60 x 10"^ s'' and/or a dissociation
constant (KD) of at most about 1.10 x 10''M, as determined by surface plasmon resonance;
(c) ErbB3, and has an off rate constant (Koff) of less than 1 x 10"* s"' and/or a dissociation constant
(KD) of less than 4.2 x 10"'^ M, as determined by surface plasmon resonance;
(d) VEGF, and has an off rate constant (Koff) of at most about 7.70 x 10'' s"' and/or a dissociation
constant (KD) of at most about 8.00 x 10'" M, as determined by surface plasmon resonance;
(e) DLL4, and has an off rate constant (Koff) of at most about 2.00 x 10"^ s'' and/or a dissociation
constant (KD) of at most about 4.14 x 10''° M, as determined by surface plasmon resonance;
(f) RON, and has an off rate constant (Koff) of at most about 6.70 x 10'^ s"' and/or a dissociation
constant (KD) of at most about 5.70 x 10'' M, as determined by surface plasmon resonance; and/or
(g) PIGF, and has an off rate constant (Koff) of at most about 2.80 x 10"^ s'' and/or a dissociation
constant (KD) of at most about 1.40 x 10'' M, as determined by surface plasmon resonance.
95. The binding protein according to 1 or 91, wherein the binding protein binds:
NGF and MTX, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting MTX comprises
three CDRs from SEQ ID NO: 88;
NGF and NKG2D, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting NKG2D comprises
three CDRs from SEQ ID NO: 90;
NGF and IGFl, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting IGF 1,2 comprises
three CDRs from SEQ ID NO: 46;
NGF and IGF2, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting IGF 1,2 comprises
three CDRs from SEQ ID NO: 46;
NGF and RON, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting RON comprises
three CDRs from SEQ ID NO: 36 or 64, respectively;
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NGF and ErbB3, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting ErbB3 comprises
three CDRs from SEQ ID NO: 72, 74, or 82 respectively;
NGF and CD-3, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting CD-3 comprises
three CDRs from SEQ ID NO: 30 or 68, respectively;
NGF and IGFR, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting IGFR comprises
three CDRs from SEQ ID NO: 48;
NGF and HGF, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting HGF comprises
three CDRs from SEQ ID NO: 50;
NGF and VEGF, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ED NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting VEGF comprises
three CDRs from SEQ ID NO: 54, 78, 80, or 84, respectively;
NGF and DLL4, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting DLL4 comprises
three CDRs from SEQ ID NO: 56;
NGF and PIGF, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting PIGF comprises
three CDRs from SEQ ID NO: 70;
NGF and CD-20, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting CD-20 comprises
three CDRs from SEQ ID NO: 28;
NGF and EGFR, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting EGFR comprises
three CDRs from SEQ ID NO: 32,62, or 76, respectively;
NGF and HER-2, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting HER-2 comprises
three CDRs from SEQ ID NO: 34;
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^B
NGF and CD-19, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting CD-I 9 comprises
three CDRs from SEQ ID NO: 38;
NGF and CD-80, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting CD-80 comprises
three CDRs from SEQ ID NO: 40;
NGF and CD-22, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting CD-22 comprises
three CDRs from SEQ ID NO: 42;
NGF and CD-40, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting CD-40 comprises
three CDRs from SEQ ID NO: 44;
NGF and c-MET, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively; and/or the heavy chain variable domain targeting c-MET comprises
three CDRs from SEQ ID NO: 52; or
NGF and NRPl, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO; 60 or 86, respectively; and/or the heavy chain variable domain targeting NRPl comprises
three CDRs from SEQ ID NO: 58 or 66, respectively.
96. The binding protein according to 3 or 92, wherein the binding protein binds:
NGF and MTX, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting MTX comprises three
CDRs from SEQ ID NO: 89;
NGF and NKG2D, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting NKG2D comprises
three CDRs from SEQ ID NO: 91;
NGF and IGFl, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting IGF 1,2 comprises three
CDRs from SEQ ID NO: 47;
NGF and IGF2, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting IGFl,2 comprises three
CDRs from SEQ ID NO: 47;
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NGF and RON, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting RON comprises three
CDRs from SEQ ID NO: 37 or 65, respectively;
NGF and ErbB3, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting ErbB3 comprises
three CDRs from SEQ ID NO: 73, 75, or 83 respectively;
NGF and CD-3, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting CD-3 comprises three
CDRs from SEQ ID NO: 31 or 69, respectively;
NGF and IGFR, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting IGFR comprises three
CDRs from SEQ ID NO: 49;
NGF and HGF, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting HGF comprises three
CDRs from SEQ ID NO: 51;
NGF and VEGF, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting VEGF comprises
three CDRs from SEQ ID NO: 55, 79, 81, or 85, respectively;
NGF and DLL4, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting DLL4 comprises three
CDRs from SEQ ID NO: 57;
NGF and PIGF, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting PIGF comprises three
CDRs from SEQ ID NO: 71;
NGF and CD-20, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting CD-20 comprises
three CDRs from SEQ ID NO: 29;
NGF and EGFR, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting EGFR comprises
three CDRs from SEQ ID NO: 33, 63, or 77, respectively;
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NGF and HER-2, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting HER-2 comprises
three CDRs from SEQ ID NO: 35;
NGF and CD-19, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting CD-19 comprises
three CDRs from SEQ ID NO: 39;
NGF and CD-80, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting CD-80 comprises
three CDRs from SEQ ID NO: 41;
NGF and CD-22, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO; 61 or 87, respectively; and/or the light chain variable domain targeting CD-22 comprises
three CDRs from SEQ ID NO: 43;
NGF and CD-40, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting CD-40 comprises
three CDRs from SEQ ID NO: 45;
NGF and c-MET, wherein the light chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting c-MET comprises
tiiree CDRs from SEQ ID NO: 53; or
NGF and NRPl, wherein the light chain variable domain targeting NGF comprises three CDRs from SEQ
ID NO: 61 or 87, respectively; and/or the light chain variable domain targeting NRPl comprises three
CDRs from SEQ ID NO: 59 or 67, respectively.
97. The binding protein according to 6,24,25,93, or 94, wherein the binding protein binds:
NGF and MTX, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting MTX comprises three CDRs from SEQ ID NO: 88, and the light chain variable domain
targeting MTX comprises three CDRs from SEQ ID NO: 89;
NGF and NKG2D, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
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Attorney Docket No.: 1016.49-304
targeting NKG2D comprises three CDRs from SEQ ID NO: 90, and the light chain variable domain
targeting NKG2D comprises three CDRs from SEQ ID NO: 91;
NGF and IGFl, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting IGFl comprises three CDRs from SEQ ID NO: 46, and the light chain variable domain targeting
IGF 1 comprises three CDRs from SEQ ID NO: 47;
NGF and IGF2, wherein the heavy chain variable domain targetmg NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting IGF2 comprises three CDRs from SEQ ID NO: 46, and the light chain variable domain targeting
IGF2 comprises three CDRs from SEQ ID NO: 47;
NGF and RON, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting RON comprises three CDRs from SEQ ID NO: 36 or 64, respectively, and the light chain
variable domain targeting RON comprises three CDRs from SEQ ID NO: 37 or 65, respectively;
NGF and ErbB3, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting ErbB3 comprises three CDRs from SEQ ID NO: 72, 74, or 82, respectively, and the light chain
variable domain targeting ErbB3 comprises three CDRs fi-om SEQ ID NO: 73,75, or 83, respectively;
NGF and CD-3, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting CD-3 comprises three CDRs from SEQ ID NO: 30 or 68, respectively, and the light chain
variable domain targeting CD-3 comprises three CDRs from SEQ ID NO: 31 or 69, respectively;
NGF and IGFR, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting IGFR comprises three CDRs from SEQ ID NO: 48, and the light chain variable domain
targeting IGFR comprises three CDRs from SEQ ID NO: 49;
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NGF and HGF, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting HGF comprises three CDRs from SEQ ID NO: 50, and the light chain variable domain targeting
HGF comprises three CDRs from SEQ ID NO: 51;
NGF and VEGF, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting VEGF comprises three CDRs from SEQ ID NO: 54, 78, 80, or 84, respectively, and the light
chain variable domain targeting VEGF comprises three CDRs from SEQ ID NO: 55,79, 81, or 85,
respectively;
NGF and DLL4, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting DLL4 comprises three CDRs from SEQ ID NO: 56, and the light chain variable domain
targeting DLL4 comprises three CDRs from SEQ ID NO: 57;
NGF and PIGF, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ED NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting PIGF comprises three CDRs from SEQ ID NO: 70, and the light chain variable domain targeting
PIGF comprises three CDRs from SEQ ID NO: 71;
NGF and CD-20, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting CD-20 comprises three CDRs from SEQ ID NO: 28, and the light chain variable domain
targeting CD-20 comprises three CDRs from SEQ ID NO: 29;
NGF and EGFR, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting EGFR comprises three CDRs from SEQ ID NO: 32, 62, or 76, respectively, and the light chain
variable domain targeting EGFR comprises three CDRs from SEQ ID NO: 33, 63, or 77, respectively;
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NGF and HER-2, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting HER-2 comprises three CDRs from SEQ ID NO: 34, and the light chain variable domain
targeting HER-2 comprises three CDRs from SEQ ID NO: 35;
NGF and CD-19, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting CD-19 comprises three CDRs from SEQ ID NO: 38, and the light chain variable domain
targeting CD-19 comprises three CDRs from SEQ ID NO: 39;
NGF and CD-80, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting CD-80 comprises three CDRs from SEQ ID NO: 40, and the light chain variable domain
targeting CD-80 comprises three CDRs from SEQ ID NO: 41;
NGF and CD-22, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting CD-22 comprises three CDRs from SEQ ID NO: 42, and the light chain variable domain
targeting CD-22 comprises three CDRs from SEQ ID NO: 43;
NGF and CD-40, wherein the heavy chain variable domam targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting CD-40 comprises three CDRs from SEQ ID NO: 44, and the light chain variable domain
targeting CD-40 comprises three CDRs from SEQ ID NO: 45;
NGF and c-MET, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting c-MET comprises three CDRs from SEQ ID NO: 52, and the light chain variable domain
targeting c-MET comprises three CDRs from SEQ ID NO: 53; or
NGF and NRPl, wherein the heavy chain variable domain targeting NGF comprises three CDRs from
SEQ ID NO: 60 or 86, respectively, and the light chain variable domain targeting NGF comprises three
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^ ^
CDRs from SEQ ID NO: 61 or 87, respectively; and/or wherein the heavy chain variable domain
targeting NRPl comprises three CDRs from SEQ ID NO: 58 or 66, respectively, and the light chain
variable domain targeting NRPl comprises three CDRs from SEQ ID NO: 59 or 67, respectively.
98. The binding protein according to claims 24,25,93, or 94, wherein the binding protein is:
DVD1307 (comprising SEQ ID NOs: 96 and 97); DVD1308 (comprising SEQ ID NOs: 98 and 99);
DVD1309 (comprising SEQ ID NOs: 100 and 101); DVD1310 (comprising SEQ ID NOs: 102 and 103);
DVD1311 (comprising SEQ ID NOs: 104 and 105); DVD1312 (comprising SEQ ID NOs: 106 and 107);
DVD1313 (comprising SEQ ID NOs: 108 and 109); DVD 1314 (comprising SEQ ID NOs: 110 and 111);
DVD1315 (comprising SEQ ID NOs: 112 and 113); DVD1316 (comprising SEQ ID NOs: 114and 115);
DVD1317 (comprising SEQ ID NOs: 116 and 117); DVD1318 (comprising SEQ ID NOs: 118 and 119);
DVD1319 (comprising SEQ ID NOs: 120 and 121); DVD1320 (comprising SEQ ID NOs: 122 and 123);
DVD1321 (comprising SEQ ID NOs: 124 and 125); DVD1322 (comprising SEQ ID NOs: 126 and 127);
DVD1323 (comprising SEQ ID NOs: 128 and 129); DVD1324 (comprising SEQ ID NOs: 130 and 131);
DVD1325 (comprising SEQ ID NOs: 132 and 133); DVD1326 (comprising SEQ ID NOs: 134 and 135);
DVD1327 (comprising SEQ ID NOs: 136 and 137); DVD1328 (comprising SEQ ID NOs: 138 and 139);
DVD1329 (comprising SEQ ID NOs: 140 and 141); DVD1330 (comprising SEQ ID NOs: 142 and 143);
DVD1331 (comprising SEQ ID NOs: 144 and 145); DVD1332 (comprising SEQ ID NOs: 146 and 147);
DVD1333 (comprising SEQ ID NOs: 148 and 149); DVD1334 (comprising SEQ ID NOs: 150 and 151);
DVD1335 (comprising SEQ ID NOs: 152 and 153); DVD1336 (comprising SEQ ID NOs: 154 and 155);
DVD1337 (comprising SEQ ID NOs: 156 and 157); DVD1338 (comprising SEQ ID NOs: 158 and 159);
DVD1339 (comprising SEQ ID NOs: 160 and 161); DVD1340 (comprising SEQ ID NOs: 162 and 163);
DVD1341 (comprising SEQ ID NOs: 164 and 165); DVD1342 (comprising SEQ ID NOs: 166 and 167);
DVD1343 (comprising SEQ ID NOs: 168 and 169); DVD1344 (comprising SEQ ID NOs: 170 and 171);
DVD1345 (comprising SEQ ID NOs: 172 and 173); DVD1346 (comprising SEQ ID NOs: 174 and 175);
DVD1347 (comprising SEQ ID NOs: 176 and 177); DVD1348 (comprising SEQ ID NOs: 178 and 179);
DVD1349 (comprising SEQ ID NOs: 180 and 181); DVD1350 (comprising SEQ ID NOs: 182 and 183);
DVD1351 (comprising SEQ ID NOs: 184 and 185); DVD1352 (comprising SEQ ID NOs: 186 and 187);
DVD1353 (comprising SEQ ID NOs: 188 and 189); DVD1354 (comprising SEQ ID NOs: 190 and 191);
DVD1357 (comprising SEQ ID NOs: 192 and 193); DVD1358 (comprising SEQ ID NOs: 194 and 195);
DVD1361 (comprising SEQ ID NOs: 196 and 197); DVD1362 (comprising SEQ ID NOs: 198 and 199);
DVD1363 (comprising SEQ ID NOs: 200 and 201); DVD1364 (comprising SEQ ID NOs: 202 and 203);
DVD1365 (comprising SEQ ID NOs: 204 and 205); DVD1366 (comprising SEQ ID NOs: 206 and 207);
DVD1367 (comprising SEQ ID NOs: 208 and 209); DVD1368 (comprising SEQ ID NOs: 210 and 211);
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DVD1369 (comprising SEQ ID NOs: 212 and 213); DVD1370 (comprising SEQ ID NOs: 214 and 215);
DVD1371 (comprising SEQ ID NOs: 216 and 217); DVD1372 (comprising SEQ ID NOs: 218 and 219);
DVD1373 (comprising SEQ ID NOs: 220 and 221); DVD1374 (comprising SEQ ID NOs: 222 and 223);
DVD1375 (comprising SEQ ID NOs: 224 and 225); DVD1376 (comprising SEQ ID NOs: 226 and 227);
DVD1377 (comprising SEQ ID NOs: 228 and 229); DVD1378 (comprising SEQ ID NOs: 230 and 231);
DVD1379 (comprising SEQ ID NOs: 232 and 233); DVD1380 (comprising SEQ ID NOs: 234 and 235);
DVD1381 (comprising SEQ ID NOs: 236 and 237); DVD1382 (comprising SEQ ID NOs: 238 and 239);
DVD1383 (comprising SEQ ID NOs: 240 and 241); DVD1384 (comprising SEQ ID NOs: 242 and 243);
DVD1385 (comprising SEQ ID NOs: 244 and 245); DVD1386 (comprising SEQ ID NOs: 246 and 247);
DVD1387 (comprising SEQ ID NOs: 248 and 249); DVD1388 (comprising SEQ ID NOs: 250 and 251);
DVD1389 (comprising SEQ ID NOs: 252 and 253); DVD1390 (comprising SEQ ID NOs: 254 and 255);
DVD1391 (comprising SEQ ID NOs: 256 and 257); DVD1392 (comprising SEQ ID NOs: 258 and 259);
DVD1393 (comprising SEQ ID NOs: 260 and 261); DVD1394 (comprising SEQ ID NOs: 262 and 263);
DVD1395 (comprising SEQ ID NOs: 264 and 265); DVD1396 (comprising SEQ ID NOs: 266 and 267);
DVD1397 (comprising SEQ ID NOs: 268 and 269); DVD1398 (comprising SEQ ID NOs: 270 and 271);
DVD1399 (comprising SEQ ID NOs: 272 and 273); DVD 1400 (comprising SEQ ID NOs: 274 and 275);
DVD1401 (comprising SEQ ID NOs: 276 and 277); DVD1402 (comprising SEQ ID NOs: 278 and 279);
DVD1403 (comprising SEQ ID NOs: 280 and 281); DVD1404 (comprising SEQ ED NOs: 282 and 283);
DVD1405 (comprising SEQ ID NOs: 284 and 285); DVbl406 (comprising SEQ ID NOs: 286 and 287);
DVD1407 (comprismg SEQ ID NOs: 288 and 289); DVD1408 (comprising SEQ ID NOs: 290 and 291);
DVD1409 (comprising SEQ ID NOs: 292 and 293); DVD1410 (comprising SEQ ID NOs: 294 and 295);
DVD1411 (comprising SEQ ID NOs: 296 and 297); DVD1412 (comprising SEQ ID NOs: 298 and 299);
DVD1413 (comprising SEQ ID NOs: 300 and 301); DVD1414 (comprising SEQ ID NOs: 302 and 303);
DVD1415 (comprising SEQ ID NOs: 304 and 305); DVD1416 (comprising SEQ ID NOs: 306 and 307);
DVD 1417 (comprising SEQ ID NOs: 3 08 and 3 09); DVD 1418 (comprising SEQ ID NOs: 310 and 311);
DVD1421 (comprising SEQ ID NOs: 312 and 313); DVD1422 (comprising SEQ ID NOs: 314 and 315);
DVD1425 (comprising SEQ ID NOs: 316 and 317); DVD1426 (comprising SEQ ID NOs: 318 and 319);
DVD1427 (comprising SEQ ID NOs: 320 and 321); DVD1428 (comprising SEQ ID NOs: 322 and 323);
DVD1429 (comprising SEQ ID NOs: 324 and 325); DVD1430 (comprising SEQ ID NOs: 326 and 327);
DVD1431 (comprising SEQ ID NOs: 328 and 329); DVD1432 (comprising SEQ ID NOs: 330 and 331);
DVD1433 (comprising SEQ ID NOs: 332 and 333); or DVD1434 (comprising SEQ ID NOs: 334 and
335).
99. The binding protein according to claims 91-94, wherein the VD1 and/or VD2 heavy chain variable
domains, if present, comprise three CDRs from SEQ ID NO: 28,30, 32,34, 36,38,40,42,44,46,48,50,
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^ 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90, respectively; and/or wherein
the VDl and/or VD2 light chain variable domains, if present, comprise three CDRs from SEQ ID NO: 29,
31,33, 35,37,39,41,43,45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85,
87, 89, or 91, respectively.
100. The binding protein according to claims 91-94, wherein the VDl and/or VD2 heavy chain variable
domains, if present, comprise SEQ ID NO: 28,30,32,34,36, 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, or 90, respectively; and/or wherein the VDl
and/or VD2 light chain variable domains, if present, comprise SEQ ID NO: 29,31, 33, 35, 37, 39,41,43,
45,47,49, 51, 53, 55, 57, 59,61, 63,65, 67,69,71,73, 75,77, 79, 81, 83, 85, 87, 89, or 91, respectively.
101. The binding protein according to claims 1,3,9,24, or 91-94, wherein the VDl and VD2 heavy chain
variable domains, if present, comprise three CDRs from SEQ ID NO: 28,30, 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, or 90, respectively; and
wherein the VDl and VD2 light chain variable domains, if present, comprise three CDRs from SEQ ID
NO: 29, 31, 33,35,37, 39,41,43,45,47,49,51, 53, 55, 57, 59, 61,63,65,67,69, 71, 73, 75, 77, 79, 81,
83, 85, 87, 89, or 91, respectively.
102. The binding protein according to claims 1,3,9,24, or 91-94, wherein the VDl and VD2 heavy chain
variable domains, if present, comprise SEQ ID NO: 28, 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, or 90, respectively; and wherein the VDl
and VD2 light chain variable domains, if present, comprise SEQ ID NO: 29,31,33,35,37, 39,41, 43,
45,47,49,51, 53,55, 57, 59,61, 63,65,67,69, 71,73, 75,77,79, 81, 83, 85, 87, 89, or 91, respectively.
103. The method according to claim 26, wherein the VDl and VD2 heavy chain variable domains, if
present, comprise SEQ ID NO: 28,30, 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, or 90, respectively; and wherein the VDl and VD2 light chain
variable domains, if present, comprise SEQ ID NO: 29, 31,33, 35,37, 39,41,43,45,47,49, 51, 53, 55,
57.59, 61,63,65,67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, or 91, respectively.
104. The binding protein according to claims 1,3,9, 24, or 91-94, wherein the (Xl)n between the first
and second light chain variable domains is not CL.
105. The method according to claim 62, wherem the (XI)n between the first and second light chain
variable domains is not CL.
106. The binding protein according to claims 1, 3, 9,24, or 91-94, wherein the binding protein inhibits
NGF with an EC50 of at most about 5.9570 nM, as measured by a TF-1 cell proliferation bioassay.
Dated this M'^ day of February 2013 ( S(r fiS^
of Anand & Anand Advocates
Agent for the Applicant