HK1239711A1

HK1239711A1 - Anti-pdgf-b antibodies and methods of use

Info

Publication number: HK1239711A1
Application number: HK17113113.6A
Authority: HK
Inventors: 马克．贝杜沙; 塞巴斯蒂安．布罗伊尔; 斯特凡．登格尔; 克里斯蒂安．加斯纳; 盖伊．乔治; 萨拜因．格吕纳; 古伊多．哈特曼; 彼得．迈克尔．许尔斯曼; 约尔格．默勒肯; 奥拉夫．蒙迪戈; 约尔格．托马斯．雷古拉; 芭芭拉魏泽尔
Original assignee: 豪夫迈‧罗氏有限公司
Priority date: 2014-11-10
Filing date: 2015-11-06
Publication date: 2018-05-11

Description

anti-PDGF-B antibodies and methods of use

Technical Field

The present invention relates to anti-PDGF-B antibodies and methods of using the same.

Background

Ocular vascular diseases, such as age-related macular degeneration (AMD) and Diabetic Retinopathy (DR), are caused by abnormal choroidal or retinal neovascularization, respectively. They are the main cause of visual loss in industrialized countries. Since the retina is composed of well-defined layers of neurons, glia, and vascular elements, relatively small disturbances, such as those observed in vascular hyperplasia or edema, can lead to significant visual function loss. Hereditary retinal degeneration, such as Retinitis Pigmentosa (RP), is also associated with vascular abnormalities, such as arteriolar stenosis and vascular atrophy. They affect up to 1 person in every 3,500 individuals and are characterized by progressive night blindness (progressive night blindness), visual field loss (visual field loss), optic nerve atrophy (optic nerve atrophy), arteriolar thinning (arterialness), and central loss of vision (central loss of vision) which usually progresses to complete blindness.

Ischemic retinopathy (ischemic retinopathies) is characterized by the loss or dysfunction of retinal blood vessels, which causes reduced blood flow and hypoxia. The retina responds to hypoxia by signaling the growth of new blood vessels, but these new vessels are often fragile and disorganized. It is the growth of these abnormal new blood vessels that poses most of the visual threat because they can leak, cause bleeding, or cause scarring that can cause retinal detachment. The current treatment for ischemic retinopathy seeks to terminate pathological blood vessel growth, but does not address the underlying ischemia that drives its growth. In addition, the standard treatment for diabetic retinopathy, an ischemic retinal neuropathy that affects millions of people, involves laser damage to a portion of the retina in an attempt to stop new blood vessel growth and maintain central vision. Several strategies have been applied to block the function of Vascular Endothelial Growth Factor (VEGF), a major promoter of vascular growth. In the short term, anti-VEGF treatment can improve vision, but it does not address the underlying ischemia, and in fact, may exacerbate this condition as it inhibits all vascular growth, including the growth of beneficial side branches. There is also a serious concern with systemic exposure to these drugs in the elderly and/or diabetic patients who may require new blood vessel growth in the ischemic brain, heart or limbs.

Typically, for ocular diseases, smaller antibody fragments, such as Fab or Fab, are often used by intravitreal application₂Because of its low serum half-life and low risk of systemic toxicity. However, such smaller fragments also typically have a lower intravitreal half-life (e.g., due to faster diffusion into serum) and must typically be administered more frequently.

Multispecific antibodies with domain substitution/exchange (CrossMabVH-VL) in one binding arm are described in WO 2009/080252 and Schaefer, w.et al, proc.natl.acad.sci.usa, 108(2011)11187-11191, which is incorporated herein by reference. They significantly reduce the by-products caused by the mismatch of the light chain against the first antigen and the wrong heavy chain against the second antigen (compared to the method without the domain exchange). However, it is not prepared completely free of by-products. The major by-products are based on the Bence-Jones-type interaction. See also Schaefer, W.et al, Proc.Natl.Acad.Sci.USA, 108(2011) 11187-; in fig. S1I in the supplementary explanation.

In WO 2014/072876 platelet derived growth factor B specific antibodies and compositions and uses thereof are reported. Multivalent antibody substances and methods for the growth factors of the VEGF/PDGF family are reported in WO 2005/087812. Vassbottn, f.s., et al (biochim. biophysis. acta. mol. cell res.1054(1990)246-249) report that monoclonal antibodies directed against the PDGFB-chain inhibit PDGF-induced DNA synthesis in C3H fibroblasts and prevent PDGF binding to its receptor.

SUMMARY

The invention provides anti-PDGF-B antibodies and methods of using the same. In a specific embodiment, the antibody is a humanized antibody.

One aspect as reported herein is an isolated antibody binding to PDGF-B, wherein said antibody is a humanized variant of a murine antibody, wherein said murine antibody comprises the amino acid sequence of SEQ ID NO: 01 and the heavy chain variable domain of SEQ ID NO: 06 light chain variable domain.

In one embodiment of all aspects reported herein, the humanized antibody further comprises in the heavy chain variable domain a tyrosine amino acid residue at position 27, an arginine amino acid residue at position 40, a histidine amino acid residue at position 43 and a tryptophan amino acid residue at position 73 (numbering according to Kabat).

In one embodiment of all aspects reported herein, said humanized antibody further comprises an alanine amino acid residue at position 78 (numbering according to Kabat) in the heavy chain variable domain.

In one embodiment of all aspects reported herein, the humanized antibody further comprises in the heavy chain variable domain a histidine amino acid residue at position 43, an alanine amino acid residue at position 71, and an alanine amino acid residue at position 78 (numbering according to Kabat).

In one embodiment of all aspects reported herein, the humanized antibody further comprises in the light chain variable domain the amino acid residue proline at position 43 and the amino acid residue arginine at position 68 (numbering according to Kabat).

In one embodiment of all aspects reported herein, the humanized antibody further comprises the amino acid residue proline at position 43 (numbering according to Kabat) in the light chain variable domain.

One aspect as reported herein is an antibody specifically binding to human PDGF-B, wherein said antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 02, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 03, and (c) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 05 of the amino acid sequence of HVR-H3.

In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 07 of HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 08 HVR-L2; and (f) comprises SEQ ID NO: 09, HVR-L3 of the amino acid sequence of seq id no.

One aspect as reported herein is an antibody that specifically binds to the same epitope of human PDGF-B as an antibody comprising: (a) comprises the amino acid sequence of SEQ ID NO: 02, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 04, (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 05, (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 07 of HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 08 HVR-L2; and (f) comprises SEQ ID NO: 09, HVR-L3 of the amino acid sequence of seq id no.

One aspect as reported herein is an antibody specifically binding to human PDGF-B, wherein said antibody comprises:

a) (i) comprises SEQ ID NO: 11, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, or HVR-H3 of the amino acid sequence of

b) (i) comprises SEQ ID NO: 16, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 19, or of the amino acid sequence of HVR-H3

c) (i) comprises SEQ ID NO: 21, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24, or of the amino acid sequence of HVR-H3

d) (i) comprises SEQ ID NO: 26, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 29, or of the amino acid sequence of HVR-H3

e) (i) comprises SEQ ID NO: 31, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 of the amino acid sequence of HVR-H3, or

f) (i) comprises SEQ ID NO: 36, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37 and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39, HVR-H3 of the amino acid sequence of seq id no.

In one embodiment, the antibody further comprises:

a) (vi) comprises SEQ ID NO: 41, (v) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 42, (vi) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 43, or of the amino acid sequence of HVR-L3

b) (vi) comprises SEQ ID NO: 45, (v) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (vi) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 47, or

c) (vi) comprises SEQ ID NO: 49, (v) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 50, (vi) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 51, or of the amino acid sequence of HVR-L3

d) (vi) comprises SEQ ID NO: 53, (v) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 54, (vi) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 55, HVR-L3.

In one embodiment, the antibody comprises:

a) comprises the amino acid sequence of SEQ ID NO: 10, or

b) Comprises the amino acid sequence of SEQ ID NO: 15, or

c) Comprises the amino acid sequence of SEQ ID NO: 20, or

d) Comprises the amino acid sequence of SEQ ID NO: 25, or

e) Comprises the amino acid sequence of SEQ ID NO: 30, or

f) Comprises the amino acid sequence of SEQ ID NO: 35, or a light chain variable domain of the amino acid sequence of seq id no.

In one embodiment, the antibody further comprises:

a) comprises the amino acid sequence of SEQ ID NO: 40, or

b) Comprises the amino acid sequence of SEQ ID NO: 44, or

c) Comprises the amino acid sequence of SEQ ID NO: 48, or

d) Comprises the amino acid sequence of SEQ ID NO: 52, or a variable light chain domain of the amino acid sequence of seq id no.

One aspect as reported herein is an antibody specifically binding to human PDGF-B, wherein said antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 57, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 60, HVR-H3.

In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 62, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 63, HVR-L2 of the amino acid sequence of seq id no; and (f) comprises SEQ ID NO: 64, HVR-L3 of the amino acid sequence of seq id no.

One aspect as reported herein is an antibody specifically binding to human PDGF-B, wherein said antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 66, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, and (c) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 69, HVR-H3 of the amino acid sequence of seq id no.

In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 71 amino acid sequence HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 72, HVR-L2 of the amino acid sequence of; and (f) comprises SEQ ID NO: 73, HVR-L3.

One aspect as reported herein is an antibody specifically binding to human PDGF-B, wherein said antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 75, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, and (c) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 78, HVR-H3 of the amino acid sequence of seq id no.

In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 80, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 81 by HVR-L2 of the amino acid sequence of seq id no; and (f) comprises SEQ ID NO: 82, HVR-L3.

One aspect as reported herein is an antibody specifically binding to human PDGF-B, wherein said antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 84, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 85, and (c) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 87 by HVR-H3.

In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 89 amino acid sequence HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 90, HVR-L2 of the amino acid sequence of seq id no; and (f) comprises SEQ ID NO: 91, HVR-L3 of the amino acid sequence of seq id no.

One aspect as reported herein is an antibody specifically binding to human PDGF-B, wherein said antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 93, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 94, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 96, HVR-H3.

In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 98, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 99, HVR-L2 of the amino acid sequence of; and (f) comprises SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no.

One aspect as reported herein is an antibody specifically binding to human PDGF-B, wherein said antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 102, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 103, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 105, HVR-H3.

In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 107, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 108, HVR-L2; and (f) comprises SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no.

One aspect as reported herein is an antibody comprising the amino acid sequence of SEQ ID NO: 10 and a heavy chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 44. SEQ ID NO: 48 and SEQ ID NO: 52.

One aspect as reported herein is an antibody comprising the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 44. SEQ ID NO: 48 and SEQ ID NO: 52.

One aspect as reported herein is an antibody comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 44. SEQ ID NO: 48 and SEQ ID NO: 52, and a light chain variable domain amino acid sequence.

One aspect as reported herein is an antibody comprising the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 44. SEQ ID NO: 48 and SEQ ID NO: group 52 light chain variable domain amino acid sequences.

One aspect as reported herein is an antibody comprising the amino acid sequence of SEQ ID NO: 30 and a heavy chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 44. SEQ ID NO: 48 and SEQ ID NO: 52.

One aspect as reported herein is an antibody comprising the amino acid sequence of SEQ ID NO: 35 and a heavy chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 44. SEQ ID NO: 48 and SEQ ID NO: 52, and a light chain variable domain amino acid sequence.

One aspect reported herein is an antibody that specifically binds human PDGF-BB and human PDGF-AB but not human PDGF-AA.

In one embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 57, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58, (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 60, (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 62, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 63, HVR-L2 of the amino acid sequence of seq id no; and (f) comprises SEQ ID NO: 64, HVR-L3 of the amino acid sequence of seq id no.

In one embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 66, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (c) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 69, (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 71 amino acid sequence HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 72, HVR-L2 of the amino acid sequence of; and (f) comprises SEQ ID NO: 73, HVR-L3.

In one embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 75, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 78, (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 81 by HVR-L2 of the amino acid sequence of seq id no; and (f) comprises SEQ ID NO: 82, HVR-L3.

In one embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 84, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 85, (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 87, (d) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 89 amino acid sequence HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 90, HVR-L2 of the amino acid sequence of seq id no; and (f) comprises SEQ ID NO: 91, HVR-L3 of the amino acid sequence of seq id no.

In one embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 93, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 94, (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 96, (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 98, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 99, HVR-L2 of the amino acid sequence of; and (f) comprises SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no.

In one embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 102, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 103, (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 105, (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 107, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 108, HVR-L2; and (f) comprises SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no.

One aspect reported herein is an antibody that specifically binds human PDGF-BB and PDGF-AB as well as PDGF-AA.

In one embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 02, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 03, and (c) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 05 of the amino acid sequence of HVR-H3..

In one embodiment, the antibody further comprises: (a) comprises the amino acid sequence of SEQ ID NO: 07 of HVR-L1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 08 HVR-L2; and (c) comprises SEQ ID NO: 09, HVR-L3 of the amino acid sequence of seq id no.

In one embodiment of all aspects reported herein, the antibody is of the subclass human IgG1 or human IgG 4.

In one embodiment of all aspects reported herein, the antibody is of the subclass human IgG1 with kappa light chain.

In one embodiment of all aspects reported herein, said antibody is a monoclonal antibody.

In one embodiment, the antibody is a bispecific antibody.

In one embodiment of all aspects, the antibody specifically binds human PDGF-B but not human PDGF-C.

In one embodiment of all aspects, the antibody specifically binds human, rat, mouse and cynomolgus PDGF-B.

In one embodiment of all aspects, the antibody inhibits the biological activity of human PDGF-B by inhibiting the binding of PDGF-B to its receptor.

In one embodiment of all aspects, the antibody inhibits the biological activity of human PDGF-B by inhibiting cell migration.

In one embodiment of all aspects, the antibody inhibits the biological activity of human PDGF-B by inhibiting PDGF-B receptor phosphorylation.

In one embodiment of all aspects, the antibody inhibits the biological activity of human PDGF-B by inhibiting cell proliferation.

One aspect as reported herein is a (isolated) nucleic acid encoding an antibody as reported herein.

One aspect as reported herein is a host cell comprising a nucleic acid as reported herein.

One aspect as reported herein is a method for producing an antibody as reported herein, said method comprising culturing a host cell as reported herein, thereby producing said antibody, and recovering said antibody from said host cell or the culture medium.

One aspect as reported herein is a pharmaceutical formulation comprising an antibody as reported herein and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical formulation further comprises an additional therapeutic agent. In one embodiment, the additional therapeutic agent is an anti-ANG 2 antibody or an anti-VEGF antibody.

One aspect as reported herein is an antibody as reported herein for use as a medicament.

One aspect as reported herein is an antibody as reported herein for use in the treatment of an ocular vascular disease, preferably for use in the treatment of macular degeneration.

The antibodies reported herein are useful for inhibiting the interaction between PDGF-B and its receptor.

The antibodies reported herein are useful for inhibiting cell migration.

The antibodies reported herein are useful for inhibiting proliferation.

One aspect as reported herein is the use of an antibody as reported herein for the preparation of a medicament.

In one embodiment, the medicament is for the treatment of ocular vascular diseases, preferably for the treatment of macular degeneration.

In one embodiment, the medicament is for inhibiting the interaction between PDGF-B and its receptor.

One aspect as reported herein is a method of treating an individual having an ocular vascular disease, preferably having macular degeneration, said method comprising administering to said individual an effective amount of an antibody as reported herein.

One aspect as reported herein is a method for inhibiting the interaction between PDGF-B and its receptor in an individual, said method comprising administering to said individual an effective amount of an antibody as reported herein, thereby inhibiting the interaction between PDGF-B and its receptor.

Detailed description of the embodiments

I. Definition of

An "acceptor human framework" for the purposes herein is a framework comprising an amino acid sequence derived from a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework of a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence alterations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K)_d) And (4) showing. Affinity can be measured by common methods known in the art, including those described herein. Specific exemplary and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody not having such alterations.

The terms "anti-PDGF-B antibody" and "antibody that binds PDGF-B" refer to antibodies that: which is capable of binding PDGF-B with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting PDGF-B. In one embodiment, the extent of binding of the anti-PDGF-B antibody to an unrelated, non-PDGF-B protein is less than about 10% of the binding of the antibody to PDGF-B as measured, for example, by ELISA or surface plasmon resonance. In certain embodiments, the antibody that binds PDGF-B has a ≤ 1 μ M,100nM or less, 10nM or less, 1nM or less, or 0.1nM or less (e.g., 10nM or less)^-8M or less, e.g. 10^-8M to 10^-10M, e.g. 10^-9M to 10^-10M) dissociation constant (KD). In certain embodiments, the anti-PDGF-B antibody binds an epitope of PDGF-B that is conserved between PDGF-B from different species.

The term "antibody" is used herein in the broadest sense and includes a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

By "antibody fragment" is meant a molecule other than an intact antibody, which molecule comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to: fv, Fab '-SH, F (ab')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from a plurality of antibody fragments.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that, like the reference antibody, has interactions with at least some of the same amino acid residues. For example, these interactions are ionic interactions between charged amino acid residues or hydrophobic interactions between hydrophobic amino acid residues.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "type" of an antibody indicates the type of constant domain or constant region that its heavy chain has. There are five major antibody types: IgA, IgD, IgE, IgG and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. Corresponds to notThe heavy chain constant domains of the same type of immunoglobulin are designated α, γ, and μ, respectively.

The term "immunoconjugate" means a covalent conjugate between an antibody and a non-antibody moiety. The non-antibody moiety may be a detection label, an effector molecule or a cytotoxic agent.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioisotope (e.g., At)²¹¹，I¹³¹，I¹²⁵，Y⁹⁰，Re¹⁸⁶，Re¹⁸⁸，Sm¹⁵³，Bi²¹²，P³²，Pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamicin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin c (mitomycin c), chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof such as nucleolytic enzymes; (ii) an antibiotic; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed below.

"effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody species. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B-cell activation.

An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or C-terminal glycyl-lysine dipeptide (Gly446Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIHPublication 91-3242.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the VH (or VL) in the following order: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full length antibody", "intact antibody" and "intact antibody" are used interchangeably herein to refer to an antibody that: having a structure substantially similar to that of a native antibody or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom (regardless of the number of passages). Progeny may not be identical to the parent cell in terms of nucleic acid content, but may contain mutations. Progeny of mutants screened or selected for the originally transformed cell to have the same function or biological activity are included herein.

A "human antibody" is an antibody that: the amino acid sequence of which corresponds to that of an antibody produced by a human or human cell or derived from an antibody of non-human origin using a repertoire of human antibodies (reptoires) or other human antibody coding sequences. The definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

A "human consensus framework" is a framework representing the amino acid residues most frequently occurring in the selection of human immunoglobulin VL or VH framework sequences. Typically, the human immunoglobulin VL or VH sequence is selected from a subset of variable domain sequences. Typically, a subset of Sequences is a subset as in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Bethesda MD (1991), NIH Publication 91-3242, volumes 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al (supra). In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al (supra).

By "humanized" antibody is meant a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of an antibody (e.g., a non-human antibody) refer to an antibody that has undergone humanization.

The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain which is hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or which forms structurally defined loops ("hypervariable loops") and/or which contains antigen-contacting residues ("antigen-contacting points"). Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

HVRs herein include:

(a) hypervariable loops (Chothia, C. and Lesk, A.M., J.mol.biol.196(1987)901-917) which are present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101 (H3);

(b) CDRs present at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2) and 95-102(H3) (Kabat, E.A. et al, Sequences of Proteins of immunological interest, 5 th edition Public Health Service, national institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242);

(c) antigen contacts present at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2) and 93-101(H3) (MacCallum et al J.mol.biol.262: 732-745 (1996)); and

(d) a combination of (a), (b), and/or (c) that comprises HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b (H1), 49-65(H2), 93-102(H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al (supra).

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods of assessing antibody purity, see, e.g., Flatman, s. et al, j.chromaogr.b 848(2007) 79-87.

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

By "isolated nucleic acid encoding an anti-PDGF-B antibody" is meant one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

The term "monoclonal antibody" as used herein means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, with the exception of possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of a monoclonal antibody preparation), such variants typically being present in minute amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of a human immunoglobulin locus, such methods and other exemplary methods of preparing monoclonal antibodies being described herein.

By "naked antibody" is meant an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in pharmaceutical formulations.

"Natural antibody" means a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, each light chain has, from N-to C-terminus, a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a constant light Chain (CL) domain. The light chains of antibodies can be classified into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

The term "package insert" is used to refer to instructions for use typically included in commercial packaging of therapeutic products, which contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or cautions for the use of such therapeutic products.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, aligned, with gaps introduced, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences to be aligned. However, for purposes herein, the use of the sequence alignment computer program ALIGN-2 results in% amino acid sequence identity values. The ALIGN-2 sequence alignment computer program was created by Genentech, inc, and the source code has been submitted with the user documentation at the us copyright Office (u.s.copyright Office, Washington d.c., 20559), which is registered under us copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems (including digital UNIX V4.0D). All alignment parameters were set by the ALIGN-2 program and were not changed.

In the case of amino acid sequence alignment using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence a relative to, with, or against a given amino acid sequence B (which may alternatively be recited as a given amino acid sequence a having or comprising a particular% amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows:

100X fraction X/Y

Wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 (in the alignment of A and B of this program), and wherein Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. Unless otherwise specifically stated, all% amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term "pharmaceutical formulation" denotes a preparation: in a form effective to render the biological activity of the active ingredient contained therein, and which does not contain additional components having unacceptable toxicity to the subject to which the formulation is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of a pharmaceutical formulation other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "PDGF-B" as used herein refers to human PDGF-B. The term includes "full length," unprocessed PDGF-B as well as any form of PDGF-B that results from processing in the cell. The term also includes naturally occurring PDGF-B variants, e.g., splice variants or allelic variants. The amino acid sequence of human PDGF-B is shown in SEQ ID NO: 110.

As used herein, "treatment" (and grammatical variants thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a treated individual, and may be performed for prophylaxis or in the course of clinical pathology. Desired therapeutic effects include, but are not limited to: preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating a disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the progression of the disease or slow the progression of the disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) typically have similar structures, with each domain comprising four conserved Framework Regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, t.j. et al. Kuby Immunology, 6 th edition, w.h.freeman and co., n.y. (2007), page 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using a library of complementary VL or VH domains, respectively, screened using VH or VL domains from antibodies that bind the antigen (see, e.g., Portolano, S. et al, J.Immunol.150(1993) 880-628; Clackson, T. et al, Nature 352(1991) 624-628).

The term "vector" as used herein denotes a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

Compositions and methods

The monoclonal antibodies of the invention specifically bind to determinant sites on the PDGF-B molecule that are involved in the receptor binding and proliferative activity of PDGF-B. The monoclonal humanized antibody does not bind PDGF-C. Specific antibodies reported herein bind to human PDGF-BB and PDGF-AB but not PDGF-AB. Other specific antibodies reported herein bind human PDGF-BB and PDGF-AB as well as PDGF-AA.

A. Exemplary anti-PDGF-B antibodies

Provided herein are various novel anti-human PDGF-B antibodies.

The first anti-PDGF-B antibody is a polypeptide having the amino acid sequence of SEQ ID NO: 01 and the VH of SEQ ID NO: 06 VL of the specification. This antibody is hereinafter referred to as mumAb. The antibodies bind human, cynomolgus monkey, rat and murine PDGF-B and inhibit the interaction between PDGF-B and its receptor.

The antibodies have the following characteristics listed in the following table.

	kd[1/s]	t1/2[min]
			mumAb	4.50E-04	26

The table below shows the storage stability of the antibodies (active concentration with reference surface).

The thermal stability of the antibodies was evaluated by determining the aggregation onset temperature (Tagg) and the melting point (Tm) (see table below).

	Tagg[℃]	Tm[℃]
			mumAb	57	62-63.5

Based on the amino acid sequences of murine mumAB (SEQ ID NO: 01 and SEQ ID NO: 06), corresponding humanized anti-PDGF-B antibodies were generated. Humanized variants of VH are based on human germline IMGT _ hVH _1_69, IMGT _ hVH _4_59 and IMGT _ hVH _3_23 in combination with human J-element germline IGHJ 5-01.

In the IMGT _ hVH _1_69 variants, a back mutation was introduced at position 27(G27Y) of framework region 1 and/or at position 40(a40R) and/or at position 43(Q43H) of framework region 2 and/or at position 73(E73T) of framework region 3.

In IMGT _ hVH _4_59, a back-mutation was introduced at position 78(F78A) of framework region 3. In IMGT _ hVH _3_23, back mutations were introduced at position 43 of framework region 2 (K43H) and at positions 71 of framework region 3 (R71A) and 78 (L78A).

Humanized variants of VL are based on the human germline IMGT _ hVK _3_20, IMGT _ hVK _4_1 and IMGT _ hVK _1_27 in combination with the human J-element germline IGKJ 2-01.

For one variant, in IMGT hVK _3_20, a back mutation was introduced at position 43 of framework region 2 (a43P) and at position 68 of framework region 3 (G68R). In IMGT hVK _1_27, a back-mutation was introduced at position 43(V43P) of framework region 3.

This antibody is represented as antibody 0044, which is a monospecific antibody, and antibody 0146, which is a bispecific CrossMab with additional ANG2 binding specificity.

The remaining antibodies were human antibodies obtained from human Ig locus transgenic rabbits. The antibodies have the binding characteristics listed in the table below.

In one embodiment, the humanized anti-PDGF-B antibody binds human, rat, mouse, and cynomolgus PDGF-B.

In the diversity activity assay, the antibodies exhibited biological activity as can be seen from the data set forth in the table below.

The kinetic binding properties of the different antibodies were determined using surface plasmon resonance techniques (see table below).

	kd[1/s]	t1/2[min]
			Antibody-0058	2.91E-04	40
Antibody-0060	2.64E-04	44
			Antibody-0064	1.35E-04	85
Antibody-0085	1.02E-04	114
			Antibody-0086	7.93E-05	146

Fab	ka[1/Ms]	kd[1/s]	KD＊[nM]	t1/2[s]
					Antibody-0106	2.02E+05	3.74E-04	2	1853
Antibody-0107	1.95E+05	3.62E-04	2	1915
					Antibody-0108	3.96E+05	7.73E-03	20	90
Antibody-0109	3.14E+05	1.36E-02	43	51
					Antibody-0110	1.30E+05	1.01E-03	8	686

The table below shows the storage stability (active concentration with reference surface) of the different antibodies.

	Tagg[℃]	Tm[℃]
			Antibody-0086	64	64.5-68

Antibody 0144 is a bispecific anti-ANG 2/PDGF-B antibody comprising the VH and VL domains of antibody 0085 as the PDGF-B binding specificity.

Antibody 0117 is a bispecific anti-VEGF/PDGF-B antibody comprising the VH and VL domains of antibody 0085 as PDGF-B binding specificity.

To determine kinetic binding values, the assay reported in example 32 was used.

ANG2	ka[1/Ms]	kd[1/s]	KD＊[nM]	t1/2[s]
					Antibody-0144	9.06E+04	1.55E-03	17	446

VEGF	ka[1/Ms]	kd[1/s]	KD＊[nM]	t1/2[s]
					Antibody-0117	2.46E+04	＜1E-06	＜0.1	-

It has been demonstrated by SPR analysis that all bispecific antibodies have the property of binding to both of their antigens simultaneously.

The bispecific antibodies exhibit binding and biological activity in cell-based assays.

In the ANG 2-specific pTie2-ELISA, the activity of antibody 0144 was 6-fold higher than the anti-ANG 2/VEGF antibody reported in WO 2014/09465.

In a VEGF-specific reporter assay, antibody 0117 has similar activity to the anti-ANG 2/VEGF antibody reported in WO 2014/09465.

It is described that antibody 0085 has the amino acid sequence of SEQ ID NO: 92-100 (binding site, HVR, VH, VL). Bispecific versions of antibody 0085 are described in the sequence SEQ ID NO: 131-, 134-, 147-, 150-, and 163-166. All of these sequences constitute aspects of the invention, either individually or in combination.

It is described that antibody 0086 has the amino acid sequence of SEQ ID NO: 101-109 (binding site, HVR, VH, VL). Bispecific versions of antibody 0086 are described in the sequence SEQ ID NO: 135, 151, 154, and 167. All of these sequences constitute aspects of the invention, either individually or in combination.

It is described that antibody 0144 has the sequence of SEQ ID NO: 147-150 (CrossMab format, 2 heavy chains, 2 light chains).

Record that antibody 0117 has the amino acid sequence of SEQ ID NO: 171-174 (CrossMab format, 2 heavy chains, 2 light chains).

Antibodies 0144 and 0145 were incubated at different pH values for 2 weeks and then determined to bind PDGF-BB and ANG2, respectively.

One aspect as reported herein is a humanized anti-human PDGF-B antibody comprising: (a) comprises the amino acid sequence of SEQ ID NO: 02, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 03 by HVR-H2; and (c) a nucleic acid comprising SEQ id no: 05 of the amino acid sequence of HVR-H3. In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence shown in SEQ ID NO: 07 of HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 08 HVR-L2; and (f) comprises SEQ ID NO: 09, HVR-L3 of the amino acid sequence of seq id no.

One aspect as reported herein is a humanized anti-human PDGF-B antibody comprising: (a) comprises the amino acid sequence of SEQ ID NO: 02, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 04, HVR-H2 of the amino acid sequence of seq id no; and (c) a nucleic acid comprising SEQ id no: 05 of the amino acid sequence of HVR-H3. In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence shown in SEQ ID NO: 07 of HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 08 HVR-L2; and (f) comprises SEQ ID NO: 09, HVR-L3 of the amino acid sequence of seq id no.

One aspect as reported herein is an anti-human PDGF-B antibody comprising: (a) comprises the amino acid sequence of SEQ ID NO: 93, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 94, HVR-H2 of the amino acid sequence of seq id no; and (c) comprises SEQ ID NO: 96, HVR-H3. In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 98, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 99, HVR-L2 of the amino acid sequence of; and (f) comprises SEQ id no: 100, and HVR-L3 of the amino acid sequence of seq id no.

In one aspect, the invention provides an anti-PDGF-B antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) comprises the amino acid sequence of SEQ ID NO: 93, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence shown in SEQ ID NO: 95 of the amino acid sequence of HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 96, HVR-H3 of the amino acid sequence of seq id no; (d) comprises SEQ ID NO: 98, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 99, HVR-L2 of the amino acid sequence of; and (f) comprises SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no.

In a preferred embodiment, the anti-PDGF-B antibody reported herein comprises: (a) comprises the amino acid sequence of SEQ ID NO: 93, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 95 of the amino acid sequence of HVR-H2; (c) comprises the amino acid sequence shown in SEQ ID NO: 96, HVR-H3 of the amino acid sequence of seq id no; (d) comprises the amino acid sequence of SEQ ID NO: 98, HVR-L1 of the amino acid sequence of seq id no; (e) comprises SEQ ID NO: 99, HVR-L2 of the amino acid sequence of; and (f) comprises SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no. In a further preferred embodiment, the anti-PDGF-B antibody reported herein comprises a heavy chain having the amino acid sequence of SEQ ID NO: 92 and a VH having the amino acid sequence of SEQ ID NO: 97, VL of an amino acid sequence of seq id no. In yet another preferred embodiment, the anti-PDGF-B antibody is a bispecific antibody.

In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) comprises the amino acid sequence of SEQ ID NO: 93, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 94, HVR-H2 of the amino acid sequence of seq id no; and (c) comprises SEQ ID NO: 96, HVR-H3. In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 96, HVR-H3. In another embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 96 and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no. In another embodiment, the antibody comprises: comprises the amino acid sequence of SEQ ID NO: 96 comprising the amino acid sequence of SEQ ID NO: 100, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 94, HVR-H2 of the amino acid sequence of seq id no. In another embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 93, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 95 of the amino acid sequence of HVR-H2; and (c) comprises SEQ ID NO: 96, HVR-H3.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VLHVR sequences selected from: (a) comprises the amino acid sequence of SEQ ID NO: 98, HVR-L1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 99, HVR-L2 of the amino acid sequence of; and (c) comprises SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no. In one embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 98, HVR-L1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 99, HVR-L2 of the amino acid sequence of; and (c) comprises SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) comprises the amino acid sequence of SEQ ID NO: 93, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 94, and (iii) a HVR-H2 comprising an amino acid sequence selected from seq id NOs: 96, HVR-H3 of the amino acid sequence of seq id no; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) comprises the amino acid sequence of SEQ ID NO: 98, (ii) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 99, and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, the invention provides an antibody comprising: (a) comprises the amino acid sequence of SEQ ID NO: 93, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 95 of the amino acid sequence of HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 96, HVR-H3 of the amino acid sequence of seq id no; (d) comprises the amino acid sequence of SEQ ID NO: 98, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 99, HVR-L2 of the amino acid sequence of; and (f) comprises a sequence selected from SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, the anti-PDGF-B antibody comprises an amino acid sequence identical to SEQ ID NO: 92 has a heavy chain variable domain (VH) sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. A VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence comprises a substitution (e.g., a conservative substitution), insertion, or deletion, but an anti-PDGF-B antibody comprising the sequence retains the ability to bind PDGF-B. In certain embodiments, SEQ ID NO: 92 of which a total of 1-10 amino acids are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-PDGF-B antibody comprises SEQ ID NO: 92, comprising a post-translational modification of said sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) comprises SEQ ID NO: 93, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 94, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 96, HVR-H3.

In another aspect, an anti-PDGF-B antibody is provided, wherein the antibody comprises an amino acid sequence identical to SEQ ID NO: 97 has a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence comprises a substitution (e.g., a conservative substitution), insertion, or deletion, but an anti-PDGF-B antibody comprising the sequence retains the ability to bind PDGF-B. In certain embodiments, SEQ ID NO: 97 to a total of 1-10 amino acids are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-PDGF-B antibody comprises SEQ id no: 97, comprising a post-translational modification of said sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from: (a) comprises the amino acid sequence of SEQ ID NO: 98, HVR-L1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence shown in SEQ ID NO: 99, HVR-L2 of the amino acid sequence of; and (c) comprises SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, there is provided an anti-PDGF-B antibody, wherein the antibody comprises a VH in any one of the embodiments provided above and a VL in any one of the embodiments provided above. In one embodiment, the antibody comprises the amino acid sequences set forth in SEQ ID NOs: 92 and SEQ ID NO: 97, including post-translational modifications of these sequences.

One aspect as reported herein is an anti-human PDGF-B antibody comprising: (a) comprises the amino acid sequence of SEQ ID NO: 102, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 103, HVR-H2 of the amino acid sequence of seq id no; and (c) comprises SEQ ID NO: 105, HVR-H3. In one embodiment, the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 107, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 108, HVR-L2; and (f) comprises SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no.

In one aspect, the invention provides an anti-PDGF-B antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) comprises the amino acid sequence of SEQ ID NO: 102, HVR-H1 of the amino acid sequence of seq id no; (b) comprises SEQ ID NO: 104, HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 105, HVR-H3; (d) comprises the amino acid sequence of SEQ ID NO: 107, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 108, HVR-L2; and (f) comprises SEQ ID NO: 109. HVR-L3.

In a preferred embodiment, the anti-PDGF-B antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 102, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 104, HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 105, HVR-H3; (d) comprises the amino acid sequence of SEQ ID NO: 107, HVR-L1; (e) comprises SEQ ID NO: 108, HVR-L2; and (f) comprises SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no. In a further preferred embodiment, the anti-PDGF-B antibody reported herein comprises a heavy chain having the amino acid sequence of SEQ ID NO: 101 and a VH having the amino acid sequence of SEQ ID NO: 106, VL of an amino acid sequence of seq id no. In yet another preferred embodiment, the anti-PDGF-B antibody is a bispecific antibody.

In one aspect, the invention provides an antibody comprising at least one, at least two or all three VHHVR sequences selected from: (a) comprises the amino acid sequence of SEQ ID NO: 102, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 103, HVR-H2 of the amino acid sequence of seq id no; and (c) comprises SEQ ID NO: 105, HVR-H3. In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 105, HVR-H3. In another embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 105 and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no. In another embodiment, the antibody comprises: comprises the amino acid sequence shown in SEQ ID NO: 105, comprising the amino acid sequence of SEQ ID NO: 109, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 103, HVR-H2. In another embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 102, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 104, HVR-H2; and (c) comprises SEQ ID NO: 105, HVR-H3.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VLHVR sequences selected from: (a) comprises the amino acid sequence of SEQ ID NO: 107, HVR-L1; (b) comprises the amino acid sequence of SEQ ID NO: 108, and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no. In one embodiment, the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 107, HVR-L1; (b) comprises the amino acid sequence of SEQ ID NO: 108, HVR-L2; and (c) comprises SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) comprises the amino acid sequence of SEQ ID NO: 102, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 103, and (iii) an HVR-H2 comprising an amino acid sequence selected from SEQ ID NOs: 105, HVR-H3; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) comprises the amino acid sequence of SEQ ID NO: 107, (ii) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 108, and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, the invention provides an antibody comprising: (a) comprises the amino acid sequence of SEQ ID NO: 102, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 104, HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 105, HVR-H3; (d) comprises the amino acid sequence of SEQ ID NO: 107, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 108, HVR-L2; and (f) comprises a sequence selected from SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, the anti-PDGF-B antibody comprises an amino acid sequence identical to SEQ ID NO: 101 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the heavy chain variable domain (VH) sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence comprises a substitution (e.g., a conservative substitution), insertion, or deletion, but an anti-PDGF-B antibody comprising the sequence retains the ability to bind PDGF-B. In certain embodiments, seq id NO: 101 with a total of 1-10 amino acids substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-PDGF-B antibody comprises SEQ ID NO: 101, including post-translational modifications of said sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) comprises the amino acid sequence of SEQ ID NO: 102, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 103, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 105, HVR-H3.

In another aspect, an anti-PDGF-B antibody is provided, wherein the antibody comprises an amino acid sequence identical to SEQ ID NO: 106 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a light chain variable domain (VL). In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence comprises a substitution (e.g., a conservative substitution), insertion, or deletion, but an anti-PDGF-B antibody comprising the sequence retains the ability to bind PDGF-B. In certain embodiments, SEQ ID NO: 106 in total 1-10 amino acids are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-PDGF-B antibody comprises seq id NO: 106, comprising a post-translational modification of said sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from: (a) comprises the amino acid sequence of SEQ ID NO: 107, HVR-L1; (b) comprises the amino acid sequence of SEQ ID NO: 108, HVR-L2; and (c) comprises SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no.

One aspect as reported herein is an antibody comprising two heavy chains and two light chains, wherein: a) the first heavy chain has seq id NO: 147, and the second heavy chain has the amino acid sequence of SEQ ID NO: 148, and a first light chain having the amino acid sequence of seq id NO: 149 and the second light chain has the amino acid sequence of SEQ ID NO: 150, or b) the antibody comprises the amino acid sequence of item a) including a post-translational modification (being a mutation in the Fc region).

In a preferred embodiment, the bispecific antibody reported herein has the mutations P329G, L234A, L235A, I253A, H310A and H434A (numbering according to Kabat index).

In another aspect, an anti-PDGF-B antibody is provided, wherein the antibody comprises a VH as in any one of the embodiments provided above, and a VL as in any one of the embodiments provided above. In one embodiment, the antibody comprises the amino acid sequences set forth in SEQ ID NOs: 101 and SEQ ID NO: 106, including post-translational modifications of those sequences.

In another aspect of the invention, the anti-PDGF-B antibody in any of the above embodiments is a monoclonal antibody.

In one embodiment, the anti-PDGF-B antibody is an antibody fragment, e.g., Fv, Fab, Fab ', scFv, diabody, or F (ab')₂And (3) fragment.

In another embodiment, the antibody is a full length antibody, e.g., a complete IgG1 antibody or other antibody types or isotypes as defined herein.

In one embodiment of all aspects reported herein, said anti-PDGF-B antibody is an effector-silenced anti-PDGF-B antibody. In one embodiment of all aspects reported herein, the anti-PDGF-B antibody is an effector-silenced anti-PDGF-B antibody and does not bind human FcRn. In one embodiment of all aspects reported herein is an anti-PDGF-B antibody of the subclass human IgG1 and has the mutations L234A, L235A, P329G, I253A, H310A and H434A (numbering according to Kabat index) in both heavy chains.

In one embodiment of all aspects reported herein, said anti-PDGF-B antibody is a bispecific antibody.

One aspect as reported herein is a bivalent, bispecific antibody comprising:

a) a first light chain and a first heavy chain of an antibody that specifically binds a first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other,

wherein the first antigen or the second antigen is human PDGF-B.

a) The antibody of (a) does not comprise the modification reported in b), and the heavy and light chains of a) are separate chains.

Among the antibodies in the step b), the antibodies,

in the light chain

The variable light chain domain VL is replaced with the variable heavy chain domain VH of the antibody,

and is

In the heavy chain

The variable heavy chain domain VH is replaced with the variable light chain domain VL of the antibody.

In one embodiment of the process of the present invention,

i) in the constant domain CL of the first light chain in a), the amino acid at position 124 (numbering according to Kabat) is substituted with a positively charged amino acid, and wherein in the constant domain CH1 of the first heavy chain in a), the amino acid at position 147 or the amino acid at position 213 (numbering according to the EU index of Kabat) is substituted with a negatively charged amino acid,

or

ii) in b) the constant domain CL of the second light chain, the amino acid at position 124 (numbering according to Kabat) is substituted with a positively charged amino acid, and wherein in b) in the constant domain CH1 of the second heavy chain, the amino acid at position 147 or the amino acid at position 213 (numbering according to Kabat EU index) is substituted with a negatively charged amino acid.

In a preferred embodiment of the process according to the invention,

i) in the constant domain CL of the first light chain in a), the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat) (in a preferred embodiment, independently substituted with lysine (K) or arginine (R)), and wherein in the constant domain CH1 of the first heavy chain in a), the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index),

or

ii) in the constant domain CL of the second light chain in b) the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in a preferred embodiment, independently substituted with lysine (K) or arginine (R)), and wherein in the constant domain CH1 of the second heavy chain in b) the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In one embodiment, in the constant domain CL of the second heavy chain, the amino acid substitutions at positions 124 and 123 are K (numbering according to the EU index of Kabat).

In one embodiment, in the constant domain CH1 of the second light chain, the amino acid substitutions at positions 147 and 213 are E (numbering according to the EU index of Kabat).

In a preferred embodiment, in the constant domain CL of the first light chain the amino acids at positions 124 and 123 are substituted with K and in the constant domain CH1 of the first heavy chain the amino acids at positions 147 and 213 are substituted with E (numbering according to the EU index of Kabat).

In one embodiment, in the constant domain CL of the second heavy chain the amino acids at positions 124 and 123 are substituted with K, and wherein in the constant domain CHl of the second light chain the amino acids at positions 147 and 213 are substituted with E, and in the domain VL of the first light chain the amino acid at position 38 is substituted with K, in the variable domain VH of the first heavy chain the amino acid at position 39 is substituted with E, in the variable domain VL of the second heavy chain the amino acid at position 38 is substituted with K, and in the variable domain VH of the second light chain the amino acid at position 39 is substituted with E (numbering according to the EU index of Kabat).

One aspect as reported herein is a bivalent, bispecific antibody comprising

b) a second light chain and a second heavy chain of an antibody that specifically binds a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other, and wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other,

wherein the first antigen or the second antigen is human PDGF-B.

a) The antibody of (a) does not comprise the modification reported in b), and the heavy and light chain of a) are separate chains.

In the antibody of b)

In the light chain

The variable light domain VL is replaced by the variable heavy domain VH of the antibody and the constant light domain CL is replaced by the constant heavy domain CH1 of the antibody; and is

In the heavy chain

The variable heavy domain VH is replaced by the variable light domain VL of the antibody and the constant heavy domain CH1 is replaced by the constant light domain CL of the antibody.

One aspect as reported herein is a bivalent, bispecific antibody comprising:

b) a second light chain and a second heavy chain of an antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other,

wherein the first antigen or the second antigen is human PDGF-B.

In the antibody of b)

In the light chain

The constant light chain domain CL is replaced by the constant heavy chain domain CH1 of the antibody; and in the heavy chain

The constant heavy chain domain CH1 was replaced by the constant light chain domain CL of the antibody.

One aspect as reported herein is a multispecific antibody comprising:

a) a full-length antibody that specifically binds a first antigen and consists of two antibody heavy chains and two antibody light chains, and

b) one, two, three or four single chain Fab fragments which specifically bind to one to four other antigens (i.e.second and/or third and/or fourth and/or fifth antigen, preferably, specifically one other antigen, i.e.second antigen),

wherein the single chain Fab fragment of b) is fused to the full length antibody of a) via a peptide linker at the C-or N-terminus of the heavy or light chain of the full length antibody,

wherein the first antigen or one of the other antigens is human PDGF-B.

In one embodiment, one or two identical single chain Fab fragments that bind to a second antigen are fused to the full length antibody via a peptide linker at the C-terminus of the heavy or light chain of the full length antibody.

In one embodiment, one or two identical single chain Fab fragments that bind to a second antigen are fused to the full length antibody via a peptide linker at the C-terminus of the heavy chain of the full length antibody.

In one embodiment, one or two identical single chain Fab fragments that bind to a second antigen are fused to the full length antibody via a peptide linker at the C-terminus of the light chain of the full length antibody.

In one embodiment, two identical single chain Fab fragments that bind to a second antigen are fused to the full length antibody by a peptide linker at the C-terminus of each heavy or light chain of the full length antibody.

In one embodiment, two identical single chain Fab fragments that bind to a second antigen are fused to the full length antibody by a peptide linker at the C-terminus of each heavy chain of the full length antibody.

In one embodiment, two identical single chain Fab fragments that bind to a second antigen are fused to the full length antibody by a peptide linker at the C-terminus of each light chain of the full length antibody.

One aspect as reported herein is a trivalent, bispecific antibody comprising:

a) a full-length antibody that specifically binds a first antigen and consists of two antibody heavy chains and two antibody light chains,

b) a first polypeptide consisting of:

ba) antibody heavy chain variable domain (VH),

or

bb) antibody heavy chain variable domain (VH) and antibody constant domain 1(CH1),

wherein the first polypeptide is fused by a peptide linker with the N-terminus of its VH domain to the C-terminus of one of the two heavy chains of the full-length antibody,

c) a second polypeptide consisting of:

ca) an antibody light chain variable domain (VL),

or

cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL),

wherein the second polypeptide is fused via a peptide linker to the C-terminus of the other of the two heavy chains of the full-length antibody at the N-terminus of the VL domain,

and is

Wherein the antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen-binding site that specifically binds a second antigen,

and is

Wherein the first antigen or the second antigen is human PDGF-B.

In one embodiment, the antibody heavy chain variable domain (VH) of the polypeptide in b) and the antibody light chain variable domain (VL) of the polypeptide in c) are linked and stabilized via interchain disulfide bridges by introducing disulfide bonds between:

i) heavy chain variable domain position 44 and light chain variable domain position 100, or

ii) heavy chain variable domain position 105 and light chain variable domain position 43, or

iii) heavy chain variable domain position 101 and light chain variable domain position 100 (always numbered according to the Kabat EU index).

Techniques for introducing disulfide bridges for stabilization are described, for example, in WO 94/029350, Rajagopal, v., et al, prot.eng (1997) 1453-59; kobayashi, H., et al, nucleic Medicine & Biology, Vol.25, (1998) 387-393; or Schmidt, M., et al, Oncogene (1999) 181711-. In one embodiment, the optional disulfide bond between the variable domains of the polypeptides of b) and c) is between heavy chain variable domain position 44 and light chain variable domain position 100. In one embodiment, the optional disulfide bond between the variable domains of the polypeptides of b) and c) is between heavy chain variable domain position 105 and light chain variable domain position 43 (always numbered according to EU index of Kabat). In one embodiment, it is preferred not to have the optional disulfide stabilized trivalent bispecific antibody between the variable domains VH and VL of the single chain Fab fragment.

One aspect as reported herein is a trispecific or tetraspecific antibody comprising:

a) a first light chain and a first heavy chain of a full-length antibody that specifically binds a first antigen, and

b) a second (modified) light chain and a second (modified) heavy chain of the full-length antibody that specifically binds a second antigen, wherein the variable domains VL and VH are replaced with each other, and/or wherein the constant domains CL and CH1 are replaced with each other, and

c) wherein one to four antigen binding peptides specifically binding to one or two other antigens (i.e., a third and/or a fourth antigen) are fused via a peptide linker to the C-or N-terminus of the light or heavy chain of a) and/or b),

wherein the first antigen or the second antigen or one of the other antigens is human PDGF-B.

In one embodiment, the trispecific or tetraspecific antibody comprises in c) one or two antigen binding peptides that specifically bind to one or two other antigens.

In one embodiment, the antigen binding peptide is selected from the group of scFv fragments and scFab fragments.

In one embodiment, the antigen binding peptide is an scFv fragment.

In one embodiment, the antigen binding peptide is a scFab fragment.

In one embodiment, the antigen binding peptide is fused to the C-terminus of the heavy chain of a) and/or b).

In one embodiment, the trispecific or tetraspecific antibody comprises in c) one or two antigen binding peptides that specifically bind to one other antigen.

In one embodiment, the trispecific or tetraspecific antibody in c) comprises two identical antigen binding peptides that specifically bind to a third antigen. In a preferred embodiment, the two identical antigen binding peptides are both fused at the C-terminus of the heavy chains of a) and b) via the same peptide linker. In a preferred embodiment, the two identical antigen binding peptides are scFv fragments or scFab fragments.

In one embodiment, the trispecific or tetraspecific antibody comprises in c) two antigen binding peptides that specifically bind to a third and a fourth antigen. In one embodiment, the two antigen binding peptides are both fused at the C-terminus of the heavy chains of a) and b) via the same peptide linker. In a preferred embodiment, the two antigen binding peptides are scFv fragments or scFab fragments.

One aspect as reported herein is a bispecific, tetravalent antibody comprising:

a) two light chains and two heavy chains of an antibody that specifically binds a first antigen (and comprises two Fab fragments),

b) two further Fab fragments of the antibody which specifically bind to a second antigen, wherein the further Fab fragments are each fused via a peptide linker to the C-or N-terminus, respectively, of the heavy chain of a),

and is

Wherein the following modifications are made in the Fab fragment:

i) in the two Fab fragments of a), or in the two Fab fragments of b), the variable domains VL and VH are replaced by each other, and/or the constant domains CL and CH1 are replaced by each other,

or

ii) in the two Fab fragments of a) the variable domains VL and VH are replaced by each other and the constant domains CL and CH1 are replaced by each other,

and is

In both Fab fragments of b) the variable domains VL and VH are replaced by each other, or the constant domains CL and CH1 are replaced by each other,

or

iii) in the two Fab fragments of a) the variable domains VL and VH are replaced by each other, or the constant domains CL and CH1 are replaced by each other,

and is

In both Fab fragments of b) the variable domains VL and VH are replaced by each other and the constant domains CL and CH1 are replaced by each other,

or

iv) in the two Fab fragments of a) the variable domains VL and VH are replaced by each other, and in the two Fab fragments of b) the constant domains CL and CH1 are replaced by each other,

or

v) in the two Fab fragments of a) the constant domains CL and CH1 are replaced by each other, and in the two Fab fragments of b) the variable domains VL and VH are replaced by each other,

wherein the first antigen or the second antigen is human PDGF-B.

In one embodiment, the additional Fab fragments are each fused via a peptide linker to the C-terminus of the heavy chain of a) or to the N-terminus of the heavy chain of a).

In one embodiment, the additional Fab fragments are each fused via a peptide linker to the C-terminus of the heavy chain of a).

In one embodiment, said further Fab fragments are each fused to the N-terminus of the heavy chain of a) via a peptide linker.

In one embodiment, the following modifications are made in the Fab fragment:

i) in both Fab fragments of a), or in both Fab fragments of b), the variable domains VL and VH are replaced by each other,

and/or

The constant domains CL and CH1 are replaced by each other.

In one embodiment, the following modifications are made in the Fab fragment:

i) in both Fab fragments of a), the variable domains VL and VH are replaced by each other,

and/or

The constant domains CL and CH1 are replaced by each other.

In one embodiment, the following modifications are made in the Fab fragment:

i) in both Fab fragments of a), the constant domains CL and CHl are replaced by each other.

In one embodiment, the following modifications are made in the Fab fragment:

i) in both Fab fragments of b), the variable domains VL and VH are replaced by each other,

and/or

The constant domains CL and CH1 are replaced by each other.

In one embodiment, the following modifications are made in the Fab fragment:

i) in both Fab fragments of b), the constant domains CL and CH1 are replaced by each other.

One aspect as reported herein is a bispecific tetravalent antibody comprising:

a) a (modified) heavy chain of a first antibody that specifically binds a first antigen and comprises a first VH-CH1 domain pair, wherein the N-terminus of a second VH-CH1 domain pair of the first antibody is fused via a peptide linker to the C-terminus of the heavy chain,

b) a) the two light chains of said first antibody,

c) (modified) heavy chain of a second antibody that specifically binds to a second antigen and comprises a first VH-CL domain pair, wherein the N-terminus of the second VH-CL domain pair of the second antibody is fused via a peptide linker to the C-terminus of the heavy chain, and

d) c) two (modified) light chains of said second antibody, each comprising a CL-CH1 domain pair,

wherein the first antigen or the second antigen is human PDGF-B.

One aspect as reported herein is a bispecific antibody comprising:

a) heavy and light chains of a first full-length antibody that specifically binds a first antigen, and

b) a heavy chain and a light chain of a second full-length antibody that specifically binds a second antigen, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker,

wherein the first antigen or the second antigen is human PDGF-B.

a) The antibody of (a) does not comprise the modification reported in b), and the heavy and light chains are separate chains.

One aspect as reported herein is a bispecific antibody comprising:

b) fv fragment, specificity thereofBinds a second antigen and comprises a VH2 domain and a VL²(ii) domains, wherein the two domains are linked to each other by a disulfide bridge,

of which only VH²Domains and VL²One of the domains is fused via a peptide linker to the heavy or light chain of a full length antibody that specifically binds to a first antigen,

wherein the first antigen or the second antigen is human PDGF-B.

In the bispecific, the heavy and light chains in a) are separate chains.

In one embodiment, VH²Domains and VL²The other of the domains is not fused via a peptide linker to the heavy or light chain of the full length antibody that specifically binds the first antigen.

In all aspects reported herein, the first light chain comprises a VL domain and a CL domain and the first heavy chain comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

In one embodiment of all aspects the antibody as reported herein is a multispecific antibody requiring heterodimerization of at least two heavy chain polypeptides, and wherein said antibody specifically binds human PDGF-B and a second non-human PDGF-B antigen.

Some methods for CH 3-modification have been described to support heterodimerization, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO2010/129304, WO 2011/90754, WO 2011/143545, WO2012/058768, WO 2013/157954, WO2013/096291, which are incorporated herein by reference. Typically, in methods known in the art, both the CH3 domain of a first heavy chain and the CH3 domain of a second heavy chain are engineered in a complementary manner such that a heavy chain comprising one engineered CH3 domain is no longer homodimerized with another heavy chain of the same structure (e.g., a CH 3-engineered first heavy chain may no longer homodimerized with another CH 3-engineered first heavy chain; and a CH 3-engineered second heavy chain may no longer homodimerized with another CH 3-engineered second heavy chain). Thus, a heavy chain comprising one engineered CH3 domain is forced to heterodimerize with another heavy chain comprising a CH3 domain engineered in a complementary manner. For this embodiment of the invention, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner by amino acid substitution, forcing the first and second heavy chains to heterodimerize, while the first and second heavy chains are no longer capable of homodimerization (e.g., for steric reasons).

The different methods known in the art to support heavy chain heterodimerization cited and included above are considered as different alternatives for the multispecific antibodies of the present invention comprising "non-intersecting Fab regions" derived from a first antibody that specifically binds a first antigen and "intersecting Fab regions" derived from a second antibody that specifically binds a second antigen, along with the specific amino acid substitutions described above for the present invention.

The CH3 domain of multispecific antibodies reported herein can be altered by the "bulge-entry-hole" technique, which is described in detail in some examples, e.g., WO 96/027011, Ridgway, J.B., et al, Protein Eng.9(1996) 617-621; and Merchant, A.M., et al, Nat.Biotechnol.16(1998) 677-. In this approach, the interaction surface of the two CH3 domains is altered to increase heterodimerization of the two heavy chains comprising the two CH3 domains. Each of the two CH3 domains (of the two heavy chains) may be "convex" while the other is "hole". Introduction of disulfide bridges further stabilized the heterodimer (Merchant, A.M., et al., Nature Biotech.16(1998) 677-.

In a preferred embodiment, the bispecific antibody reported herein comprises a T366W mutation in the CH3 domain of the "bulge chain" and a T366S, L368A, Y407V mutation (numbering according to the kabat eu index) in the CH3 domain of the "well chain". Additional interchain disulfide bridges between the CH3 domains can also be used (Merchant, a.m., et al, Nature biotech.16(1998) 677-. Thus, in another preferred embodiment, the multispecific antibody reported herein comprises Y349C and T366W mutations in one of the two CH3 domains and E356C, T366S, L368A and Y407V mutations in the other of the two CH3 domains, or the multispecific antibody reported herein comprises Y349C and T366W mutations in one of the two CH3 domains and S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain form interchain disulfide bridges) (numbering according to the Kabat index).

However, other projection-in-hole techniques as described in EP 1870459 a1 may alternatively or additionally be applied. In one embodiment, the multispecific antibody reported herein comprises R409D and K370E mutations in the CH3 domain of the "bulge chain" and D399K and E357K mutations in the CH3 domain of the "well chain" (numbering according to the Kabat EU index).

In one embodiment, the multispecific antibody reported herein comprises a T366W mutation in the CH3 domain of the "bulge chain", and T366S, L368A and Y407V mutations in the CH3 domain of the "well chain", and further R409D and K370E mutations in the CH3 domain of the "bulge chain" and D399K and E357K mutations in the CH3 domain of the "well chain" (numbering according to the Kabat EU index).

In one embodiment, the multispecific antibody reported herein comprises Y349C and T366W mutations in one of the two CH3 domains and S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains, or the multispecific antibody reported herein comprises Y349C and T366W mutations in one of the two CH3 domains and S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains, and further comprises R409D and K370E mutations in the CH3 domain of the "bulge chain" and D399K and E357K mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index).

In addition to the "bulge-entry-hole technique", other techniques for modifying the CH3 domain of the heavy chain of a multispecific antibody, thereby forcing heterodimerization, are known in the art. These techniques, in particular those described in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO2010/129304, WO 2011/90754, WO 2011/143545, WO2012/058768, WO 2013/157954 and WO2013/096291, are considered herein as alternatives to the "bulge-entry-hole technique" in combination with the multispecific antibodies reported herein.

In one embodiment of the multispecific antibody reported herein, the method described in EP 1870459 is used to support heterodimerization of the first and second heavy chains of the multispecific antibody. This approach is based on the introduction of oppositely charged amino acids at specific amino acid positions in the CH3/CH 3-domain-interface between the two heavy chains, i.e. the first and second heavy chains.

Thus, this embodiment relates to a multispecific antibody as reported herein, wherein in the tertiary structure of said antibody the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface between the respective antibody CH3 domains, wherein the respective amino acid sequences of the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain comprise a set of amino acids located within said interface in the tertiary structure of the antibody, respectively, wherein a first amino acid from the set of amino acids located within the interface in the CH3 domain of one heavy chain is substituted with a positively charged amino acid and a second amino acid from the set of amino acids located within the interface in the CH3 domain of the other heavy chain is substituted with a negatively charged amino acid. The multispecific antibodies described in this embodiment are also referred to herein as "CH 3(+/-) -engineered multispecific antibodies" (where the abbreviation "+/-" indicates oppositely charged amino acids introduced into the respective CH3 domain).

In one embodiment of the CH3(+/-) -engineered multispecific antibody reported herein, the positively charged amino acid is selected from K, R and H, and the negatively charged amino acid is selected from E or D.

In one embodiment of the CH3(+/-) -engineered multispecific antibody reported herein, the positively charged amino acid is selected from K and R and the negatively charged amino acid is selected from E or D.

In one embodiment of the CH3(+/-) -engineered multispecific antibody reported herein, the positively charged amino acid is K and the negatively charged amino acid is E.

In one embodiment of the CH3(+/-) -engineered multispecific antibody reported herein, in the CH3 domain of one heavy chain the amino acid R at position 409 is substituted with D and the amino acid K at position is substituted with E, and in the CH3 domain of the other heavy chain the amino acid D at position 399 is substituted with K and the amino acid E at position 357 is substituted with K (numbering according to the EU index of Kabat).

In one embodiment of the multispecific antibody reported herein, the heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody is supported using the method described in WO 2013/157953. In one embodiment of the multispecific antibody reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted with K and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted with D (numbering according to the Kabat EU index). In another embodiment of the multispecific antibody reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted with K, the amino acid L at position 351 is substituted with K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted with D (numbering according to the Kabat EU index).

In one embodiment of the multispecific antibody reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted with K, the amino acid L at position 351 is substituted with K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted with D (numbering according to the Kabat EU index). In addition, at least one of the following substitutions is included in the CH3 domain of the other heavy chain: amino acid Y at position 349 is substituted with E, amino acid Y at position 349 is substituted with D and amino acid L at position 368 is substituted with E (numbering according to the EU index of Kabat). In one embodiment, amino acid L at position 368 is substituted with E (numbered according to the Kabat EU index).

In one embodiment of the multispecific antibody reported herein, the heterodimerization of the first and second heavy chains of the multispecific antibody is supported using the method described in WO 2012/058768. In one embodiment of the multispecific antibody reported herein, in the CH3 domain of one heavy chain the amino acid L at position 351 is substituted with Y and the amino acid Y at position 407 is substituted with a, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted with a and the amino acid K at position 409 is substituted with F (numbering according to the EU index of Kabat). In another embodiment, in addition to the aforementioned substitutions, in the CH3 domain of the other heavy chain at least one of the amino acids at the following positions is substituted: positions 411 (initially T), 399 (initially D), 400 (initially S), 405 (initially F), 390 (initially N) and 392 (initially K) (numbered according to the Kabat EU index). Preferred substitutions are:

-amino acid T at position 411 is substituted with an amino acid selected from N, R, Q, K, D, E and W (numbering according to the EU index of Kabat),

-amino acid D at position 399 is substituted with an amino acid selected from R, W, Y and K (numbering according to the Kabat EU index),

-amino acid S at position 400 is substituted with an amino acid selected from E, D, R and K (numbering according to the EU index of Kabat),

-amino acid F at position 405 is substituted with an amino acid selected from I, M, T, S, V and W (numbering according to the Kabat EU index);

-amino acid N at position 390 is substituted with an amino acid selected from R, K and D (numbering according to the Kabat EU index); and

-amino acid K at position 392 is replaced by an amino acid selected from V, M, R, L, F and E (numbering according to the Kabat EU index).

In another embodiment of the multispecific antibody reported herein (engineered as described in WO 2012/058768), in the CH3 domain of one heavy chain the amino acid L at position 351 is substituted with Y and the amino acid Y at position 407 is substituted with a, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted with V and the amino acid K at position 409 is substituted with F (numbering according to the EU index of Kabat). In another embodiment of said multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid Y at position 407 is substituted with a, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted with a and the amino acid K at position 409 is substituted with F (numbering according to the kabat eu index). In the last preceding embodiment, in the CH3 domain of the other heavy chain, amino acid K at position 392 is substituted with E, amino acid T at position 411 is substituted with E, amino acid D at position 399 is substituted with R and amino acid S at position 400 is substituted with R (numbering according to the EU index of Kabat).

In one embodiment of the multispecific antibody reported herein, the method described in WO 2011/143545 is used to support heterodimerization of a first heavy chain and a second heavy chain of the multispecific antibody. In one embodiment of the multispecific antibody reported herein, an amino acid modification (numbering according to the EU index of Kabat) is introduced at position 368 and/or 409 in the CH3 domains of both heavy chains.

In one embodiment of the multispecific antibody reported herein, the method described in WO 2011/090762 is used to support heterodimerization of a first heavy chain and a second heavy chain of the multispecific antibody. WO 2011/090762 relates to amino acid modifications according to the "bulge-entry-hole" technique. In one embodiment of said CH3(KiH) -engineered multispecific antibody reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted with W and in the CH3 domain of the other heavy chain the amino acid Y at position 407 is substituted with a (numbering according to the Kabat EU index). In another embodiment of said CH3(KiH) -engineered multispecific antibody reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted with Y and in the CH3 domain of the other heavy chain the amino acid Y at position 407 is substituted with T (numbering according to the Kabat EU index).

In one embodiment of the multispecific antibody reported herein, which is of the IgG2 isotype, the method described in WO201I/090762 is used to support heterodimerization of a first heavy chain and a second heavy chain of the multispecific antibody.

In one embodiment of the multispecific antibody reported herein, the method described in WO 2009/089004 is used to support heterodimerization of a first heavy chain and a second heavy chain of the multispecific antibody. In one embodiment of the multispecific antibody reported herein, in the CH3 domain of one heavy chain the amino acid K or N at position 392 is substituted with a negatively charged amino acid (in a preferred embodiment with E or D, in a preferred embodiment with D), and in the CH3 domain of the other heavy chain the amino acid D at position 399, the amino acid E or D at position 356 or the amino acid E at position 357 is substituted with a positively charged amino acid (in a preferred embodiment with K or R, in a preferred embodiment with K, in a preferred embodiment with the amino acid at position 399 or 356 with K) (numbering according to the EU index of Kabat). In another embodiment, in addition to the foregoing substitutions, in the CH3 domain of one heavy chain, amino acid K or R at position 409 is substituted with a negatively charged amino acid (in a preferred embodiment, with E or D, and in a preferred embodiment, with D) (numbering according to the Kabat EU index). In even another embodiment, in addition or alternatively to the aforementioned substitutions, in the CH3 domain of one heavy chain, amino acid K at position 439 and/or amino acid K at position 370 are substituted independently of each other with a negatively charged amino acid (in a preferred embodiment, with E or D, in a preferred embodiment, with D) (numbering according to the Kabat EU index).

In one embodiment of the multispecific antibody reported herein, the method described in WO 2007/147901 is used to support heterodimerization of a first heavy chain and a second heavy chain of the multispecific antibody. In one embodiment of the multispecific antibody reported herein, in the CH3 domain of one heavy chain the amino acid K at position 253 is substituted with E, the amino acid D at position 282 is substituted with K, and the amino acid K at position 322 is substituted with D, and in the CH3 domain of the other heavy chain the amino acid D at position 239 is substituted with K, the amino acid E at position 240 is substituted with K, and the amino acid K at position 292 is substituted with D (numbering according to the EU index of Kabat).

In one embodiment of the multispecific antibody reported herein, the method described in WO 2007/110205 is used to support heterodimerization of a first heavy chain and a second heavy chain of the multispecific antibody.

In one embodiment of all aspects and embodiments reported herein, the multispecific antibody is a bispecific antibody or a trispecific antibody. In a preferred embodiment of the invention, the multispecific antibody is a bispecific antibody.

In one embodiment of all aspects reported herein, said antibody is a bivalent or trivalent antibody. In one embodiment, the antibody is a bivalent antibody.

In one embodiment of all aspects reported herein, the multispecific antibody has the constant domain structure of an antibody of the IgG class. In another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the subclass human IgG1 or of the subclass human IgG1 with mutations L234A and L235A. In another embodiment of all aspects reported herein, said multispecific antibody is characterized in that said multispecific antibody is of the subclass human IgG 2. In another embodiment of all aspects reported herein, said multispecific antibody is characterized in that said multispecific antibody is of the subclass human IgG 3. In another embodiment of all aspects reported herein, said multispecific antibody is characterized in that said multispecific antibody is of the subclass human IgG4, or of the subclass human IgG4 with the additional mutation S228P. In another embodiment of all aspects reported herein, said multispecific antibody is characterized in that said multispecific antibody is of the subclass human IgG1 or of the subclass human IgG 4. In another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG1 subclass with mutations L234A and L235A (numbering according to the Kabat EU index). In another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG1 subclass (numbered according to the Kabat EU index) with mutations L234A, L235A and P329G. In another embodiment of all aspects reported herein, said multispecific antibody is characterized in that said multispecific antibody is of the human IgG4 subclass with mutations S228P and L235E (numbered according to the kabat eu index). In another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG4 subclass with mutations S228P, L235E and P329G (numbering according to the Kabat EU index).

In one embodiment of all aspects reported herein, the antibody comprising a heavy chain comprising the CH3 domain specified herein comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of all aspects reported herein, the antibody comprising a heavy chain comprising a CH3 domain specified herein comprises an additional C-terminal glycine residue (G446, numbering according to the EU index of Kabat).

In another aspect, an anti-PDGF-B antibody according to any one of the above embodiments can bind any of the features as described in sections 1-5 below, alone or in combination.

1. Affinity of antibody

In certain embodiments, an antibody provided herein has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, or ≦ 0.1nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M-10^-10M, e.g. 10^-9M-10^-10M) dissociation constant (KD).

Methods for determining KD values are described in the examples below.

When in useIn surface plasmon resonance measurements, alternatively, the KD values can be measured as follows: use ofOr(BIAcore, Inc., Piscataway, NJ) was performed at 25 ℃ on-10 Response Units (RU) immobilized antigen CM5 chips. In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5. mu.g/mL (. about.0.2. mu.M) with 10mM sodium acetate (pH 4.8) and then injected at a flow rate of 5. mu.L/min to obtain approximately 10 Response Units (RU) of conjugated protein. After the injection of antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, polysorbate 20 (TWEEN-20) at 0.05% was injected at 25 ℃ at a flow rate of about 25 μ L/min^TM) Two-fold serial dilutions (0.78nM to 500nM) of Fab in surfactant pbs (pbst). Using a simple one-to-one Langmuir (Langmuir) binding model (Evaluation software version 3.2), calculating the binding rate (k) by matching the binding and dissociation sensorgrams simultaneously_a) And dissociation rate (k)_d). Equilibrium dissociation constant (KD) is calculated as the ratio k_d/k_a(see, e.g., Chen, Y. et al, J.Mol.biol.293(1999) 865-881). If the on-rate exceeds 10, as determined by the above surface plasmon resonance⁶M^-1s^-1The dissociation rate can then be determined by using a fluorescence quenching technique in a spectrometer, such as a stop-flow equipped spectrophotometer (Aviv Instruments) or 8000-series SLM-AMINCO with a stirred cuvette^TMThe increase or decrease in fluorescence emission intensity of 20nM anti-antigen antibody (Fab form) in PBS (pH 7.2) was measured at 25 ℃ in the presence of increased antigen concentration measured by a spectrophotometer (thermospectonic) (excitation 295 nM; emission 340nM, 16nM band pass).

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to: fab, Fab ', Fab ' -SH, F (ab ')₂Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson, p.j. et al, nat. med.9(2003) 129-. For reviews on scFv fragments, see, e.g., Plueckthun, a., at: in The Pharmacology of Monoclonal Antibodies (Pharmacology of Monoclonal Antibodies), Vol.113, edited by Rosenburg and Moore, Springer-Verlag, New York (1994), p.269-315; see also WO 93/16185; US5,571,894 and US5,587,458. Relating to Fab and F (ab') comprising binding salvage receptor binding epitope residues and having increased half-life in vivo₂See US5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 0404097; WO 1993/01161; hudson, P.J., et al, nat. Med.9(2003) 129-; and Holliger, P. et al, Proc. Natl. Acad. Sci. USA 90(1993) 6444-. Triabodies and tetrabodies are also described in Hudson, p.j. et al, nat. med.9(2003) 129-.

Single domain antibodies are antibody fragments that: it comprises all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US6,248,516).

Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies as described herein and production by recombinant host cells (e.g., e.

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US4,816,567; and Morrison, S.L. et al, Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855). In one embodiment, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another embodiment, the chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains that: wherein the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody, and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro, J.C. and Fransson, J., Front.biosci.13(2008)1619-1633, and are further described, for example, in Riechmann, I.et al, Nature332(1988) 323-329; queen, C. et al, Proc. Natl. Acad. Sci. USA 86(1989) 10029-10033; US5, 821,337, US 7,527,791, US6,982,321, and US 7,087,409; kashmiri, s.v. et al, Methods36(2005)25-34 (specificity determining region (SDR) transplantation is described); padlan, E.A., mol.Immunol.28(1991)489-498 (describing "surface reconstruction"); dall' Acqua, w.f. et al, Methods36 (2005)43-60 (describing "FR shuffling"); and Osbourn, J. et al, Methods36 (2005)61-68 and Klimka, A. et al, Br.J. cancer83(2000)252- "260 (describing the" guided selection "protocol for FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best fit" approach (see, e.g., Sims, M.J. et al, J.Immunol.151(1993) 2296-; framework regions derived from consensus sequences of human antibodies from a specific subset of light or heavy chain variable regions (see, e.g., Carter, P. et al, Proc. Natl. Acad. Sci. USA 89 (1992)) 4285-; human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, j.c. and Fransson, j., front. biosci.13(2008) 1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca, M. et al, J.biol.chem.272(1997) 10678-.

4. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for PDGF-B and the other is for any other antigen. In certain embodiments, the bispecific antibody can bind two different epitopes of PDGF-B. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing PDGF-B. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305(1983)537-540, WO 93/08829, and Traunecker, A. et al., EMBO J.10(1991)3655-3659), and "bulge-entry-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by: engineering electrostatic steering effects (engineering electrostatic steering effects) for the preparation of antibody Fc-heterodimer molecules (WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan, M. et al, Science 229(1985) 81-83); bispecific antibodies were produced using leucine zippers (see, e.g., Kostelny, s.a. et al, j.immunol.148(1992) 1547-; the use of the "diabody" technique to prepare bispecific antibody fragments (see, e.g., Holliger, p. et al, proc. natl. acad. sci. usa 90(1993) 6444-; and the use of single-chain fv (sFv) dimers (see, e.g., Gruber, M et al, J.Immunol.152(1994) 5368-5374); and making a trispecific antibody as described, for example, in Tutt, a. et al, j.immunol.147(1991) 60-69).

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "Octopus antibodies" (see, e.g., US 2006/0025576).

Antibodies or fragments herein also include "dual action fabs" or "DAFs" comprising an antigen binding site that binds PDGF-B as well as another distinct antigen (see, e.g., US 2008/0069820).

The antibodies or fragments herein also include specific antibodies described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO 2010/145793.

5. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion of residues and/or insertion of residues and/or substitution of residues from the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

a) Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in the table below under the heading of "preferred substitutions". More substantial variations are provided in table 1 under the heading "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

Watch (A)

Amino acids can be grouped according to common side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp, Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions require the exchange of a member of one of these classes for a member of the other class.

One class of substitutional variants involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study has an improvement (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and/or has certain biological properties of the parent antibody that are substantially retained. An exemplary substitution variant is an affinity matured antibody, which can be conveniently produced, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such alterations may be made in HVR "hotspots" (i.e., residues encoded by codons that undergo mutation with high frequency during somatic maturation (see, e.g., Chowdhury, P.S., Methods mol. biol.207(2008) 179) 196), and/or residues that contact antigen, and testing the resulting variant VH or VL for binding affinity A targeted approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly frequently targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such alterations do not substantially reduce the antibody's ability to bind antigen. For example, conservative changes that do not substantially reduce binding affinity may be made in HVRs (e.g., conservative substitutions as provided herein). Such changes may be, for example, outside of the antigen contacting residues of the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

One useful method for identifying antibody residues or regions that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham, B.C. and Wells, J.A., Science 244(1989) 1081-1085. In this method, a residue or set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of an antibody with an enzyme (e.g., an enzyme directed against ADEPT) or a polypeptide that increases the serum half-life of the antibody.

b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

Where the antibody comprises an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides that are typically attached via an N-bond to Asn297 of the CH2 domain of the Fc region (see, e.g., Wright, a. and Morrison, s.l., TIBTECH 15(1997) 26-32). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified in order to produce antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by: the average amount of fucose within the sugar chain at Asn297 is calculated relative to the sum of all sugar structures (e.g., complex structures, hybrid structures, and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008/077546). Asn297 denotes an asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 may also be located ± 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, US 2003/0157108; US 2004/0093621. Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki, a. et al, j.mol.biol.336(2004) 1239-; Yamane-Ohnuki, N. et al, Biotech.Bioeng.87(2004) 614-622. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka, J. et al., Arch, biochem. Biophys.249(1986) 533-.

Antibody variants may also be provided with bisected oligosaccharides, for example, where the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; US6,602,684; and US 2005/0123546. Antibody variants having at least one galactose residue in an oligosaccharide linked to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and in WO 1999/22764.

c) Fc-region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In certain embodiments, the present invention contemplates antibody variants with some, but not all, effector functions, which make them desirable candidates for the following applications: where the in vivo half-life of the antibody is important and certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays may be performed to demonstrate a reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells (i.e., NK cells) for mediating ADCC express Fc (RIII) only, while monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. ravech, J.V. and Kinet, J.P., Annu.Rev.Immunol.9(1991) on page 464 of 457. non-limiting examples of in vitro assays to assess ADCC activity of molecules of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I.et al., Proc.Natl.Acad.Sci.USA 83(1986) 7059. Acone and Hellstrom, I.et al., Proc.Natl.Acad.Sci.82 (1985) 9 one) (see, e.g., Brgeymann, Ex. J.Acad, Sci.USA 82(1985) 1502) for mediating ADCC, see, E.J.Pro.Pro.Miq.J.Pro.Acad.O.No. 1. Polyp.Pro.Pro.Pro.A.A.A. Miq.A.A. 7, see, C.A. Miq.A. 7, A. Miq.A. 7, for alternative assays to flow A. Mi^TMNon-radioactive cytotoxicity assays (Celltechnology, Inc. mountain View, CA; and CytoTox)Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule may be assessed in vivo, for exampleIn animal models, such as those disclosed in Clynes, R. et al, Proc. Natl. Acad. Sci. USA 95(1998) 652-. C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity (see, e.g., C1q and C3C binding ELISA in WO2006/029879 and WO 2005/100402). To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro, H. et al., J.Immunol. methods 202(1996) 163-. FcRn binding and in vivo clearance/half-life determinations can also be made using methods known in the art (see, e.g., Petkova, s.b. et al, int. immunol.18 (2006: 1759-.

Antibodies with reduced effector function include those with substitutions of one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (US 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (US 7,332,581).

Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., US6,737,056; WO2004/056312, and Shields, r.l. et al, j.biol.chem.276(2001) 6591-.

In certain embodiments, an antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues)).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US6,194,551, WO 99/51642, and Idusogie, e.e., et al, j.hnmunol.164(2000) 4178-.

Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus are described in US 2005/0014934 (Guyer, r.l. et al, j.immunol.117(1976) 587-. Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, a substitution of residue 434 of the Fc region (US 7,371,826).

For further examples of Fc region variants, see also Duncan, A.R. and Winter, G., Nature 322(1988) 738-740; US5,648,260; US5,624,821; and WO 94/29351.

d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thiomabs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the substituted residue occurs at an accessible site of the antibody. By replacing those residues with cysteine, the reactive thiol group is thereby located at an accessible site of the antibody and can be used to conjugate the antibody to other moieties (such as a drug moiety or linker-drug moiety) to produce an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies may be produced as described, for example, in US 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymer or random copolymers) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers for derivatization may be determined based on the following considerations: including, but not limited to, the particular characteristics or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam, N.W. et al, Proc. Natl. Acad. Sci. USA 102(2005) 11600-. The radiation may be of any wavelength, and includes, but is not limited to, wavelengths such as: it does not harm normal cells, but it heats the non-protein moiety to a temperature that kills cells in the vicinity of the antibody-non-protein moiety.

B. Recombinant methods and compositions

Antibodies can be produced using recombinant methods and compositions (e.g., as described in US4,816,567). In one embodiment, isolated nucleic acids are provided that encode the anti-PDGF-B antibodies described herein. The nucleic acid may encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and/or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acids are provided. In another embodiment, host cells comprising the nucleic acids are provided. In one such embodiment, the host cell comprises the following vectors (e.g., that have been used to carry transformant transformation): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a nucleic acid comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-PDGF-B antibody is provided, wherein the method comprises culturing the host cell provided above comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally, recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-PDGF-B antibodies, the nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be made in bacteria, especially when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US5,648,237, US5,789,199, and US5,840,523. (see also Charlton, K.A., in: Methods in Molecular Biology, Vol.248, Lo, B.K.C. (eds.), HumanaPress, Totowa, NJ (2003), pp.245-. After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gemgross, T.U., nat. Biotech.22(2004) 1409-; and Li, H. et al, nat. Biotech.24(2006) 210-.

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous strains of baculovirus have been identified which can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts (see, e.g., U.S. Pat. No. 5,959,177, U.S. Pat. No. 6,040,498, U.S. Pat. No. 6,420,548, U.S. Pat. No. 7,125,978, and U.S. Pat. No. 6,417,429 (describing PLANTIBODIES for antibody production in transgenic plants)^TMTechnique)).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 or 293 cells, described in, e.g., Graham, f.l. et al, j.gen virol.36(1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, described in, e.g., Mather, J.P., biol. reprod.23(1980) 243-252); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells described in, for example, Mather, J.P. et al, AnnalS N.Y.Acad.Sci.383(1982) 44-68; MRC 5 cells; and FS 4. other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G et al, Proc.Natl.Acad.Sci.USA 77 (1980)) 4216-and myeloma cell lines, such as Y0, NS0 and Sp 2/0. for a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A.M., Methohol, Molecular, Hugma 255, Towa, Voluo et al, Voluwa, Volua.

C. Measurement of

The anti-PDGF-B antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. Exemplary assays are reported in the examples.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-PDGF-B antibody as reported herein conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioisotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs including, but not limited to, maytansinoids (maytansinoids) (see US5,208,020, US5,416,064, and EP 0425235B 1); auristatins (auristatins) such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see US5,635,483, US5,780,588, and US 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman, L.M. et al, Cancer Res.53(1993) 3336-3342; and Lode, H.N. et al, Cancer Res.58(1998) 2925-2928); anthracycline antibiotics (anthracyclines) such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz, F. et al, curr. Med. chem.13(2006) 477-523; Jeffrey, S.C. et al, Bioorg. Med. chem.Lett.16(2006) 358-362; Torgov, M.Y. et al, bioconjugate. chem.16(2005) 717-721; Nagy, A. et al, Proc. Natl. Acad. Sci.USA 97(2000) 829-834; Dubowchik, G.M. et al, Bioorg. chem. 12(2002) Chetters 12(2002) 1539, King 1529, H. D. 3545; 4325. J. chem. 4325); methotrexate (methotrexate); vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel (paclitaxel), larotaxel (larotaxel), tesetaxel (tesetaxel) and otetaxel (ortataxel); trichothecene (trichothecene); and CC 1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, the enzymatically active toxin or fragment thereof includes, but is not limited to, diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, phytolacca americana protein (PAPI, PAPII and PAP-S), momordica charantia (mordica charrantia) inhibitor, curcin (curcin), crotin (crotin), saponaria officinalis (sapanonaria officinalis) inhibitor, gelonin (gelonin), mitogellin (mitogellin), restrictocin (restrictocin), phenomycin (phyromycin), trichothecin (trichothecin) and trichothecene toxin (thecene toxin).

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom, thereby forming a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At²¹¹、I¹³¹、I¹²⁵、y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. When a radioconjugate is used for detection, it may include a radioactive atom, such as TC, for scintillation studies^99mOr I¹²³Or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 (again), iodine-131, steel-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

Conjugates of the antibody and cytotoxic agent can be prepared with a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate) and a di-active fluorine compound (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, ricin immunotoxins may be prepared as described in Vitetta, E.S. et al, Science 238(1987) 1098-1104. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triamine pentaacetate (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug in the cell. For example, acid-sensitive linkers, peptidase-sensitive linkers, light-sensitive linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari, R.V et al, Cancer Res.52(1992)127- "131; U.S. Pat. No. 5,208,020).

Immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with commercially available cross-linking agents (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a) including, but not limited to: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SLAB, sulfo-SMCC, and sulfo-SMPB and SVSB (succinimidyl- (4-vinylsulfone) benzoate).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-PDGF-B antibodies provided herein can be used to detect the presence of PDGF-B in a biological sample. The term "detecting" as used herein includes quantitative or qualitative detection.

In one embodiment, anti-PDGF-B antibodies for use in diagnostic or detection methods are provided. In another aspect, a method of detecting the presence of PDGF-B in a biological sample is provided. In certain embodiments, the method comprises: the biological sample is contacted with an anti-PDGF-B antibody as described herein under conditions that allow the anti-PDGF-B antibody to bind to PDGF-B, and it is tested whether a complex is formed between the anti-PDGF-B antibody and PDGF-B. Such methods may be in vitro or in vivo. In one embodiment, an anti-PDGF-B antibody is used to select subjects eligible for therapy with the anti-PDGF-B antibody, for example where PDGF-B is a biomarker for patient selection.

In certain embodiments, labeled anti-PDGF-B antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent labels, chromogenic labels, electron density labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detectable (such as enzymes or ligands), for example, by enzymatic reactions or molecular interactions. Exemplary markers include, but are not limited to: radioisotope³²P、¹⁴C、¹²⁵I、³H and¹³¹fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferases (e.g. firefly luciferase and bacterial luciferase (U.S. Pat. No.4,737,456)), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase(s) (oxidase (R) (R))E.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase) coupled with enzymes that utilize hydrogen peroxide to oxidize dye precursors (such as HR, lactoperoxidase, or microaxidase), biotin/avidin, spin labeling, phage labeling, stable free radicals, and the like.

F. Pharmaceutical preparation

Pharmaceutical formulations of anti-PDGF-B antibodies as described herein can be prepared in lyophilized formulations or as an aqueous solution by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. (eds.) (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly (vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers also include interstitial (injectable) drug dispersions herein, such as soluble, neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein,such as rhuPH20(Baxter International, Inc.). Certain exemplary shasegps, including rhuPH20, and methods of use are described in US 2005/0260186 and US 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases (such as chondroitinase).

An exemplary lyophilized antibody formulation is described in US6,267,958. Aqueous antibody formulations include those described in US6,171,586 and WO 2006/044908, the latter formulations including histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an anti-ANG 2 antibody or an anti-VEGF antibody. Such active ingredients are suitably present in combination in an amount effective for the intended purpose.

The active ingredient may be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions, for example, prepared by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. (eds.) (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

G. Therapeutic methods and compositions

Any of the anti-PDGF-B antibodies provided herein can be used in a method of treatment.

In one aspect, anti-PDGF-B antibodies for use as a medicament are provided. In other aspects, anti-PDGF-B antibodies are provided for use in the treatment of ocular vascular diseases, preferably macular degeneration. In certain embodiments, anti-PDGF-B antibodies are provided for use in methods of treatment. In certain embodiments, the invention provides an anti-PDGF-B antibody for use in a method of treating an individual having an ocular vascular disease, preferably macular degeneration, the method comprising administering to the individual an effective amount of an anti-PDGF-B antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In other embodiments, the invention provides anti-PDGF-B antibodies for use in inhibiting angiogenesis. In certain embodiments, the invention provides an anti-PDGF-B antibody for use in a method of inhibiting angiogenesis in an individual, the method comprising administering to the individual an effective amount of the anti-PDGF-B antibody, thereby inhibiting angiogenesis. An "individual" according to any of the embodiments above is preferably a human.

In another aspect, the invention provides the use of an anti-PDGF-B antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of an ocular vascular disease, preferably macular degeneration. In another embodiment, the medicament is for use in a method of treating a vascular disease of the eye, preferably macular degeneration, the method comprising administering to an individual having a vascular disease of the eye, preferably macular degeneration, an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In another embodiment, the medicament is for inhibiting angiogenesis. In another embodiment, the medicament is for use in a method of inhibiting angiogenesis in an individual, the method comprising administering to the individual an effective amount of the medicament, thereby inhibiting angiogenesis. An "individual" according to any of the embodiments above may be a human.

In another aspect, the present invention provides a method for treating ocular vascular disease, preferably macular degeneration. In one embodiment, the method comprises administering to an individual having such an ocular vascular disease, preferably macular degeneration, an effective amount of an anti-PDGF-B antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the embodiments above may be a human.

In another aspect, the invention provides a method for inhibiting angiogenesis in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-PDGF-B antibody, thereby inhibiting angiogenesis. In one embodiment, the "individual" is a human.

In another aspect, the invention provides a pharmaceutical formulation comprising any one of the anti-PDGF-B antibodies provided herein, e.g., for use in any one of the methods of treatment described above. In one embodiment, the pharmaceutical formulation comprises any one of the anti-PDGF-B antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any one of the anti-PDGF-B antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

The antibodies of the invention may be used in therapy, alone or in combination with other agents. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-VEGF antibody or an anti-ANG 2 antibody.

Such combination therapies noted above include combined administration (where two or more therapeutic agents are included in the same formulation or in separate formulations) and separate administration, in which case administration of the antibody of the invention may occur prior to, concurrently with, and/or subsequent to administration of one or more additional therapeutic agents. In one embodiment, administration of the anti-PDGF-B antibody and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

The antibodies of the invention (and any additional therapeutic agent) may be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing can be by any suitable route, e.g., by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing schedules include, but are not limited to, single or multiple administrations at different time points, bolus administration, and pulse infusions are contemplated herein.

The antibodies of the invention are formulated, dosed and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the time regimen of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally used with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosage and by the routes of administration described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. Suitably the antibody is administered to the patient at once or over a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.5mg/kg-10mg/kg) of the antibody may be an initial candidate dose for administration to a patient, e.g., whether by one or more divided administrations, or by continuous infusion. A typical daily dose may be in the range of about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is usually continued until the desired suppression of disease symptoms occurs. An exemplary dose of antibody is in the range of about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or any combination thereof) may be administered to a patient. Such a dose may be administered intermittently (e.g., weekly or every three weeks) (e.g., such that the patient receives from about two to about twenty doses, or, for example, about six doses of antibody). An initial higher loading dose (loading dose) may be administered followed by one or more lower doses. However, other dosage regimens may be useful. The progress of the treatment is readily monitored by conventional techniques and assays.

It will be appreciated that any of the above formulations or methods of treatment may be achieved using the immunoconjugates of the invention in place of or in addition to anti-PDGF-B antibodies.

Article of manufacture

In another aspect of the invention, there is provided an article of manufacture containing materials for the treatment, prevention and/or diagnosis of the disorders described above. The article comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, Intravenous (IV) solution bags, and the like. The container may be made of a variety of materials, such as glass or plastic. The container holds the composition alone or in combination with another composition effective to treat, prevent and/or diagnose the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is for use in treating a selected condition. Further, the article may comprise: (a) a first container comprising a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition comprises another cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

It will be appreciated that any of the above preparations may include an immunoconjugate of the invention in place of or in combination with an anti-PDGF-B antibody.

Specific embodiments

1. An antibody that specifically binds human PDGF-B, wherein the antibody is a monoclonal antibody comprising SEQ ID NO: 01 and the heavy chain variable domain of SEQ ID NO: 06 of the light chain variable domain.

2. A humanized antibody that specifically binds human PDGF-B, wherein the humanized antibody comprises: (a) comprises the amino acid sequence shown in SEQ ID NO: 02, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 03, and (c) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 05 of the amino acid sequence of HVR-H3.

3. A humanized antibody that specifically binds human PDGF-B, wherein the humanized antibody comprises: (a) comprises the amino acid sequence shown in SEQ ID NO: 02, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 04, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 05 of the amino acid sequence of HVR-H3.

4. The humanized antibody of any one of embodiments 2-3, wherein the humanized antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 07 (b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 08 HVR-L2; and (c) comprises SEQ ID NO: 09, HVR-L3 of the amino acid sequence of seq id no.

5. An antibody that specifically binds human PDGF-B comprising: (a) comprises the amino acid sequence of SEQ ID NO: 93, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 94, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 96, HVR-H3.

6. An antibody that specifically binds human PDGF-B comprising: (a) comprises the amino acid sequence of SEQ ID NO: 93, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 95, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 96, HVR-H3.

7. The antibody of any one of embodiments 5-6, wherein the antibody further comprises: (d) comprises SEQ ID NO: 98, (e) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 99, and (f) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no.

8. An antibody that specifically binds human PDGF-B comprising: (a) comprises the amino acid sequence of SEQ ID NO: 102, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 103, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 105, HVR-H3.

9. An antibody that specifically binds human PDGF-B comprising: (a) comprises the amino acid sequence of SEQ ID NO: 102, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 104, and (c) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 105, HVR-H3.

10. The antibody of any one of embodiments 8-9, wherein the antibody further comprises: (d) comprises the amino acid sequence of SEQ ID NO: 107, (e) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 108, and (f) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no.

11. An antibody that specifically binds to the same epitope as the antibody of any one of embodiments 1-4, or an antibody that specifically binds to the same epitope as the antibody of any one of embodiments 5-7, or an antibody that specifically binds to the same epitope as the antibody of any one of embodiments 8-10.

12. The antibody of any one of embodiments 1-11, wherein the antibody is of the subclass human IgG1 or of the subclass human IgG 4.

13. The antibody of any one of embodiments 1-12, wherein the antibody is of the human IgG1 subclass with kappa light chains.

14. The antibody of any one of embodiments 1-13, wherein the antibody is a monoclonal antibody.

15. Comprises the amino acid sequence of SEQ ID NO: 92 and the heavy chain variable domain amino acid sequence of SEQ ID NO: 97, or an antibody comprising the light chain variable domain amino acid sequence of SEQ ID NO: 101 and SEQ id no: 106, and a light chain variable domain amino acid sequence.

16. The antibody of any one of embodiments 1-15, wherein the antibody is a bispecific antibody.

17. The antibody of any one of embodiments 1-16, wherein the antibody specifically binds human PDGF-B but not human PDGF-C.

18. The antibody of any one of embodiments 1-4, wherein the antibody specifically binds human PDGF-BB, human PDGF-AB, and human PDGF-AA.

19. The antibody of any one of embodiments 1-17, wherein the antibody blocks the biological activity of human PDGF-B by inhibiting the binding of human PDGF-B to its receptor.

20. The antibody of any one of embodiments 1-19, wherein the antibody is a bivalent, bispecific antibody comprising:

wherein the first antigen or the second antigen is human PDGF-B.

21. The antibody of embodiment 20, wherein the antibody comprises:

or

22. The antibody of any one of embodiments 20-21, wherein the antibody comprises the amino acids at positions 124 and 123 substituted with K (numbering according to the EU index of Kabat) in the constant domain CL of the second heavy chain.

23. The antibody of any one of embodiments 20-22, wherein the antibody comprises the amino acids at positions 147 and 213 (numbering according to the EU index of Kabat) substituted with E in constant domain CH1 of the second light chain.

24. The antibody according to any one of embodiments 20-23, wherein the antibody comprises the amino acids at positions 124 and 123 substituted with K in the constant domain CL of the first light chain and the amino acids at positions 147 and 213 substituted with E in the constant domain CH1 of the first heavy chain (numbering according to the EU index of Kabat).

25. The antibody of any one of embodiments 20-24, wherein the antibody comprises the amino acids at positions 124 and 123 substituted with K in the constant domain CL of the second heavy chain, and wherein the amino acids at positions 147 and 213 are substituted with E in the constant domain CH1 of the second light chain, and the amino acid at position 38 is substituted with K in the variable domain VL of the first light chain, the amino acid at position 39 is substituted with E in the variable domain VH of the first heavy chain, the amino acid at position 38 is substituted with K in the variable domain VL of the second heavy chain, and the amino acid at position 39 is substituted with E in the variable domain VH of the second light chain (numbering according to the Kabat EU index).

26. The antibody of any one of embodiments 1-19, wherein the antibody is a bivalent, bispecific antibody comprising:

b) a second light chain and a second heavy chain of an antibody that specifically binds a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other, and wherein the constant domains CL and CHl of the second light chain and the second heavy chain are replaced with each other,

wherein the first antigen or the second antigen is human PDGF-B.

27. The antibody of any one of embodiments 1-19, wherein the antibody is a bivalent, bispecific antibody comprising:

wherein the first antigen or the second antigen is human PDGF-B.

28. The antibody of any one of embodiments 1-19, wherein the antibody is a multispecific antibody comprising:

wherein the first antigen or one of the other antigens is human PDGF-B.

29. The antibody of any one of embodiments 1-19, wherein the antibody is a trivalent bispecific antibody comprising:

b) a first polypeptide consisting of:

ba) antibody heavy chain variable domain (VH),

or

bb) antibody heavy chain variable domain (VH) and antibody constant domain 1(CH 1).

c) a second polypeptide consisting of:

ca) an antibody light chain variable domain (VL),

or

and is

Wherein the first antigen or the second antigen is human PDGF-B.

30. The antibody of embodiment 29, wherein the antibody heavy chain variable domain (VH) of the polypeptide in b) and the antibody light chain variable domain (VL) of the polypeptide in c) are linked and stabilized via an interchain disulfide bridge by introducing a disulfide bond between:

31. The antibody of any one of embodiments 1-19, wherein the antibody is a trispecific or tetraspecific antibody comprising:

32. The antibody of any one of embodiments 1-19, wherein the antibody is a bispecific tetravalent antibody comprising:

and is

Wherein the following modifications are made in the Fab fragment:

or

and is

In both Fab fragments of b), the variable domains VL and VH are replaced by each other,

or the constant domains CL and CH1 are substituted for each other,

or

and is

or

v) in the two Fab fragments of a), the constant domains CL and CH1 are replaced by one another,

and in both Fab fragments of B) the variable domains VL and VH are replaced by each other, wherein the first antigen or the second antigen is human PDGF-B.

33. The antibody of any one of embodiments 1-19, wherein the antibody is a bispecific tetravalent antibody comprising:

b) a) the two light chains of said first antibody,

c) a (modified) heavy chain of a second antibody that specifically binds to a second antigen and comprises a first VH-CL domain pair, wherein the N-terminus of the second VH-CL domain pair of the second antibody is fused via a peptide linker to the C-terminus of the heavy chain, and

wherein the first antigen or the second antigen is human PDGF-B.

34. The antibody of any one of embodiments 1-19, wherein the antibody is a bispecific antibody comprising:

wherein the first antigen or the second antigen is human PDGF-B.

35. The antibody of any one of embodiments 1-19, wherein the antibody is a bispecific antibody comprising:

b) fv fragment specifically binding to a second antigen and comprising VH²Domains and VL²(ii) domains, wherein the two domains are linked to each other by a disulfide bridge,

wherein the first antigen or the second antigen is human PDGF-B.

36. The antibody of any one of embodiments 1-35, wherein the antibody comprises a first Fc-region polypeptide and a second Fc-region polypeptide, and

wherein

i) The first Fc-region polypeptide is selected from the group comprising:

-a human IgG1 Fc-region polypeptide,

-a human IgG2 Fc-region polypeptide,

-a human IgG3 Fc-region polypeptide,

-a human IgG4 Fc-region polypeptide,

a human IgG1 Fc-region polypeptide having the mutations L234A, L235A,

a human IgG1 Fc-region polypeptide having the mutations Y349C, T366S, L368A, Y407V,

human IgG1 Fc-region polypeptide having the mutations S354C, T366S, L368A, Y407V

Human IgG1 Fc-region polypeptide having the mutations L234A, L235A, Y349C, T366S, L368A, Y407V,

human IgG1 Fc-region polypeptide having the mutations L234A, L235A, S354C, T366S, L368A, Y407V,

a human IgG1 Fc-region polypeptide having the mutation P329G,

a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G,

a human IgG1 Fc-region polypeptide having the mutations P329G, Y349C, T366S, L368A, Y407V,

a human IgG1 Fc-region polypeptide having the mutations P329G, S354C, T366S, L368A, Y407V,

human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G, Y349C, T366S, L368A, Y407V,

human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G, S354C, T366S, L368A, Y407V,

a human IgG4 Fc-region polypeptide having the mutations S228P, L235E,

a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, P329G,

a human IgG4 Fc-region polypeptide having the mutations Y349C, T366S, L368A, Y407V,

human IgG4 Fc-region polypeptides having the mutations S354C, T366S, L368A, Y407V,

human IgG4 Fc-region polypeptide having the mutations S228P, L235E, Y349C, T366S, L368A, Y407V,

human IgG4 Fc-region polypeptide having the mutations S228P, L235E, S354C, T366S, L368A, Y407V,

a human IgG4 Fc-region polypeptide having the mutation P329G,

a human IgG4 Fc-region polypeptide having the mutations P329G, Y349C, T366S, L368A, Y407V,

a human IgG4 Fc-region polypeptide having the mutations P329G, S354C, T366S, L368A, Y407V,

human IgG4 Fc-region polypeptide having the mutations S228P, L235E, P329G, Y349C, T366S, L368A, Y407V,

human IgG4 Fc-region polypeptide having the mutations S228P, L235E, P329G, S354C, T366S, L368A, Y407V,

human IgG1, IgG2 or IgG4 with the mutation K392D, and

human IgG3 with the mutation N392D,

and is

ii) a second Fc-region polypeptide selected from the group comprising:

-a human IgG1 Fc-region polypeptide,

-a human IgG2 Fc-region polypeptide,

-a human IgG3 Fc-region polypeptide,

-a human IgG4 Fc-region polypeptide,

a human IgG1 Fc-region polypeptide having the mutations L234A, L235A,

a human IgG1 Fc-region polypeptide having the mutations S354C, T366W,

a human IgG1 Fc-region polypeptide having the mutations Y349C, T366W,

a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, S354C, T366W,

a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, Y349C, T366W,

a human IgG1 Fc-region polypeptide having the mutation P329G,

a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G,

a human IgG1 Fc-region polypeptide having the mutations P329G, S354C, T366W,

a human IgG1 Fc-region polypeptide having the mutations P329G, Y349C, T366W,

a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G, S354C, T366W,

a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G, Y349C, T366W,

a human IgG4 Fc-region polypeptide having the mutations S228P, L235E,

a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, P329G,

a human IgG4 Fc-region polypeptide having the mutations S354C, T366W,

a human IgG4 Fc-region polypeptide having the mutations Y349C, T366W,

a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, S354C, T366W,

a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, Y349C, T366W,

a human IgG4 Fc-region polypeptide having the mutation P329G,

a human IgG4 Fc-region polypeptide having the mutations P329G, S354C, T366W,

a human IgG4 Fc-region polypeptide having the mutations P329G, Y349C, T366W,

a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, P329G, S354C, T366W,

a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, P329G, Y349C, T366W,

human IgG1 with mutations D399K, D356K and/or E357K, and

human IgG2, IgG3 or IgG4 with mutations D399K, E356K and/or E357K.

37. The antibody of any one of embodiments 1-35, wherein the antibody comprises a first Fc-region polypeptide and a second Fc-region polypeptide, and

wherein

i) The first Fc-region polypeptide is a human IgG1 Fc-region polypeptide and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide, or

ii) the first Fc-region polypeptide is a human IgG1 Fc-region polypeptide having the mutations L234A, L235A and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, or

iii) the first Fc-region polypeptide is a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G, and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G, or

iv) the first Fc-region polypeptide is a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, S354C, T366W, and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, Y349C, T366S, L368A, Y407V, or

v) the first Fc-region polypeptide is a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G, S354C, T366W, and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide having the mutations L234A, L235A, P329G, Y349C, T366S, L368A, Y407V, or

vi) the first Fc-region polypeptide is a human IgG4 Fc-region polypeptide and the second Fc-region polypeptide is a human IgG4 Fc-region polypeptide, or

vii) the first Fc-region polypeptide is a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, and the second Fc-region polypeptide is a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, or

viii) the first Fc-region polypeptide is a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, P329G and the second Fc-region polypeptide is a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, P329G, or

ix) the first Fc-region polypeptide is a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, S354C, T366W, and the second Fc-region polypeptide is a human IgG4 Fc-region polypeptide having the mutations S228P, L235E, Y349C, T366S, L368A, Y407V, or

x) the first Fc-region polypeptide is a human IgG4 Fc-region polypeptide having mutations S228P, L235E, P329G, S354C, T366W, and the second Fc-region polypeptide is a human IgG4 Fc-region polypeptide having mutations S228P, L235E, P329G, Y349C, T366S, L368A, Y407V.

38. The antibody of any one of embodiments 1-37, wherein the antibody comprises a first Fc-region polypeptide and a second Fc-region polypeptide, and

wherein the antibody comprises the following combination of mutations in the first Fc-region polypeptide and in the second Fc-region polypeptide:

i) I253A, H310A, and H435A, or

ii) H310A, H433A, and Y436A, or

iii) L251D, L314D, and L432D, or

iv) a combination of i) to iii).

39. The antibody of any one of embodiments 1-37, wherein the antibody comprises a first Fc-region polypeptide and a second Fc-region polypeptide, and wherein

a) The first and second Fc-region polypeptides are each of the subclasses human IgG1 or human IgG4 (derived from human origin), and comprise one or both of the following mutations in the first Fc-region polypeptide selected from: i) I253A, group H310A and H435A, or ii) group H310A, H433A and Y436A, or iii) group L251D, L314D and L432D (numbered according to the Kabat EU index numbering system) and comprising in the second Fc-region polypeptide one or two mutations selected from the group comprising mutations L251D, I253A, H310A, L314D, L432D, H433A, H435A and Y436A (numbered according to the Kabat EU index numbering system), such that all mutations in the first and second Fc-region polypeptides, when taken together, result in the inclusion in the variant (human) IgG class Fc-region of the following mutations: i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D,

or

b) The first and second Fc-region polypeptides are both of the subclass human IgG1 or human IgG4 (i.e., derived from human origin), and both comprise the mutations I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D, or combinations thereof (numbered according to the Kabat EU index numbering system) in the Fc-regions, whereby either all mutations are present in the first or second Fc-region polypeptides, or one or both mutations are present in the first Fc-region polypeptide and one or both mutations are present in the second Fc-region polypeptide, such that when all mutations in the first and second Fc-region polypeptides occur simultaneously, they result in the inclusion in the Fc-regions of: i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D,

or

c) The first and second Fc-region polypeptides are both of the human IgG1 or human IgG4 subclass (i.e., derived from human origin) and comprise the mutations I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D (numbered according to the Kabat EU index numbering system) in the first and second Fc-region polypeptides or the mutant combination I253A/H310A/H435A in the first Fc-region polypeptide and the mutant combination H310A/H433A/Y436A in the second Fc-region polypeptide (numbered according to the Kabat EU index numbering system).

40. The antibody of any one of embodiments 1-37, wherein the antibody comprises a first Fc-region polypeptide and a second Fc-region polypeptide, and wherein

a) The first variant Fc-region polypeptide is derived from a first parent IgG species Fc-region polypeptide and the second variant Fc-region polypeptide is derived from a second parent IgG species Fc-region polypeptide, wherein the first parent IgG species Fc-region polypeptide is the same or different from the second parent IgG species Fc-region polypeptide, and

b) the first variant Fc-region polypeptide differs from the second variant Fc-region polypeptide at one or more amino acid residues in addition to those amino acid residues at which the first parent IgG species Fc-region polypeptide differs from the second parent IgG species Fc-region polypeptide, and

c) the IgG species Fc-region comprising the first variant Fc-region polypeptide and the second variant Fc-region polypeptide has an affinity for a human Fc-receptor that is different from the affinity of the IgG species Fc-region comprising the first parent IgG species Fc-region polypeptide of a) and the second parent IgG species Fc-region polypeptide of a),

wherein the first Fc-region polypeptide or the second Fc-region polypeptide or both Fc-region polypeptides independently of each other comprise one of the following mutations or combinations of mutations:

-T307H, or

-Q311H, or

-E430H, or

-N434H, or

-T307H and Q311H, or

-T307H and E430H, or

-T307H and N434A, or

-T307H and N434H, or

-T307Q and Q311H, or

-T307Q and E430H, or

-T307Q and N434H, or

-T307H and Q311H and E430H and N434A, or

-T307H and Q311H and E430H and N434H, or

-T307H and Q311H and E430H and N434Y, or

-T307Q and Q311H and E430H and N434A, or

-T307Q and Q311H and E430H and N434H, or

-T307Q and Q311H and E430H and N434Y, or

-T307Q and V308P and N434Y and Y436H, or

-T307H and M252Y and S254T and T256E, or

-T307Q and M252Y and S254T and T256E, or

Q311H and M252Y and S254T and T256E, or

E430H and M252Y and S254T and T256E, or

N434H and M252Y and S254T and T256E, or

-T307H and Q311H and M252Y and S254T and T256E, or

-T307H and E430H and M252Y and S254T and T256E, or

-T307H and N434A and M252Y and S254T and T256E, or

-T307H and N434H and M252Y and S254T and T256E, or

-T307Q and Q311H and M252Y and S254T and T256E, or

-T307Q and E430H and M252Y and S254T and T256E, or

-T307Q and N434H and M252Y and S254T and T256E, or

-T307H and Q311H and E430H and N434A and M252Y and S254T and T256E, or

-T307H and Q311H and E430H and N434H and M252Y and S254T and T256E, or

-T307H and Q311H and E430H and N434Y and M252Y and S254T and T256E, or

-T307Q and Q311H and E430H and N434A and M252Y and S254T and T256E, or

-T307Q and Q311H and E430H and N434H and M252Y and S254T and T256E, or

-T307Q and Q311H and E430H and N434Y and M252Y and S254T and T256E, or

-T307Q and V308P and N434Y and Y436H and M252Y and S254T and T256E.

41. The antibody of any one of embodiments 1-37, wherein the antibody comprises a first Fc-region polypeptide and a second Fc-region polypeptide,

and wherein the first Fc-region polypeptide comprises the mutations Y349C, T366S, L368A and Y407V (pore-chain) and the second Fc-region polypeptide comprises the mutations S354C and T366W (bulge-chain), and wherein the first Fc-region polypeptide (pore-chain) comprises the following mutations:

i) I253A or I253G, and

ii) L314A or L314G or L314D,

and wherein the first Fc-region polypeptide is linked to the second Fc-region polypeptide by one or more disulfide bridges,

and wherein the CH 3-domain of the first polypeptide and the CH 3-domain of the second polypeptide both bind protein A or neither (numbered according to the Kabat EU index).

42. The antibody of embodiment 41, wherein the antibody comprises the following mutations:

i) I253A or I253G, and

ii) L314A or L314G or L314D, and

iii) T250Q, and/or

iv) T256E or T256A.

43. The antibody of any one of embodiments 41-42, wherein the antibody comprises the following mutations:

i) I253A or I253G, and

ii) L314A or L314G or L314D, and

iii) optionally, a) T250Q, and/or T256E or T256A, and

iv) a) L251A or L251G or L251D, and/or b) H310A or H310G.

44. The antibody of any one of embodiments 41-43, wherein the antibody comprises the following mutations:

i) I253A or I253G, and

ii) L314A or L314G or L314D, and

iii) a) T250Q, and/or T256E or T256A, and

iv) a) L251A or L251G or L251D, and/or b) H310A or H310G,

v) optionally, a) T307A or T307H or T307Q or T307P, and/or b) Q311H, and/or c) M252Y, and/or d) S254T.

45. The antibody of any one of embodiments 41-44, wherein the antibody comprises the following mutations:

i) T250Q, and/or

ii) M252Y, and/or

iii) S254T, and/or

iv) T256E or T256A, and/or

v) T307A or T307H or T307Q or T307P, and/or

vi)Q311H。

46. The antibody according to any one of embodiments 1-45 for use as a medicament.

47. The antibody of any one of embodiments 1-45 for use in treating an ocular vascular disease.

48. Use of an antibody according to any one of embodiments 1 to 45 for the treatment of an ocular disease, in particular an ocular vascular disease.

49. The antibody of any one of embodiments 1-45 for use in treating an ocular disease.

50. The antibody according to any one of embodiments 1-45 for use in the treatment of an ocular disease, in particular an ocular vascular disease.

51. A method of treating an individual having an ocular vascular disease, the method comprising administering to the individual an effective amount of the antibody of any one of embodiments 1-45.

52. A pharmaceutical formulation comprising the antibody of any one of embodiments 1-45.

53. A pharmaceutical formulation for treating an ocular vascular disease comprising the antibody of any one of embodiments 1-45.

54. Use of an antibody according to any one of embodiments 1-45 in the manufacture of a medicament for the treatment of an ocular vascular disease.

55. A method of treating a patient having an ocular vascular disease by administering to a patient in need of such treatment an antibody of any one of embodiments 1-45.

56. The pharmaceutical formulation of any one of embodiments 52-53, wherein the antibody is administered by intravitreal application.

57. The administration of any one of embodiments 55-56, wherein the administration is intravitreal application.

58. A nucleic acid encoding the antibody of any one of embodiments 1-45.

59. A cell comprising one or more nucleic acids encoding an antibody of any one of embodiments 1-45.

60. A method for making the antibody of any one of embodiments 1-45, wherein the method comprises the steps of:

a) optionally transfecting a mammalian cell with one or more nucleic acids encoding an antibody of any one of embodiments 1-45,

b) culturing the cell, thereby expressing the antibody, and

c) recovering the antibody from the cell or the culture medium, thereby producing the antibody.

V. examples

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced, given the general description provided above.

Example 1

Immunization

For IMmunization of mice (NMRI mice) and rabbits (human Ig locus transgenic rabbits), time protocols based on RIMMS ("Rapid IMmunization, Multiple Sites") were used. The antigen was human PDGF-BB (Cell Signaling Tech.).

Example 2

Determination of serum Titers of anti-PDGF-B antibodies

Human recombinant PDGF-B was immobilized on 96-well NUNCMaxisorb plates (100. mu.l/well) at 2.5. mu.g/ml (mouse) or 1.0. mu.g/ml (rabbit) in PBS, followed by: blocking the plate with 200. mu.l/well of 2% CroteinC in PBS; serial dilutions of antiserum in PBS containing 0.5% CroteinC were applied in duplicate at 100 μ l/well; mouse sera were detected with HRP-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch) diluted 1: 16,000 in PBS with 0.5% Crotein C, or rabbit sera were detected with biotinylated goat anti-human kappa antibody (Southern Biotech) diluted 1: 5,000 and HRP-conjugated streptavidin conjugated diluted 1: 8,000 in PBS with 0.5% Crotein C, 100. mu.l/well. For all steps, the plate at 37 degrees C temperature 1h incubation. Between all steps, plates were washed 3 times with PBS containing 0.05% tween 20. Signals were visualized by adding 100. mu.l/well of soluble BM Blue POD substrate (Roche diagnostics GmbH, Mannheim, Germany); and stopped by adding 100. mu.l/well of 1M HCl. Absorbance was read at 450nm with 690nm as a reference. Titers were defined as the dilutions of antisera that resulted in half of the maximal signal.

Example 3

B cell cloning from rabbits

Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMC)

Blood samples were collected from three immunized rabbits. Whole blood containing EDTA was diluted two-fold with 1x PBS (PAA, Pasching, austria) and then density centrifuged using mammalian lymphocytes (lymphocyte) according to the manufacturer's instructions (cedarlane laboratories, Burlington, Ontario, canada). PBMCs were washed twice with 1x PBS.

EL-4B5 medium

RPMI 1640(Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2mM glutamine, 1% penicillin/streptomycin solution (PAA, Pasching, Austria), 2mM sodium pyruvate, 10mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05mM beta-mercaptoethanol (Gibco, Paisley, Scotland) was used.

Flat-plate coating

Sterile cell culture 6-well plates were coated overnight at 4 ℃ with PBGF-BB (2. mu.g/ml) or mixtures of PDGF-AA and PDGF-CC proteins (1. mu.g/ml PDGF-AA and PDGF-CC) in a carbonic acid buffer (0.1M sodium bicarbonate, 34mM disodium bicarbonate, pH 9.55). Plates were washed three times with sterile PBS prior to use.

Consumption of macrophageCell/monocyte

Half of the PBMCs of the blood samples were seeded on sterile 6-well plates (cell culture grade) to deplete macrophages and monocytes by non-specific adhesion.

The remaining 50% of the PBMCs were seeded in plates previously coated with a mixture of PDGF-AA and PDGF-CC proteins to remove B cells binding to these proteins and remove macrophages and monocytes in one step.

Each well was filled up to 4ml of medium and up to 6X10⁶PBMCs from immunized rabbits were allowed to bind in an incubator at 37 ℃ for 1 hour.

Cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen panning step.

Enrichment of B cells on PDGF BB protein

6-well tissue culture plates coated with PDGF-BB protein were inoculated up to 6X10⁶PBL/4ml medium and allowed to bind in the incubator at 37 ℃ for 1 hour. Nonadherent cells were carefully removed by washing the wells 1-2 times with 1 xPBS. The remaining adherent cells were detached by treatment with trypsin in an incubator at 37 ℃ for 10 minutes. Trypsin digestion was stopped with EL-4B5 medium. Cells were kept on ice prior to immunofluorescent staining.

Immunofluorescent staining and flow cytometry

Single cell sorting was performed using anti-IgG FITC (AbD Serotec, Dusseldorf, Germany). For surface staining, cells from depletion and enrichment steps were incubated with FITC-conjugated anti-IgG antibody in PBS and incubated for 45 minutes at 4 ℃ in the dark. After staining, PBMCs were washed twice with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Prior to FACS analysis, propidium iodide (BDPharmingen, San Diego, Calif., USA) was added at a concentration of 5. mu.g/ml to distinguish dead from live cells.

Single cell sorting was performed using Becton Dickinson FACSAria equipped with computer and FACSDiva software (BD Biosciences, USA).

B-cell culture

Briefly, single sorted rabbit B-cells were plated in 96-well plates with 200. mu.l/well of a solution containing Pansorbin cells (1: 100,000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymocyte supernatant (Microcoat, Bernarid, Germany), and gamma-irradiated murine EL-4B5 thymoma cells (2.5 × 10⁴Individual cells/well) was cultured in an incubator at 37 ℃ for 7 days. Supernatants from B-cell cultures were removed for screening, and the remaining cells were immediately collected and frozen at-80 ℃ in 100. mu.l RLT buffer (Qiagen, Hilden, Germany).

Example 4

Hybridoma production

Cell culture

The mouse myeloma cell line P3x63-ag8.653 was used as a fusion partner to generate mouse-mouse hybridomas. Cells were thawed about 14 days prior to fusion and cultured in the presence of 8-azaguanine. Every 3-4 days, cells were distributed (split) and adjusted to 1-2x 10⁵Concentration of individual cells/m.

Cell fusion

Materials:

mouse hybridoma Medium (RPMI 1640(PAN), FBS ultra-Low-IgG, 2mM L-Glutamine, 1mM sodium pyruvate, NEAA, Nutridoma-CS with muIL-6, HAZ (SIGMA, # A9666)), adjusted to Room Temperature (RT)

RPMI 1640 medium (37 ℃ C.)

RPMI 1640 medium (4 ℃ C.)

PEG(37℃)

Prior to the fusion, the fusion is carried out,myeloma cells were approximately centrifuged (250rpm, 7 min.). The cell pellet was resuspended in culture medium. For a spleen, about 1-5 x10 is required⁷And (4) cells.

For cell fusion, about 5: 1 lymphocytes should be used: the proportion of myeloma cells. P3X 63-Ag8.653-cells were resuspended in 50ml RPMI 1640 medium and centrifuged (250rpm, 7 min.). The supernatant was removed. Cells were then added to splenocytes.

During the fusion process, the temperature was adjusted to 37 ℃ in a water bath.

To the cells was added a PEG solution dropwise (37 ℃).

The fusion mixture was incubated in an incubator at 37 ℃ for 15-120 minutes. The fusion mixture was then centrifuged (250rpm, 7min.) and resuspended in 1200. mu.l of resuspension medium. 100 μ l of the cell suspension was added to 50ml of semi-solid hybridoma medium, homogenized, and 4ml was added to each well of a 6-well plate. After 9-13 days of culture, individual clones were picked.

Example 5

Culture of hybridomas

About 5x10⁶Individual cells were suspended in 50ml Hyclone medium. The culture mixture was incubated for 96 hours. Then, 75ml Hyclone medium was added. The culture was continued for 7 days. If viability drops below 40%, the cell suspension is filtered through a 0.22 μ filter and purified using filtrate.

Example 6

Cloning of B-cells

PCR amplification of V-domains

Total RNA was prepared from B-cell lysates (resuspended in RLT buffer-Qiagen-Cat.N. 79216) using the NucleoSpin 8/96 RNA kit (Macherey & Nagel; 740709.4, 740698) following the manufacturer's protocol. RNA was eluted with 60. mu.l RNase-free water. cDNA was generated using 6. mu.l of RNA by reverse transcriptase reaction using Superscript III first strand synthesis supersxture (Invitrogen 18080-400) and oligo dT-primer according to the manufacturer's instructions. All steps were performed on a Hamilton ML Star system. Immunoglobulin heavy and light chain variable regions (VH and VL) were amplified using 4. mu.l of cDNA with AccuPrime super mix (Invitrogen 12344-040) in a final volume of 50. mu.l, using primers rbHC. up and rbHC. do for the heavy chain of wild type rabbit B-cells, rbLC. up and rbLC. do for the light chain, and BcPCR _ FHLC _ leader. fw and BcPCR _ huCkappa. rev for the light chain of transgenic rabbit B-cells. All forward primers are specific for the signal peptide (of VH and VL respectively) and the reverse primers are specific for the constant region (of VH and VL respectively). The PCR conditions for RbVH + RbVL are as follows: hot start at 94 ℃ for 5 minutes; 35 cycles: 94 ℃ 20s, 70 ℃ 20s, 68 ℃ 45s, and a final extension at 68 ℃ for 7 minutes. PCR conditions for HuVL were as follows: hot start at 94 ℃ for 5 minutes; 40 cycles: 94 ℃ 20s, 52 ℃ 20s, 68 ℃ 45s, and a final extension at 68 ℃ for 7 minutes.

Mu.l of 50. mu.l of the PCR solution was loaded onto 48E-gel 2% (Invitrogen G8008-02). Positive PCR reactions were purified using the NucleoSpin Extract II kit (Macherey & Nagel; 740609250) according to the manufacturer's protocol and eluted with 50. mu.l of elution buffer. All purification steps were performed on a Hamilton ML Starlet system.

Example 7

Recombinant expression of rabbit monoclonal bivalent antibodies

For recombinant expression of the rabbit monoclonal bivalent antibody, PCR products encoding VH or VL were cloned as cDNA into an expression vector by the bulge cloning method (RS Haun et al, BioTechniques13(1992) 515-. The expression vector comprises an expression cassette consisting of: a 5 'CMV promoter comprising intron a, and a 3' BGH polyadenylation sequence. In addition to the expression cassette, the plasmid contains a pUC-derived origin of replication for plasmid amplification in E.coli and a beta-lactamase gene conferring ampicillin resistance. Three variants of the basic plasmid were used: a plasmid comprising a rabbit IgG constant region designed to accept a VH region; both plasmids contain a rabbit or human kappa LC constant region that hosts the VL region.

Linear expression plasmids encoding the kappa or gamma constant regions and VL/VH inserts were amplified by PCR using overlapping primers.

The purified PCR product was incubated with T4 DNA-polymerase, which produced single-stranded protrusions. The reaction was terminated by adding dCTP.

In the next step, the plasmid and insert are combined and incubated with recA, which induces site-specific recombination. The recombinant plasmid was transformed into E.coli. The next day, growing colonies were picked and the correct recombinant plasmid was detected by plasmid preparation, restriction analysis and DNA-sequencing.

For antibody expression, isolated HC and LC plasmids were transiently co-transfected into HEK293 cells, and supernatants were collected after 1 week.

Example 8

Recombinant expression of rabbit monoclonal monovalent antibodies

To express the selected candidates recombinantly as monoclonal monovalent antibodies, the rabbit constant regions of all VH chains were transformed into human constant regions encompassing the bulge-mutations in the CH3 fragment. For VL chains derived from rabbit wild-type B-cells, rabbit C.kappa.constant regions were transformed to human. The immunoglobulin heavy and light chain variable regions were amplified in a final volume of 50. mu.l using 4. mu.l cDNA of the selected candidate with an AccuPrime super mix (Invitrogen 12344-040) with the forward primer specific for the signal peptide and the reverse primer specific for the CDR3-J region with a consensus sequence (20bp) (at the 3' end) homologous to the human constant regions (of VH and VL, respectively). The PCR conditions for VH and VL chain amplification were as follows: hot start at 94 ℃ for 5 minutes; 35 cycles: 94 ℃ 20s, 68 ℃ 20s, 68 ℃ 45s, and a final extension at 68 ℃ for 7 minutes.

PCR-products encoding VH or VL are cloned as cDNA into expression vectors by the overhang cloning method (RSHaun et al, BioTechniques13(1992) 515-518; MZ Li et al, Nature Methods 4(2007) 251-256). The expression vector comprises an expression cassette consisting of: a 5 'CMV promoter comprising intron a and a 3' BGH polyadenylation sequence. In addition to the expression cassette, the plasmid contains a pUC-derived origin of replication for plasmid amplification in E.coli and a beta-lactamase gene conferring ampicillin resistance. Two variants of the basic plasmid were used: one plasmid contained a human IgG constant region designed to accept the newly amplified VH chain and the second plasmid contained a human kappa LC constant region that accepts the VL chain.

Example 9

Human PDGF-BB binding ELISA

Binding using enzyme-linked immunosorbent assay (ELISA) based techniquesAnd (6) analyzing. The antigen human PDGF-BB (Cell Signaling, Cat. No. 8921BF) was immobilized on 384-well microtiter plates (Thermo Scientific, Cat. No.464718) at a concentration of 125ng/mL (25. mu.L in PBS, 0.5% BSA and 0.05% tween). Each of the following steps was followed by 3 routine washes (90 μ L PBS, with dispersion and aspiration): 1) and (3) sealing: saturating the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detecting antibody, at a dilution of 1: 3000(ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or alternatively for murine antibody ECL anti-mouse IgG-POD; NA 9310V). The optical density was determined at 370nm 20-30 minutes after addition of the substrate 3, 3 ', 5, 5' -tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). EC was calculated using a four parameter logistic model using GraphPad Prism 6.0 software₅₀。

Example 10

Cynomolgus monkey PDGF-BB binding ELISA

Binding assays were performed using enzyme-linked immunosorbent assay (ELISA) based techniques. The antigen human PDGF-BB was immobilized at a concentration of 125ng/mL (25. mu.L in PBS, 0.5% BSA and 0.05% Tween) on 384-well microtiter plates (Thermo Scientific, Cat. No. 464718). Each of the following steps was followed by 3 routine washes (90 μ L PBS, 0.5% BSA, 0.05% tween, performed with dispersion and aspiration): 1) and (3) sealing: saturating the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detecting antibody, at a dilution of 1: 3000(ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or alternatively for murine antibody ECL anti-mouse IgG-POD; NA 9310V). The optical density was determined at 370nm 20-30 minutes after addition of the substrate 3, 3 ', 5, 5' -tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). EC was calculated using a four parameter logistic model using GraphPad Prism 6.0 software₅₀。

Example 11

Murine PDGF-BB binding ELISA

Binding assays were performed using enzyme-linked immunosorbent assay (ELISA) based techniques. The antigen murine PDGF-BB (Peprotech 315-18) was immobilized on 384-well microtiter plates (Thermo Scientific, Cat. No.464718) at a concentration of 125ng/mL (25. mu.L in PBS, 0.5% BSA and 0.05% tween). Each of the following steps was followed by 3 routine washes (90 μ L PBS, 0.5% BSA, 0.05% tween, performed with dispersion and aspiration): 1) and (3) sealing: saturating the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detecting antibody, at a dilution of 1: 3000(ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or alternatively for murine antibody ECL anti-mouse IgG-POD; NA 9310V). The optical density was determined at 370nm 20-30 minutes after addition of the substrate 3, 3 ', 5, 5' -tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). EC was calculated using a four parameter logistic model using GraphPad Prism 6.0 software₅₀。

Example 12

Rat PDGF-BB binding ELISA

Binding assays were performed using enzyme-linked immunosorbent assay (ELISA) based techniques. Antigen rat PDGF-BB (R)&D, 520-BB) was fixed at a concentration of 125ng/mL (25. mu.L in PBS, 0.5% BSA and 0.05% Tween) on 384-well microtiter plates (Thermo Scientific, Cat. No. 464718). Each of the following steps was followed by 3 routine washes (90 μ L PBS, 0.5% BSA, 0.05% tween, performed with dispersion and aspiration): 1) and (3) sealing: saturating the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detecting antibody, at a dilution of 1: 3000(ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or alternatively for murine antibody ECL anti-mouse IgG-POD; NA 9310V). The optical density was determined at 370nm 20-30 minutes after addition of the substrate 3, 3 ', 5, 5' -tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). GraphPad Prism 6 was used.0 software uses four-parameter logic model to calculate EC₅₀。

Example 13

Human PDGF-AA binding ELISA

Binding assays were performed using enzyme-linked immunosorbent assay (ELISA) based techniques. The antigen human PDGF-AA (Peprotech, Cat. No. AF-100-13A) was immobilized on 384-well microtiter plates (Thermo Scientific, Cat. No.464718) at a concentration of 125ng/mL (25. mu.L in PBS, 0.5% BSA and 0.05% Tween). Each of the following steps was followed by 3 routine washes (90 μ L PBS, 0.5% BSA, 0.05% tween, performed with dispersion and aspiration): 1) and (3) sealing: saturating the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detecting antibody, at a dilution of 1: 3000(ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or alternatively for murine antibody ECL anti-mouse IgG-POD; NA 9310V). The optical density was determined at 370nm 20-30 minutes after addition of the substrate 3, 3 ', 5, 5' -tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). EC was calculated using a four parameter logistic model using GraphPad Prism 6.0 software₅₀。

Example 14

Human PDGF-CC binding ELISA

Binding assays were performed using enzyme-linked immunosorbent assay (ELISA) based techniques. The antigen human PDGF-CC (PeprotechAF-100-00C) was immobilized on 384-well microtiter plates (Thermo Scientific, Cat. No.464718) at a concentration of 125ng/mL (25. mu.L in PBS, 0.5% BSA and 0.05% tween). Each of the following steps was followed by 3 routine washes (90 μ L PBS, 0.5% BSA, 0.05% tween, performed with dispersion and aspiration): 1) and (3) sealing: saturating the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detection of antibody, dilution 1: 3000(ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-P)OD, NA933V or alternatively for murine antibodies ECL anti-mouse IgG-POD; NA 9310V). The optical density was determined at 370nm 20-30 minutes after addition of the substrate 3, 3 ', 5, 5' -tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). EC was calculated using a four parameter logistic model using GraphPad Prism 6.0 software₅₀。

Example 15

Protein-protein interaction inhibition assay: human PDGF-B: human PDGF-BB receptor

Protein-protein interaction inhibition assays of human PDGF-BB and the human PDGF-BB receptor were performed using enzyme-linked immunosorbent assay (ELISA) based techniques. Human Fc-labeled PDGF-BB receptor protein (RnD, Cat. No.385-PR-100) was immobilized at a concentration of 750. mu.g/mL (25. mu.L in PBS, 0.5% BSA and 0.05% Tween) on 384-well microtiter plates (Thermo Scientific Cat. No. 464718). Each of the following steps was followed by 3 routine washes (90 μ L PBS, with dispersion and aspiration): 1) a blocking step to saturate unbound surface (1 hour, 2% BSA); 2) 15 μ L of increasing concentrations of anti-PDGF-BB antibody was incubated with 15 μ L of 75nM biotinylated human PDGF-BB (Cell Signaling, Cat. No.8921BF) in a volume of 30 μ L for 1 hour; 3) detection was achieved using peroxidase-labeled streptavidin (Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 11089153001). The optical density was determined at 370nm 20-30 minutes after addition of the substrate 3, 3 ', 5, 5' -tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). IC calculation Using the four parameter logic model Using GraphPad Prism 6.0 software₅₀。

Example 16

Antibody purification from murine hybridomas

The hybridoma supernatants containing the antibodies were filtered and purified by two chromatography steps. PBS (1 mMKH) was used₂PO₄，10mM Na₂HPO₄137mM NaCl, 2.7mM KCl) (pH 7.4) in balance with HiTrap Protein G (GEHealthcare) and the antibody was captured by affinity chromatography. Unbound proteins were removed by washing with equilibration buffer and the antibody was recovered with 25mM citrate buffer (pH 3.0) and neutralized to pH6.0 with 1M Tris-base (pH 9.0) immediately after elution. Used in Superdex 200^TMSize exclusion chromatography on (GE Healthcare) was used as the second purification step. The size exclusion chromatography was performed in 20mM histidine buffer, 0.14M NaCl, pH 6.0. The antibody-containing solution was concentrated and stored at-80 ℃ using an Ultrafree-CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, Mass.).

The hybridoma supernatants containing the antibodies were filtered and purified by two chromatography steps. The supernatant was mixed with 50% v/v 2M glycine, pH 8.6, 600mM NaCl and captured by affinity chromatography using HiTrapapMabSelectSuRe (GE healthcare) equilibrated with 1M glycine, pH 8.6, 300mM NaCl. Unbound proteins were removed by washing with equilibration buffer and the antibody was recovered with 100mM citrate buffer (pH 2.8) and neutralized to pH6.0 with 1M Tris-base (pH8.5) immediately after elution. Used in Superdex 200^TMSize exclusion chromatography on (GE Healthcare) was used as the second purification step. The size exclusion chromatography was performed in 20mM histidine buffer, 0.14M NaCl, pH 6.0. The antibody-containing solution was concentrated and stored at-80 ℃ using an Ultrafree-CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, Mass.).

Example 17

PDGF-BB binding surface plasmon resonance spectroscopy

Binding analysis was performed using a BIAcore B4000 System (GE Healthcare) with a surface plasmon resonance spectroscopy based technique. The antigen human PDGF-BB (Cell Signaling, Cat. No.8921BF) was immobilized on a C1 sensor chip (GE Healthcare, Cat. No. BR-1005-35) at a concentration of 1. mu.g/mL (in PBS, 0.1% BSA, 0.05% Tween) using amine coupling chemistry. Application at 10mM HEPES pH7.2, increased concentration of anti-PDGF-BB antibody in 150mM NaCl. Apparent k was calculated using the read times for the 180 second association period and the 600 second dissociation period_aAnd apparent k_d. Calculation of apparent K Using BIAcoreT200 v2.0 fitting software_D(avidity ).

Example 18

Cynomolgus monkey PDGF-BB binding surface plasmon resonance spectroscopy determination

Binding analysis was performed using a BIAcore B4000 System (GE Healthcare) with a surface plasmon resonance spectroscopy based technique. The antigen cynomolgus monkey PDGF-BB was immobilized on a C1 sensor chip (GE Healthcare, Cat. No. BR-1005-35) at a concentration of 1. mu.g/mL (in PBS, 0.1% BSA, 0.05% tween) using amine coupling chemistry. An increasing concentration of anti-PDGF-BB antibody in 10mM HEPES pH 7.2, 150mM NaCl was used. Apparent k was calculated using the read times for the 180 second association period and the 600 second dissociation period_aAnd apparent k_d. Calculation of apparent K Using BIAcore T200 v2.0 fitting software_D(avidity ).

Example 19

Transfection and transient expression of humanized antibodies in HEK cells

Transient expression of the antibody was performed in suspension-adapted HEK293F (FreeStyle293-F cells; Invitrogen) cells using the Transfection agent 293-free (Transfection Reagent 293-free, Novagen).

In 125ml shake flasks (at 37 ℃, 7% CO)₂85% humidity, 135rpm incubation/shaking) the cells have been passaged at least four times by dilution (volume 30 ml).

Cells were propagated to 3X10 in a 250ml volume⁵Individual cells/ml. After three days, cells were distributed and 7 x10⁵The density of each cell/ml is newly inoculated to 1 literIn a 250ml volume in a shake flask. Transfection will occur at a cell density of about 1.4-2.0X10⁶24 hours after each cell/ml.

Prior to transfection, 250. mu.g of plasmid-DNA (122. mu.g light chain and 128. mu.g heavy chain) were diluted in a final volume of 10ml with pre-heated (water bath; 37 ℃) Opti-MEM (Gibco). The solution was gently mixed and incubated at room temperature for no more than 5 minutes. Then, 333.3. mu.l of 293-free transfection reagent was added to the DNA-OptiMEM-solution. Mix gently and incubate at room temperature for 15-20 minutes. The entire volume of the mixture was added to a 1L shake flask with a 250ml HEK-cell-culture volume.

At 37 ℃ 7% CO₂85% humidity, 135rpm incubation/shaking for 6 or 7 days.

The supernatant was collected by a first centrifugation step at 2,000rpm, 4 ℃, 10 minutes. The supernatant was then transferred to a new centrifuge bottle and centrifuged a second time at 4,000rpm for 20 minutes at 4 ℃. The cell-free supernatant was then filtered through a 0.22 μm bottle top filter (bottle-top-filter) and stored in a refrigerator (-20 ℃).

Example 20

Purification of antibodies from HEK supernatants

The antibody-containing culture supernatant was filtered and purified by two chromatography steps. PBS (1 mMKH) was used₂PO₄，10mM Na₂HPO₄HiTrap MabSelectSuRe (GEHealthcare) equilibrated with 137mM NaCl, 2.7mM KCl) (pH 7.4) captures the antibody by affinity chromatography. Unbound proteins were removed by washing with equilibration buffer and the antibody was recovered with 100mM citrate buffer (pH 2.8) and neutralized to pH6.0 with 1M Tris-base (pH 9.0) immediately after elution. Used in Superdex 200^TMSize exclusion chromatography on (GE Healthcare) was used as the second purification step. The size exclusion chromatography was performed in 20mM histidine buffer, 0.14M NaCl, pH 6.0. The antibody-containing solution was centrifuged and filtered using Ultrafree-CL equipped with Biomax-SK membranes (Millipore, Billerica, Mass.)The vessel parts were concentrated and stored at-80 ℃.

Example 21

Analysis of antibody preparations

The protein concentration of the antibody preparation was determined by measuring the Optical Density (OD) at 280nm using a molar extinction coefficient calculated based on the amino acid sequence.

The purity and integrity of the antibodies were analyzed by CE-SDS using LabChip GX II (PerkinElmer) with a Protein expression Chip (Protein Express Chip) and an HT Protein expression Kit (HTProtein Express Reagents Kit).

High performance SEC using TSK-GEL QC-PAK GFC 300 with 2 × PBS, pH7.4 as running buffer, or by using Biosuite high resolution SEC,5 μm analytical size exclusion column (Waters GmbH) with 200mMK₂HPO₄/KH₂PO₄High performance SEC as running buffer, 250mM KCl, pH 7.0, to determine aggregate content of the antibody preparation.

Example 22

Fab fragments were prepared from the antibody and analyzed:

5mg of antibody (about 1mg/ml in 20mM histidine, 140mM NaCl, pH 6.0) was incubated with 90. mu. l L-cysteine solution (Merck Millipore; 250mM in 20mM histidine, 140mM NaCl, pH 6.0) and 12. mu.l papain (Roche Life Science; 3.2U/mg antibody) at 37 ℃ for 120 minutes. After cleavage, PBS (1mM KH) was used₂PO₄，10mM Na₂HPO₄Affinity chromatography of HiTrap MabSelectSuRe (GEHealthcare) equilibrated at 137mM NaCl, 2.7mM KCl) (pH 7.4) removed intact IgG and Fc fragments. Subsequently, it was used in Superdex 200^TMLarge on (GEHealthcare)Small exclusion chromatography was used as a second purification step to further purify the flowthrough for MabSelectSuRe chromatography. The size exclusion chromatography was performed in 20mM histidine buffer, 0.14M NaCl, pH 6.0. The solution containing the Fab fragments was concentrated and stored at-80 ℃ using an Ultrafree-CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, Mass.).

The protein concentration of the Fab-fragments was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated on the basis of the amino acid sequence.

The purity and integrity of the Fab-fragments were analyzed by SDS-PAGE (NuPAGE 4-12% Bis-Tris gel, Invitrogen) in the presence and absence of reducing agent (5mM 1.4-dithiothreitol) and staining with Simply Blue Safe Stain (Invitrogen).

Aggregate content of Fab preparations was determined by high performance SEC using Superdex 20010/300 GL analytical size exclusion column (GE Healthcare) using 2x PBS, pH7.4 as running buffer.

Example 23

3T3 cell proliferation ELISA

To determine the proliferation inhibition, BrdU colorimetric assay (Roche diagnostics GmbH, Mannheim, Germany, # 11647229001) was used according to the manufacturer's manual. In this assay, 5ng/ml of human PDGF-BB and antibody were used.

Example 24

phospho-PDGF-R beta (Tyr751) sandwich ELISA

10,000 3T 3-cells/well were cultured in DMEM-medium supplemented with 10% newborn bovine serum for 24 hours. Then, the cells were cultured in Hunger's medium (DMEM-medium with 0.5% newborn bovine serum) for 24 hours.

The antibody (1. mu.g/ml) was pre-incubated for 2 hours in medium containing 5ng/ml PDGF-BB prior to addition. 3T3 cells were cultured in Hunger's medium with 5ng/ml of human PDGF-BB and antibody for 10 minutes.

Cells were washed 4 times with PBS prior to lysis. Cell lysis was performed in 100. mu.l lysis buffer.

The lysis solution was incubated overnight at 4 ℃ in wells of a 96-well plate coated with rabbit anti-PDGF-R β antibody. Wells were then washed 4 times with PBS. After 1 hour incubation with mouse anti-phospho-PDGF receptor beta antibody, wells were washed 4 times with washing buffer. For detection, wells were incubated with 100 μ l of TMB substrate solution for 30 minutes. The reaction was stopped by adding 100. mu.l of stop solution. Absorbance was determined at 450 nm.

Example 25

Cell migration assay

CIM-plate 16(ACEA Biosciences Inc., n. 05665817001, pore size membrane 8 μm) was used.

Flow path

Primary human retinal pericytes ((ACBRI 183 CellSystems, immortalized with hTERT)) were starved overnight (in CellSystems CSC serum-free medium without growth factors (b ℃ SF-4 ZR-500-S)) before use (50-70% confluency).

Plate preparation

An upper chamber: (50. mu.l medium + 100. mu.l cell suspension)

Both sides were coated with 20. mu.g/ml fibronectin (Sigma n ℃ F0895-2mg) in PBS:

on the sensor side (bottom side) of each well 40 μ l fibronectin solution was added and the upper chamber was incubated in a fume hood for 30 min. The fibronectin solution was carefully aspirated from the sensor side, avoiding touching the electrodes. UC is flipped so that the aperture side is up and the sensor side is down. 50 μ l fibronectin solution was added to the wells and incubated in a fume hood at Room Temperature (RT) for 30 minutes. Carefully suck the solution from the inside of the well. Add 50. mu.l of CSC serum-free medium without growth factors to the wells.

A lower chamber: 160 μ l/well your sample to be tested (1X concentrated)

All dilutions were in CellSystems CSC serum-free medium without growth factors (n ℃ SF-4 ZR-500-S); incubating the sample/antibody mixture in a fume hood for 2 hours; add 160. mu.l/well of sample; assembly of UC and LC using CIM-plate 16 assembly tool; CIM-plate 16 was placed in an incubator at 37 ℃ for 1 hour to equilibrate; placing CIM-plate 16 on an RTCADP analyzer; background measurements were initiated in RTCA software by starting step 1.

Cell preparation

Cells were stripped with cell dissociation buffer and washed at 1.5x10⁵Individual cells/ml were resuspended in CellSystems CSC serum-free medium without growth factor (n ℃ SF-4 ZR-500-S); CIM-plate 16 was removed from RTCA analyzer and 15,000 primary human retinal pericytes ((ACBRI 183 CellSystems), immortalized with hTERT)/well/100 μ Ι in basal medium (Cell Systems CSC serum-free medium without growth factor n ° SF-4ZR-500-S) were added to UC; the plate was returned to the RTCA analyzer and measurement was immediately started for 10-20 hours.

Example 26

Cell proliferation assay

Cell proliferation assay in CellTiter 96 Aqueous One solution (Promega, G3580).

Primary human retinal pericytes ((ACBRI 183 CellSystems), immortalized with hTERT), 2500 cells/well/100. mu.l

Culture medium

Growth medium ═ CSC complete medium, culture booster (ACBRI n ° 4Z ═ 500)

Determination of culture Medium-CSC culture serum Medium without growth factor (ACBRI n ℃ SF-4ZR-500-S)

Balbc3T3, 5000 cells/well/100 μ l

Culture medium

Growth medium DMEM (Gibco n ° 41966), 10% NBCS (newborn bovine serum)

Assay medium-DMEM (Gibco n ° 41966), 0.4% NBCS

Proliferation assay

Cells were seeded in growth medium 24-48 hours prior to assay when cells reached 80-90% confluence, cells were harvested with trypsin solution, cells were plated at 0.25 × 10⁵Individual cell/ml (or 0.5 × 10)⁵Individual cells/ml) were resuspended in growth medium. To each well of a 96-well plate, 100. mu.l of the cell suspension was added. Incubate at 37 ℃ for 24 hours. Growth medium was removed and 100 μ l of assay medium was added to each well. Incubate for 24 hours. To each well 100 μ l of standard or sample (2X concentrated, diluted in assay medium) was added. At 37 with 5% CO₂Incubate for 72 hours. Add 40. mu.l of dye solution to the cells and incubate at 37 ℃. Read at 490nm at different times (1h to 8 h).

Example 27

anti-PDGF-BB antibody kinetic screening

Binding of anti-PDGF-BB antibodies to human PDGF-BB was studied by surface plasmon resonance using the BIACORE T200 instrument (GE Healthcare). Approximately 20 Resonance Units (RU) of recombinant human PDGF-BB (5. mu.g/ml; ordering code 220-BB; R & D Systems) were coupled to Series S C1 chips (GE Healthcare BR-1005-35) at pH4.0 using the amine coupling kit supplied by GE Healthcare. The running buffer used for immobilization was HBS-N pH7.4(10mM HEPES, 150mM NaCl, pH7.4, GE Healthcare). For the following kinetic characterization, the running and dilution buffer was HBS-P pH7.4(10mM HEPES, 150mM NaCl, 0.05% surfactant P20, pH7.4, GEHealthcare). The flow cell was set to 25 ℃ and the sample block was set to 12 ℃ and primed twice with running buffer.

Association was measured by injecting anti-PDGF-BB antibody in solution at concentrations of 30nM and 3nM for 30 seconds at a flow rate of 100. mu.l/min. The dissociation period was monitored up to 600 seconds and initiated by switching from the sample solution to running buffer. By injecting 0.85% H twice in succession at a flow rate of 5. mu.l/min₃PO₄The (phosphoric acid) solution was washed for 60 seconds to regenerate the surface. Bulk refractive index (bulk refractive index) differences were corrected by subtracting the responses from the blank surface. Blank injections (double reference) were also subtracted. To calculate K_DAnd other kinetic parameters, using the Langmuir 1: 1 model.

Example 28

anti-PDGF-BB Fab kinetic characterization

Binding of anti-PDGF-BB Fab samples to human PDGF-BB was studied by surface plasmon resonance using the BIACORE T200 instrument (GE Healthcare). Approximately 50 Resonance Units (RU) of recombinant human PDGF-BB (0.5. mu.g/ml; ordering code 220-BB; R & D Systems) were coupled to Series S CM3 chips (GE Healthcare BR-1005-36) at pH4.0 using the amine coupling kit supplied by GE Healthcare. The running buffer used for immobilization was HBS-N pH7.4(10mM HEPES, 150mM NaCl, pH7.4, GE Healthcare). For the following kinetic characterization, the running and dilution buffer was HBS-P pH7.4(10mM HEPES, 150mM NaCl, 0.05% surfactant P20, pH7.4, GE Healthcare). The flow cell was set to 25 ℃ and the sample block was set to 12 ℃ and primed twice with running buffer.

Association was measured by injecting different concentrations (starting at 300nM, successive 1: 3 dilutions) of anti-PDGF-BB Fab in solution at a flow rate of 30. mu.l/min for 180 seconds. MonitoringThe dissociation period was up to 900 seconds and was initiated by switching from the sample solution to running buffer. By using 0.85% H at a flow rate of 5. mu.l/min₃PO₄The (phosphoric acid) solution was washed for 60 seconds to regenerate the surface. The bulk refractive index difference was corrected by subtracting the reaction from the blank surface. Blank injections (double reference) were also subtracted. To calculate KD and other kinetic parameters, a langmuir 1: 1 model was used.

Example 29

Chemical degradation test

Samples were split into triplicate aliquots and re-buffered in 20mM His/His HCl, 140mM NaCl, pH6.0 or in PBS, respectively, stored at 40 ℃ (His/NaCl) or 37 ℃ (PBS). The control sample was stored at-80 ℃.

After incubation, samples were analyzed for relative active concentration (BIAcore), aggregation (SEC) and disruption (capillary electrophoresis or SDS-PAGE) and compared to untreated controls.

Example 30

Thermal stability

Samples were prepared at a concentration of 1mg/mL in 20mM histidine/histidine hydrochloride, 140mM NaCl, pH6.0, transferred to optical 384-well plates by centrifugation through 0.4 μm filter plates, and covered with paraffin oil. The hydraulic radius was repeatedly measured by dynamic light scattering on a DynaPro plate reader (Wyatt) while heating the sample from 25 ℃ to 80 ℃ at a rate of 0.05 ℃/min.

Alternatively, the samples were transferred to a 10 μ L microcuvette array and static light scattering data and fluorescence data upon excitation with a 266nm laser were recorded with an Optim1000 instrument (avacainc.) while heating them from 25 ℃ to 90 ℃ at a rate of 0.1 ℃/min.

The onset of aggregation temperature is defined as the temperature at which the hydraulic radius (DLS) or scattered light intensity (Optim1000) begins to increase.

Melting temperature is defined as the inflection point in the plot of fluorescence intensity versus wavelength.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention. The functional content of all patent and scientific literature cited herein is expressly incorporated by reference in its entirety.

Example 31

Preparation and purification of bispecific antibodies

Transient expression of bispecific antibodies was performed in suspension-adapted HEK293F (FreeStyle293-F cells; Invitrogen) cells after transfection of DNA with the transfection reagent 293-free (Novagen).

After thawing in a 125ml shake flask (incubation/shaking at 37 ℃, 7% CO2, 85% humidity, 135 rpm), the cells were passaged by dilution at least four times (volume 30ml) every third or fourth day. By mixing cells at 3X10⁵The cells were expanded by seeding the cells at a cell density of one cell/ml in 250ml of medium. After three days, cells were distributed and counted at 2x 10⁵The density of individual cells/ml was freshly inoculated in 500ml of medium. After four days, cells were distributed and 7 x10⁵The cells/ml were freshly inoculated in 1 liter of medium (incubated/shaken at 37 ℃, 7% CO2, 85% humidity, 110 rpm). After 24 hours, at about 1.4-2.0X10⁶Transfection was performed at a cell density of individual cells/ml.

Prior to transfection, 1000. mu.g of plasmid-DNA (2X 250. mu.g of light chain-encoding plasmid DNA and 2X 250. mu.g of heavy chain-encoding plasmid DNA) were diluted in pre-heated (water bath; 37 ℃) Opti-MEM (Gibco) with a final volume of 40 ml. The solution was gently mixed and incubated at room temperature for no more than 5 minutes. Then, 1333. mu.l of 293-free transfection reagent was added to the DNA-Opti-MEM-solution. The mixture was gently mixed and incubated at room temperature for 15-20 minutes. The entire volume of the mixture was carefully added to 1 liter of HEK-cell culture. Cells were further cultured at 37 ℃, 7% CO2, 85% humidity with shaking at 110rpm for 7 days.

After 7 days, the supernatant was collected by a first centrifugation step at 2000rpm, 4 ℃ for 10 minutes. The supernatant was then transferred to a new centrifuge bottle for a second centrifugation step at 4000rpm, 4 ℃ for 20 minutes. The cell-free supernatant was filtered through a 0.22 μm filter (Millipore) and stored in a refrigerator (-20 ℃) before starting the purification step.

The antibody-containing culture supernatant was filtered and purified by at least two chromatographic steps. Antibodies were captured by affinity chromatography using CaptureSelect prepacked column IgG-CH1(life technologies, #494320005) equilibrated with PBS (1 mkh2PO4, 10mM Na2HPO4, 137mM NaCl, 2.7mM KCl) (pH 7.4). Unbound proteins were removed by washing with equilibration buffer and the antibody was recovered with 25mM citrate buffer (pH 3.0) and neutralized to pH6.0 with 1m tris-base (pH 9.0) immediately after elution.

Used in Superdex 200^TMSize exclusion chromatography on (GE Healthcare) was used as the second purification step. The size exclusion chromatography was performed in 20mM histidine buffer, 0.14M NaCl, pH 6.0. The antibody-containing solution was concentrated and stored at-80 ℃ using an Ultrafree-CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, Mass.).

The anti-PDGF-B/ANG 2 antibody (clone 0144-. Ammonium sulfate was added to the antibody-containing solution to a final concentration of 1M. The solution was applied to a Butyl Sepharose 4 Fast Flow (GE Healthcare) column equilibrated with 1M ammonium sulfate, 35mM acetate, pH 5.6. The antibody was eluted with a linear gradient (0-100%) with 35mM acetate (pH 5.6). The antibody-containing fractions were pooled and applied to size exclusion chromatography.

The protein concentration of the antibody preparation was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence.

The purity and integrity of the antibodies were analyzed by CE-SDS using LabChip GX II (Perkinelmer) with a protein expression chip and HT protein expression kit.

Using the BioSuite high-resolution SEC,5 μm analytical size exclusion column (Waters GmbH), the aggregate content of the antibody preparation was determined by high performance SEC using 200mM K2HPO4/KH2PO4, 250mM KCl, pH 7.0 as running buffer.

Antibody 0144 is a polypeptide comprising SEQ ID NO: 117(VH) and SEQ ID NO: 118(VL) and the ANG2 binding site of seq id NO: 92(VH) and SEQ ID NO: 97(VL) PDGF-B binding site CrosMab antibody.

Antibody 0117 is a polypeptide comprising SEQ ID NO: 119(VH) and SEQ ID NO: 120(VL) and the VEGF binding site of seq id NO: 92(VH) and SEQ ID NO: 97(VL) PDGF-B binding site CrosMab antibody.

Antibody 0145 is a fusion protein comprising SEQ ID NO: 119(VH) and SEQ ID NO: 120(VL) and the ANG2 binding site of seq id NO: 101(VH) and SEQ ID NO: 106(VL) of the PDGF-B binding site.

Antibody 0146 is a fusion protein comprising SEQ ID NO: 117(VH) and SEQ ID NO: 118(VL) and the ANG2 binding site of seq id NO: 121(VH) and SEQ ID NO: 122(VL) of PDGF-B binding site.

Example 32

Bispecific antibody kinetic characterization

PDGF-BB

Binding of the bispecific anti-PDGF-BB/ANG 2 antibody to human PDGF-BB was studied by surface plasmon resonance using a BIACORE T200 instrument (GE Healthcare). Approximately 50 Resonance Units (RU) of recombinant human PDGF-BB (0.5. mu.g/ml; ordering code 220-BB; R & DSsystems) were coupled to Series S CM3 chips (GE Healthcare BR-1005-36) at pH4.0 using the amine coupling kit supplied by GE Healthcare. The running buffer used for immobilization was HBS-N pH7.4(10mM HEPES, 150mM NaCl, pH7.4, GE Healthcare). For the following kinetic characterization, the running and dilution buffer was HBS-P pH7.4(10mM HEPES, 150mM NaCl, 0.05% surfactant P20, pH7.4, GE Healthcare). The flow cell was set to 25 ℃ and the sample block was set to 12 ℃ and primed twice with running buffer.

Association was measured by injecting different concentrations (starting at 300nM, successive 1: 3 dilutions) of bispecific antibody in solution at a flow rate of 30. mu.l/min for 180 seconds. The dissociation period was monitored up to 900 seconds and initiated by switching from the sample solution to running buffer. By using 0.85% H at a flow rate of 5. mu.l/min₃PO₄The (phosphoric acid) solution was washed for 60 seconds to regenerate the surface. The bulk refractive index difference was corrected by subtracting the reaction from the blank surface. Blank injections (double reference) were also subtracted. To calculate KD and other kinetic parameters, a langmuir 1: 1 model was used.

ANG2

Binding of bispecific antibodies to human ANG 2-RBD-mouse Fc-region fusions was studied by surface plasmon resonance using BIACORE T200 instrument (GE Healthcare). Approximately 4000RU of anti-mouse Fc-region antibody (10. mu.g/ml anti-mouse (Fc) antibody) was coupled to Series SCM5 chips (GE Healthcare BR-1005-30) at pH 5.0 using the amine coupling kit supplied by GE Healthcare. HBS-N (10mM HEPES, 150mM NaCl pH7.4, GE Healthcare) was used as running buffer during the immobilization step. For the following kinetic characterization, the sample and running buffer was HBS-P (10mM HEPES, 150mM NaCl pH7.4, 0.05% surfactant P20; GEHealthcare). The flow cell was set to 25 ℃ and the sample block was set to 12 ℃ and primed twice with running buffer before kinetic characterization.

Human ANG 2-RBD-murine Fc-region fusions were captured by injection of 1. mu.g/ml solution at a flow of 5. mu.l/min for 30 seconds. Association was measured by injecting different concentrations (starting at 300nM, successive 1: 3 dilutions) of bispecific antibody in solution at a flow rate of 90. mu.l/min for 90 seconds. The dissociation period was monitored up to 600 seconds and initiated by switching from the sample solution to running buffer. All surfaces were regenerated by washing with 3M MgCl2 solution at a flow rate of 5. mu.l/min for 60 seconds. The bulk refractive index difference was corrected by subtracting the reaction obtained from the surface of anti-mouse IgG antibody (Fc). Blank injections (double reference) were also subtracted. To calculate KD and other kinetic parameters, a langmuir 1: 1 model was used.

VEGF

Binding of bispecific antibodies to human VEGF isoform 121 was studied by surface plasmon resonance using BIACORE T200 instrument (GE Healthcare). Anti-hexa-histidine antibodies were conjugated to CM5 chips (GE Healthcare BR-1005-30) using the amine coupling kit supplied by GE Healthcare, according to the manufacturer's instructions. HBS-N (10mM HEPES, 150mM NaCl pH7.4, GE Healthcare) was used as running buffer during the immobilization step. For the following kinetic characterization, the sample and running buffer was HBS-P (10mM HEPES, 150mM NaCl pH7.4, 0.05% surfactant P20; GE Healthcare). The flow cell was set to 25 ℃ and the sample block was set to 12 ℃ and primed twice with running buffer before kinetic characterization.

Human VEGF isoform 121 containing a histidine tag was captured by injecting the solution at a flow rate of 5. mu.l/min for 30 seconds. Association was measured by injecting different concentrations (starting at 300nM, successive 1: 3 dilutions) of bispecific antibody in solution at a flow rate of 90. mu.l/min for 90 seconds. The dissociation period was monitored up to 600 seconds and initiated by switching from the sample solution to running buffer. All surfaces were regenerated by washing with 3M MgCl2 solution at a flow rate of 5. mu.l/min for 60 seconds. The bulk refractive index difference was corrected by subtracting the reaction obtained from the anti-hexa-histidine antibody surface. Blank injections (double reference) were also subtracted. To calculate KD and other kinetic parameters, a langmuir 1: 1 model was used.

Sequence listing

<110> Haofmai Roche Ltd

<120> anti-PDGF-B antibodies and methods of use

<130>P32415

<150>EP14192519.8

<151>2014-11-10

<160>174

<170>PatentIn version 3.5

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Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

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Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

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Trp Ile Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met

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Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Gln Thr Thr Gln Asp Phe Asp Ser Trp Gly Gln Gly Thr Leu Val

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Thr Val Ser Ser

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<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H1-004

<400>26

Gly Tyr Thr Phe Ser Ser Tyr

1 5

<210>27

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H2s-004

<400>27

Gly Ser Gly

1

<210>28

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H2-004

<400>28

Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210>29

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H3-004

<400>29

Thr Thr Gln Asp Phe Asp Ser

1 5

<210>30

<211>116

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 VH-005

<400>30

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Ala Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gln Thr Thr Gln Asp Phe Asp Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>31

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H1-005

<400>31

Gly Tyr Thr Phe Ser Ser Tyr

1 5

<210>32

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H2s-005

<400>32

Gly Ser Gly

1

<210>33

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H2-005

<400>33

Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ser

<210>34

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H3-005

<400>34

Thr Thr Gln Asp Phe Asp Ser

1 5

<210>35

<211>116

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 VH-006

<400>35

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Val

35 40 45

Ser Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Asn Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gln Thr Thr Gln Asp Phe Asp Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>36

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H1-006

<400>36

Gly Tyr Thr Phe Ser Ser Tyr

1 5

<210>37

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H2s-006

<400>37

Gly Ser Gly

1

<210>38

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H2-006

<400>38

Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

15 10 15

Gly

<210>39

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-H3-006

<400>39

Thr Thr Gln Asp Phe Asp Ser

1 5

<210>40

<211>111

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 VL-001

<400>40

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Ile Tyr

20 25 30

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>41

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L1-001

<400>41

Ser Glu Ser Val Asp Ile Tyr Gly Tyr Ser Phe

1 5 10

<210>42

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L2-001

<400>42

Arg Ala Ser

1

<210>43

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L3-001

<400>43

Ser Asn Glu Asp Pro Arg

1 5

<210>44

<211>111

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 VL-002

<400>44

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Ile Tyr

20 25 30

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>45

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L1-002

<400>45

Ser Glu Ser Val Asp Ile Tyr Gly Tyr Ser Phe

1 5 10

<210>46

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L2-002

<400>46

Arg Ala Ser

1

<210>47

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L3-002

<400>47

Ser Asn Glu Asp Pro Arg

1 5

<210>48

<211>111

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 VL-003

<400>48

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Ile Tyr

20 2530

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>49

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L1-003

<400>49

Ser Glu Ser Val Asp Ile Tyr Gly Tyr Ser Phe

1 5 10

<210>50

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L2-003

<400>50

Arg Ala Ser

1

<210>51

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L3-003

<400>51

Ser Asn Glu Asp Pro Arg

1 5

<210>52

<211>111

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 VL-004

<400>52

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr IleThr Cys Arg Ala Ser Glu Ser Val Asp Ile Tyr

20 25 30

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>53

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L1-004

<400>53

Ser Glu Ser Val Asp Ile Tyr Gly Tyr Ser Phe

1 5 10

<210>54

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L2-004

<400>54

Arg Ala Ser

1

<210>55

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0044 HVR-L3-004

<400>55

Ser Asn Glu Asp Pro Arg

1 5

<210>56

<211>120

<212>PRT

<213>Homo sapiens

<400>56

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Thr Phe Ser Ser Phe

20 25 30

Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ala Gly Gly Gly Ile Thr His Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Asp Ile Tyr Ser Asp Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>57

<211>5

<212>PRT

<213> Intelligent (Homo sapiens)

<400>57

Ser Phe Trp Met Thr

1 5

<210>58

<211>6

<212>PRT

<213> Intelligent people

<400>58

Ser Ala Gly Gly Gly Ile

1 5

<210>59

<211>17

<212>PRT

<213> Intelligent people

<400>59

Ser Ile Ser Ala Gly Gly Gly Ile Thr His Tyr Pro Asp Ser Val Lys

1 5 10 15

Asp

<210>60

<211>11

<212>PRT

<213> Intelligent people

<400>60

Ser Gly Gly Asp Ile Tyr Ser Asp Phe Asp Tyr

1 5 10

<210>61

<211>107

<212>PRT

<213> Intelligent people

<400>61

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>62

<211>11

<212>PRT

<213> Intelligent people

<400>62

Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu Asn

1 5 10

<210>63

<211>7

<212>PRT

<213> Intelligent people

<400>63

Ala Ala Ser Ser Leu Gln Ser

1 5

<210>64

<211>9

<212>PRT

<213> Intelligent people

<400>64

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210>65

<211>120

<212>PRT

<213> Intelligent people

<400>65

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ala Gly Gly Gly Ile Thr His Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Asp Ile Tyr Ser Asp Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>66

<211>5

<212>PRT

<213> Intelligent people

<400>66

Ser Phe Trp Met Thr

1 5

<210>67

<211>6

<212>PRT

<213> Intelligent people

<400>67

Ser Ala Gly Gly Gly Ile

1 5

<210>68

<211>17

<212>PRT

<213> Intelligent people

<400>68

Ser Ile Ser Ala Gly Gly Gly Ile Thr His Tyr Pro Asp Ser Val Lys

1 5 10 15

Asp

<210>69

<211>11

<212>PRT

<213> Intelligent people

<400>69

Ser Gly Gly Asp Ile Tyr Ser Asp Phe Asp Tyr

1 5 10

<210>70

<211>107

<212>PRT

<213> Intelligent people

<400>70

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>71

<211>11

<212>PRT

<213> Intelligent people

<400>71

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210>72

<211>7

<212>PRT

<213> Intelligent people

<400>72

Thr Thr Ser Ser Leu Gln Ser

1 5

<210>73

<211>9

<212>PRT

<213> Intelligent people

<400>73

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210>74

<211>126

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0060 VH

<400>74

Glu Val Gln Leu Leu Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Ala Gly Gly Gly Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Thr Gly Arg Phe Thr Ser Ser Arg Asp Asn Tyr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Arg Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Tyr Pro Gly Tyr Asn Ser Asp Thr Tyr Tyr Tyr Asp Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210>75

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0060 HVR-H1

<400>75

Ser Tyr Ala Met Ser

1 5

<210>76

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0060 HVR-H2s

<400>76

Ser Ala Gly Gly Gly Tyr

1 5

<210>77

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0060 HVR-H2

<400>77

Gly Ile Ser Ala Gly Gly Gly Tyr Ile Tyr Tyr Ala Asp Ser Val Thr

1 5 10 15

Gly

<210>78

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0060 HVR-H3

<400>78

Phe Tyr Pro Gly Tyr Asn Ser Asp Thr Tyr Tyr Tyr Asp Gly Met Asp

1 5 10 15

Val

<210>79

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0060 VL kappa

<400>79

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp PheAla Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>80

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0060 HVR-L1

<400>80

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210>81

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0060 HVR-L2

<400>81

Ala Ala Ser Ser Leu Gln Ser

1 5

<210>82

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0060 HVR-L3

<400>82

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210>83

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0064 VH

<400>83

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

6570 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Ser Cys

85 90 95

Ala Glu Ser Gly Gly Ala Thr Asp Tyr Leu Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>84

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0064 HVR-H1

<400>84

Ser Tyr Trp Met Ser

1 5

<210>85

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0064 HVR-H2s

<400>85

Ser Asp Gly Gly Gly Thr

1 5

<210>86

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0064 HVR-H2

<400>86

Thr Ile Ser Asp Gly Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>87

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0064 HVR-H3

<400>87

Ser Gly Gly Ala Thr Asp Tyr Leu Phe Asp Tyr

1 5 10

<210>88

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0064 VL kappa

<400>88

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>89

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0064 HVR-L1

<400>89

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210>90

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0064 HVR-L2

<400>90

Ala Ala Ser Ser Leu Gln Ser

1 5

<210>91

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0064 HVR-L3

<400>91

Gln Gln Ser Tyr Ile Thr Pro Leu Thr

1 5

<210>92

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0085 VH

<400>92

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>93

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0085 HVR-H1

<400>93

Ser Tyr Trp Met Ser

1 5

<210>94

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0085 HVR-H2s

<400>94

Ser Asp Gly Gly Gly Leu

1 5

<210>95

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0085 HVR-H2

<400>95

Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val Lys

1 510 15

Gly

<210>96

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0085 HVR-H3

<400>96

Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr

1 5 10

<210>97

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0085 VL kappa

<400>97

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ala Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>98

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0085 HVR-L1

<400>98

Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu Asn

1 5 10

<210>99

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0085 HVR-L2

<400>99

Ala Ala Ser Ser Leu Gln Ser

1 5

<210>100

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0085 HVR-L3

<400>100

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210>101

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0086 VH

<400>101

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>102

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0086

<400>102

Ser Tyr Trp Met Ser

1 5

<210>103

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0086 HVR-H2s

<400>103

Ser Asp Gly Gly Gly Leu

1 5

<210>104

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0086 HVR-H2

<400>104

Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>105

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0086 HVR-H3

<400>105

Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr

1 5 10

<210>106

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0086 VL kappa

<400>106

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>107

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0086 HVR-L1

<400>107

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210>108

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0086 HVR-L2

<400>108

Ala Ala Ser Ser Leu Gln Ser

1 5

<210>109

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFB-0086 HVR-L3

<400>109

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210>110

<211>231

<212>PRT

<213> Intelligent people

<400>110

Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg

1 5 10 15

Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met

20 25 30

Leu Ser Asp His Ser Ile Arg Ser Asp Pro Gly Glu Glu Asp Gly Ala

35 40 45

Glu Leu Asp Leu Asn Met Thr Arg Ser His Ser Gly Gly Glu Leu Glu

50 55 60

Ser Leu Ala Arg Gly Arg Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu

65 70 75 80

Pro Ala Met Ile Ala Glu Cys Lys Thr Arg Thr Glu Val Phe Glu Ile

85 90 95

Ser Arg Arg LeuIle Asp Arg Thr Asn Ala Asn Phe Leu Val Trp Pro

100 105 110

Pro Cys Val Glu Val Gln Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn

115 120 125

Val Gln Cys Arg Pro Thr Gln Val Gln Leu Arg Pro Val Gln Val Arg

130 135 140

Lys Ile Glu Ile Val Arg Lys Lys Pro Ile Phe Lys Lys Ala Thr Val

145 150 155 160

Thr Leu Glu Asp His Leu Ala Cys Lys Cys Glu Thr Val Ala Ala Ala

165 170 175

Arg Pro Val Thr Arg Ser Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys

180 185 190

Thr Pro Gln Thr Arg Val Thr Ile Arg Thr Val Arg Val Arg Arg Pro

195 200 205

Pro Lys Gly Lys His Arg Lys Phe Lys His Thr His Asp Lys Thr Ala

210 215 220

Leu Lys Glu Thr Leu Gly Ala

225 230

<210>111

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223>rbHC.up

<400>111

aagcttgcca ccatggagac tgggctgcgc tggcttc 37

<210>112

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223>rbHCf.do

<400>112

ccattggtga gggtgcccga g 21

<210>113

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223>rbLC.up

<400>113

aagcttgcca ccatggacay gagggccccc actc 34

<210>114

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223>rbLC.do

<400>114

cagagtrctg ctgaggttgt aggtac 26

<210>115

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>BcPCR_FHLC_leader.fw

<400>115

atggacatga gggtccccgc 20

<210>116

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223>BcPCR_huCkappa.rev

<400>116

gatttcaact gctcatcaga tggc 24

<210>117

<211>129

<212>PRT

<213> Artificial sequence

<220>

<223>Ang2 LC10 wt + G114A, S360P, T28N, T30A (HC) + D50T (LC) VH

<400>117

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Ala Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Pro Gly Tyr Tyr Tyr

100 105 110

Pro Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser

<210>118

<211>110

<212>PRT

<213> Artificial sequence

<220>

<223>Ang2 LC10 wt + G114A, S360P, T28N, T30A (HC) + D50T (LC) VL

<400>118

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Thr Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser

100 105 110

<210>119

<211>123

<212>PRT

<213> Artificial sequence

<220>

<223>VEGF_LC VHVL cross LC kappa

<400>119

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>120

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223>VEGF_LC10 VHVL cross IgG HC LALAPGAAA knob

<400>120

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>121

<211>116

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFang2-0044 VH

<400>121

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Val Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gln Thr Thr Gln Asp Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210>122

<211>112

<212>PRT

<213> Artificial sequence

<220>

<223>PDGFang2-0044 VL

<400>122

Asp Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ile Tyr

20 25 30

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210>123

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0044 HC1

<400>123

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>124

<211>446

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0044 HC2

<400>124

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Val Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gln Thr Thr Gln Asp Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro

245 250 255

Glu ValThr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>125

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0044 LC1

<400>125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr

100 105 110

Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>126

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0044 LC2

<400>126

Asp Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ile Tyr

20 25 30

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 5560

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>127

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0058 HC1

<400>127

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>128

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0058 HC2

<400>128

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ala Gly Gly Gly Ile Thr His Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Asp Ile Tyr Ser Asp Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>129

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0058 LC1

<400>129

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr

100 105 110

Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>130

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0058 LC2

<400>130

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 9095

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>131

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0085 HC1

<400>131

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val AspVal Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>132

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0085 HC2

<400>132

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys AsnVal Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>133

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0085 LC1

<400>133

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr

100 105 110

Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>134

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0085 LC2

<400>134

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ala Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>135

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0086 HC1

<400>135

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 1015

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

SerLeu Ser Leu Ser Pro Gly Lys

435 440

<210>136

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0086 HC2

<400>136

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 9095

Ala Glu Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>137

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0086 LC1

<400>137

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr

100 105 110

Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>138

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 wt-PDGF-0086 LC2

<400>138

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>139

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0146 HC1

<400>139

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Thr Ser Asp Arg ProSer Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>140

<211>446

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0146 HC2

<400>140

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Val Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gln Thr Thr Gln Asp Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>141

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0146 LC1

<400>141

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Ala Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser IleSer Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Pro Gly Tyr Tyr Tyr

100 105 110

Pro Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>142

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0146 LC2

<400>142

Asp Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ile Tyr

20 25 30

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 7075 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>143

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut-PDGF-0058 HC1

<400>143

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Thr Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

TrpVal Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>144

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut-PDGF-0058 HC2

<400>144

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Trp Met Thr Trp Val Arg Gln Ala ProGly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ala Gly Gly Gly Ile Thr His Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Asp Ile Tyr Ser Asp Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>145

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut-PDGF-0058 LC1

<400>145

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Ala Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Pro Gly Tyr Tyr Tyr

100 105 110

Pro Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>146

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut-PDGF-0058 LC2

<400>146

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>147

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0144 HC1

<400>147

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Thr Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>148

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0144 HC1

<400>148

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

6570 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>149

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0144 LC1

<400>149

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Ala Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Pro Gly Tyr Tyr Tyr

100 105 110

Pro Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>150

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0144 LC2

<400>150

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ala Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>151

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0145 HC1

<400>151

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

HisTrp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Thr Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>152

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0145 HC2

<400>152

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>153

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0145 LC1

<400>153

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Ala Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Pro Gly Tyr Tyr Tyr

100 105 110

Pro Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>154

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0145 LC2

<400>154

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>155

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut-PDGF-0044 HC1

<400>155

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Thr Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro CysPro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>156

<211>446

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut-PDGF-0044 HC2

<400>156

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 510 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Val Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gln Thr Thr Gln Asp Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser ThrTyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>157

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut-PDGF-0044 LC1

<400>157

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Ala Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Ser Ser Pro Gly Tyr Tyr Tyr

100 105 110

Pro Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>158

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut-PDGF-0044 LC2

<400>158

Asp Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ile Tyr

20 25 30

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 9095

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>159

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0058 HC1

<400>159

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Thr Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>160

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0058 HC2

<400>160

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ala Gly Gly Gly Ile Thr His Tyr Pro Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Asp Ile Tyr Ser Asp Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>161

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0058 LC1

<400>161

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Ala Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Ser Ser Pro Gly Tyr Tyr Tyr

100 105 110

Pro Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>162

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0058 LC2

<400>162

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>163

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0085 HC1

<400>163

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

15 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Thr Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser ThrTyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>164

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0085 HC2

<400>164

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>165

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0085 LC1

<400>165

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Ala Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Ser Ser Pro Gly Tyr Tyr Tyr

100 105 110

Pro Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>166

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0085 LC2

<400>166

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ala Ile Thr Cys Arg Ala Ser Gln SerIle Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>167

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0086 HC1

<400>167

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Thr Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

305 310 315 320

Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

340 345 350

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>168

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0086 HC2

<400>168

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>169

<211>236

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0086 LC1

<400>169

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Ala Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Ser Ser Pro Gly Tyr Tyr Tyr

100 105 110

Pro Ala Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>170

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223>CrossMab Ang2 mut5-PDGF-0086 LC2

<400>170

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser ProVal Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>171

<211>439

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0117 HC1

<400>171

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr TyrCys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro Gly Lys

435

<210>172

<211>450

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0117 HC2

<400>172

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Gly Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Gly Gly Tyr Thr Asp Trp Leu Phe Gly Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

ValVal Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440445

Gly Lys

450

<210>173

<211>230

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0117 LC1

<400>173

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 9095

Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala

115 120 125

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

130 135 140

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

145 150 155 160

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

165 170 175

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

180 185 190

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

195 200 205

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

210 215 220

Phe Asn Arg Gly Glu Cys

225 230

<210>174

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> antibody 0117 LC2

<400>174

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ala Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. An antibody that specifically binds human PDGF-B, wherein the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 93, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 94, (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 96, (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 98, (e) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 99, and (f) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 100, and HVR-L3 of the amino acid sequence of seq id no.

2. An antibody that specifically binds human PDGF-B, wherein the antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 102, (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 103, (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 105, (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 107, (e) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 108, and (f) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 109, or a fragment thereof, and HVR-L3 of the amino acid sequence of seq id no.

3. The antibody of any one of claims 1-2, wherein the antibody is of the subclass human IgG1 or human IgG 4.

4. The antibody of any one of claims 1-3, wherein the antibody blocks the biological activity of human PDGF-B by inhibiting the binding of human PDGF-B to a human PDGF receptor.

5. A pharmaceutical formulation comprising the antibody of any one of claims 1-4 and optionally a pharmaceutically acceptable carrier.

6. The pharmaceutical formulation of claim 5, further comprising an additional therapeutic agent selected from an anti-ANG 2 antibody and an anti-VEGF antibody.

7. The antibody according to any one of claims 1-4 for use as a medicament.

8. Use of an antibody according to any one of claims 1 to 4 in the manufacture of a medicament.

9. Use according to any one of claims 7 and 8, wherein the medicament is for the treatment of ocular vascular diseases, preferably for the treatment of macular degeneration.

10. The use according to any one of claims 7-9, wherein the medicament is for inhibiting the interaction between PDGF-B and the PDGF-B receptor.