HK1239711B - Anti-pdgf-b antibodies and methods of use - Google Patents

Description

Anti-PDGF-B antibodies and methods of use

Field of the Invention

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. They are the main causes of vision loss in industrialized countries. Because the retina is composed of well-defined neurons, glial and vascular element layers, relatively small disturbances, such as those observed in vascular proliferation or edema, can lead to significant loss of visual function. Hereditary retinal degeneration, such as retinitis pigmentosa (RP), is also associated with vascular abnormalities, such as arteriolar stenosis and vascular atrophy. They affect 1 person in every 3,500 individuals and are characterized by progressive night blindness, visual field loss, optic nerve atrophy, arteriolar thinning and usually progress to complete central vision loss (central loss of vision).

Ischemic retinopathies are characterized by the loss or dysfunction of retinal blood vessels, which causes reduced blood flow and oxygen deprivation. The retina responds to the lack of oxygen by generating signals to grow new blood vessels, but these new vessels are typically fragile and disorganized. Because they can leak, cause bleeding, or lead to scarring that can cause retinal detachment, it is the growth of these abnormal new vessels that poses the greatest threat to vision. Current treatments for ischemic retinopathies seek to stop the pathological blood vessel growth, but do 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 destroying a portion of the retina with a laser in an attempt to stop new blood vessel growth and preserve central vision. Some strategies have been used to block the function of vascular endothelial growth factor (VEGF), a major promoter of blood vessel growth. In the short term, anti-VEGF treatments can improve vision, but they do not address the underlying ischemia and, in fact, can worsen the condition by inhibiting all blood vessel growth, including the growth of beneficial collaterals. There are also serious concerns about systemic exposure to these drugs in the elderly and/or diabetic patients, who may require new blood vessel growth in ischemic brain, heart, or limbs.

Typically, for ocular diseases, smaller antibody fragments, such as Fab or _Fab2 , are used via intravitreal administration because they have a low serum half-life and a lower risk of systemic toxicity. However, such smaller fragments also typically have a lower intravitreal half-life (e.g., due to faster diffusion into the serum) and must generally be administered more frequently.

WO 2009/080252 and Schaefer, W. et al., Proc. Natl. Acad. Sci. USA, 108 (2011) 11187-11191 (which are incorporated herein by reference) describe multispecific antibodies with domain replacement/exchange (CrossMab VH-VL) in one binding arm. They significantly reduce byproducts caused by the mispairing of a light chain for a first antigen with an incorrect heavy chain for a second antigen (compared to methods without such domain exchange). However, their preparation is not completely free of byproducts. The main byproducts are based on Bence-Jones-type interactions. See also Schaefer, W. et al., Proc. Natl. Acad. Sci. USA, 108 (2011) 11187-11191; in Figure S1I in the Supplementary Notes.

WO 2014/072876 reports antibodies specific for platelet-derived growth factor B, compositions thereof, and uses thereof. WO 2005/087812 reports multivalent antibodies and methods for VEGF/PDGF family growth factors. Vassbotn, F.S., et al. (Biochim. Biophys. Acta. Mol. Cell Res. 1054 (1990) 246-249) report that monoclonal antibodies against the PDGF B chain inhibit PDGF-induced DNA synthesis in C3H fibroblasts and prevent PDGF from binding to its receptor.

Overview

The present 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 that binds to PDGF-B, wherein said antibody is a humanized variant of a murine antibody, wherein said murine antibody comprises the heavy chain variable domain of SEQ ID NO: 01 and the light chain variable domain of SEQ ID NO: 06.

In one embodiment of all aspects as 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 (according to Kabat numbering).

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

In one embodiment of all aspects as 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 (according to Kabat numbering).

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

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

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

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

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

One aspect as reported herein is an antibody that specifically binds to human PDGF-B, wherein the antibody comprises:

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

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

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

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

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

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

In one embodiment, the antibody further comprises:

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

b) (vi) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 45, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 46, (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 47, or

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

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

In one embodiment, the antibody comprises:

a) a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 10, or

b) a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 15, or

c) a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 20, or

d) a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 25, or

e) a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 30, or

f) a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 35.

In one embodiment, the antibody further comprises:

a) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 40, or

b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 44, or

c) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 48, or

d) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 52.

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

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

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

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

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

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

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

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

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

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

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

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

One aspect as reported herein is an antibody comprising a heavy chain variable domain amino acid sequence of SEQ ID NO: 10 and a light 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 a heavy chain variable domain amino acid sequence of SEQ ID NO: 15 and a light 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 a heavy chain variable domain amino acid sequence of SEQ ID NO: 20 and a light 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 a heavy chain variable domain amino acid sequence of SEQ ID NO: 25 and a light 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 a heavy chain variable domain amino acid sequence of SEQ ID NO: 30 and a light 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 a heavy chain variable domain amino acid sequence of SEQ ID NO: 35 and a light 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 are antibodies that specifically bind to human PDGF-BB and human PDGF-AB but not to human PDGF-AA.

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

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

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

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

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

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

One aspect as reported herein are antibodies that specifically bind to human PDGF-BB and PDGF-AB as well as PDGF-AA.

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

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

In one embodiment of all aspects as reported herein the antibody is of human IgG1 subclass or of human IgG4 subclass.

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

In one embodiment of all aspects as reported herein the 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 monkey 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 phosphorylation of the PDGF-B receptor.

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 an (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 the preparation of an antibody as reported herein, said method comprising culturing a host cell as reported herein, such that said antibody is produced, 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-ANG2 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 the use in the treatment of an ocular vascular disease, preferably for the treatment of macular degeneration.

The antibodies reported herein are used to inhibit the interaction between PDGF-B and its receptor.

The antibodies reported herein are used to inhibit cell migration.

The antibodies reported herein are used to inhibit proliferation.

One aspect as reported herein is the use of the antibodies as reported herein in the preparation of a medicament.

In one embodiment, the medicament is for use in the treatment of an ocular vascular disease, 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 for treating an individual with an ocular vascular disease, preferably with macular degeneration, comprising administering to said individual an effective amount of an antibody as reported herein.

One aspect as reported herein is a method of 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 embodiment of the invention

I. Definition

For the purposes of this article, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from 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 changes. 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 the VL human immunoglobulin framework sequence or the 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 the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant ( _Kd ). Affinity can be measured by common methods known in the art, including those described herein. Specific exemplary and illustrative embodiments for measuring binding affinity are described below.

"Affinity matured" antibodies are those with one or more alterations in one or more hypervariable regions (HVRs) which result in improvements in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such alterations.

The terms "anti-PDGF-B antibody" and "antibody that binds to PDGF-B" refer to an antibody that is capable of binding to PDGF-B with sufficient affinity to allow the antibody to be used as a diagnostic and/or therapeutic agent targeting PDGF-B. In one embodiment, the extent of binding of the anti-PDGF-B antibody to unrelated, non-PDGF-B proteins 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 to PDGF-B has a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹⁰ M, e.g., 10 ⁻⁹ M to 10 ⁻¹⁰ M). In certain embodiments, the anti-PDGF-B antibody binds to an epitope of PDGF-B that is conserved between PDGF-B from different species.

The term "antibody" herein is used in the broadest sense and includes various 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.

"Antibody fragment" refers to a molecule other than an intact antibody that 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, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from multiple antibody fragments.

An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that interacts with at least some of the same amino acid residues as the reference antibody. 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 "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . The heavy chain constant domains corresponding to the different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

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

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C); C), chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; 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 class. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B-cell activation.

An "effective amount" of an agent (eg, 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 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 carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or C-terminal glycyl-lysine dipeptide (Gly446Lys447) in the Fc region may be present or absent. Unless otherwise noted herein, the numbering of the amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest (sequences of proteins of interest to immunology), 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH publication 91-3242.

"Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of a variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

The terms "full length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to antibodies that have a structure substantially similar to a native antibody structure or have heavy chains that contain an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. 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 completely identical to the parent cell in terms of nucleic acid content, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source utilizing human antibody repertoires or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

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

"Humanized" antibody refers to 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 the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain antigen contact residues ("antigen contact points"). Generally, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

The HVRs in this article include:

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

(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, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.);

(c) antigenic contact points 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), comprising 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 in the variable domain (eg, FR residues) 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., cattle, sheep, cats, dogs, and horses), primates (e.g., humans 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 by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-PDGF-B antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

As used herein, the term "monoclonal antibody" refers to an antibody derived from a colony of substantially homologous antibodies, that is, each antibody constituting the colony is identical and/or in conjunction with identical epitopes, except for possible variant antibodies (for example, containing naturally occurring mutations or producing in the production process of monoclonal antibody products), such variants are generally present in trace amounts. Different from the polyclonal antibody products generally comprising different antibodies for different determinants (epitopes), each monoclonal antibody of monoclonal antibody products is directed to a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody derives from the feature of a substantially homologous antibody colony, and should not be construed as needing to produce the antibody by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by various techniques, including, but not limited to, hybridoma method, recombinant DNA method, phage display method, and the method using all or part of a transgenic animal comprising human immunoglobulin locus, and such method and other exemplary methods for preparing monoclonal antibodies are described herein.

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

"Natural antibodies" refers to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded together. From N- to C-, each heavy chain has a variable region (VH), also referred to as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-, each light chain has a variable region (VL), also referred to as a variable light domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences, introducing gaps if necessary to achieve the maximum percent sequence identity, and not considering 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 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 algorithm required to achieve maximum alignment over the full length of the sequences being compared. However, for the purposes of this article, the sequence alignment computer program ALIGN-2 is used to generate % 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 user documentation to the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and do not change.

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 can alternatively be stated as a given amino acid sequence A having or comprising a specific % amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows:

100 × fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 (in that program's alignment of A and B), and where 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 equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, 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" means a preparation that is in such form as to render the biological activity of the active ingredient contained therein effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation is to be administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-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 resulting from intracellular processing. 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 variations thereof such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desired therapeutic effects include, but are not limited to: preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development 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 binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally 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, 6th ed., W.H. Freeman and Co., N.Y. (2007), p. 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to specific antigens can be isolated by using the VH or VL domain from an antigen-binding antibody to screen a library of complementary VL or VH domains, respectively (see, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

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

II. Compositions and Methods

The monoclonal antibodies of the present invention specifically bind to determinant sites on the PDGF-B molecule that are involved in PDGF-B's receptor binding and proliferative activity. The humanized monoclonal antibodies do not bind to PDGF-C. The specific antibodies reported herein bind to human PDGF-BB and PDGF-AB but not PDGF-AB. Other specific antibodies reported herein bind to 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 novel murine anti-human PDGF-B antibody having a VH of SEQ ID NO: 01 and a VL of SEQ ID NO: 06. This antibody is hereinafter referred to as a mumAb. This antibody binds to human, cynomolgus monkey, rat, and murine PDGF-B and inhibits the interaction between PDGF-B and its receptor.

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

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 temperature (Tm) (see table below).

Tagg[℃] Tm[℃] mumAb 57 62-63.5

Based on the amino acid sequence of the murine mumAb (SEQ ID NO: 01 and SEQ ID NO: 06), the corresponding humanized anti-PDGF-B antibodies were generated. The humanized variants of VH were based on human germlines IMGT_hVH_1_69, IMGT_hVH_4_59 and IMGT_hVH_3_23 in combination with human J-element germline IGHJ5-01.

In the IMGT_hVH_1_69 variant, back mutations were introduced at position 27 (G27Y) of framework region 1 and/or position 40 (A40R) and/or position 43 (Q43H) of framework region 2 and/or 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 (K43H) of framework region 2 and at positions 71 (R71A) and 78 (L78A) of framework region 3.

Humanized variants of VL were based on human germlines IMGT_hVK_3_20, IMGT_hVK_4_1 and IMGT_hVK_1_27 in combination with human J-element germline IGKJ2-01.

For one variant, in IMGT_hVK_3_20, back mutations were introduced at position 43 (A43P) of framework region 2 and position 68 (G68R) of framework region 3. In IMGT_hVK_1_27, one back mutation was introduced at position 43 (V43P) of framework region 3.

The antibodies are denoted as Antibody 0044 as a monospecific antibody and Antibody 0146 as a bispecific CrossMab with an additional ANG2 binding specificity.

The remaining antibodies are human antibodies obtained from rabbits transgenic for the human Ig locus. The antibodies have the binding properties listed in the table below.

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

In the multivariate activity assay, the antibodies exhibited biological activities as can be seen from the data listed in the table below.

Surface plasmon resonance was used to determine the kinetic binding properties of the different antibodies (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 of different antibodies (active concentration with reference surface).

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

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

Antibody 0144 is a bispecific anti-ANG2/PDGF-B antibody that comprises the VH and VL domains of antibody 0085 as PDGF-B binding specificities.

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

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 -

SPR analysis has demonstrated that all bispecific antibodies have the property of binding to their two antigens simultaneously.

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

In the ANG2-specific pTie2-ELISA, antibody 0144 was 6 times more active than the anti-ANG2/VEGF antibody reported in WO 2014/09465.

In a VEGF-specific reporter assay, antibody 0117 had similar activity to the anti-ANG2/VEGF antibody reported in WO 2014/09465.

Antibody 0085 is described as having the sequences of SEQ ID NOs: 92-100 (binding site, HVR, VH, VL). Bispecific forms of antibody 0085 are described in SEQ ID NOs: 131-134, 147-150, and 163-166. All of these sequences, alone or in combination, constitute various aspects of the present invention.

Antibody 0086 is described as having the sequences of SEQ ID NOs: 101-109 (binding site, HVR, VH, VL). Bispecific forms of antibody 0086 are described in SEQ ID NOs: 135-138, 151-154, and 167-167. All of these sequences, alone or in combination, constitute various aspects of the present invention.

Antibody 0144 is described as having the sequence of SEQ ID NOs: 147-150 (CrossMab format, 2 heavy chains, 2 light chains).

Antibody 0117 is described as having the sequence of SEQ ID NOs: 171-174 (CrossMab format, 2 heavy chains, 2 light chains).

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

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

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

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

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

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

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

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

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 the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 93, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 94, and (iii) HVR-H3 comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 96; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 98, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 99, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 100.

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

In another aspect, an anti-PDGF-B antibody comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 92. A VH sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the reference sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions, but an anti-PDGF-B antibody comprising said sequence retains the ability to bind to PDGF-B. In certain embodiments, a total of 1-10 amino acids in SEQ ID NO: 92 are substituted, inserted, and/or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, an anti-PDGF-B antibody comprises the VH sequence in SEQ ID NO: 92, including post-translational modifications of said sequence. In a specific embodiment, VH comprises one, two or three HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 93, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 94, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 96.

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

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

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

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

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

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VHHVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 102; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 103; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 105. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 105. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 105 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 109. In another embodiment, the antibody comprises: HVR-H3 comprising the amino acid sequence of SEQ ID NO: 105, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 109, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 103. In another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 102; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 105.

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

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 the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 102, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 103, and (iii) HVR-H3 comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 105; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 108, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 109.

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

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

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

One aspect as reported herein is an antibody comprising two heavy chains and two light chains, wherein: a) the first heavy chain has the amino acid sequence of SEQ ID NO: 147, the second heavy chain has the amino acid sequence of SEQ ID NO: 148, the first light chain has 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 which is a mutation in the Fc region.

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

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

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

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

In another embodiment, the antibody is a full-length antibody, eg, an intact IgG1 antibody or other antibody class or isotype as defined herein.

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

In one embodiment of all aspects as reported herein the 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 to a first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to 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,

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

The antibody in a) does not comprise the modifications reported in b), and the heavy and light chains in a) are separated chains.

In the antibody in b),

In the light chain

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

and

In the heavy chain

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

In one embodiment,

i) in the constant domain CL of the first light chain in a), the amino acid at position 124 (according to Kabat numbering) 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 (according to the Kabat EU index) is substituted with a negatively charged amino acid,

or

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

In a preferred embodiment,

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) (according to Kabat numbering) (in a preferred embodiment, it is 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) (according to the Kabat EU index numbering),

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) (according to Kabat numbering) (in a preferred embodiment, it is 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) (according to the Kabat EU index numbering).

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 (numbering according to the Kabat EU index).

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

In one embodiment, in the constant domain CL of the second heavy chain, the amino acids at positions 124 and 123 are substituted by K, and wherein in the constant domain CH1 of the second light chain, the amino acids at positions 147 and 213 are substituted by E, and in the domain VL of the first light chain, the amino acid at position 38 is substituted by K, in the variable domain VH of the first heavy chain, the amino acid at position 39 is substituted by E, in the variable domain VL of the second heavy chain, the amino acid at position 38 is substituted by K, and in the variable domain VH of the second light chain, the amino acid at position 39 is substituted by E (numbering according to the Kabat EU index).

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 to a first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to 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,

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

The antibody in a) does not comprise the modifications reported in b), and the heavy and light chains in a) are separated chains.

In the antibody in b)

In the light chain

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

In the heavy chain

The variable heavy chain domain VH is replaced by the variable light chain domain VL of said antibody, and the constant heavy chain domain CH1 is replaced by the constant light chain domain CL of said 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 to a first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to 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,

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

The antibody in a) does not comprise the modifications reported in b), and the heavy and light chains in a) are separated chains.

In the antibody in b)

In the light chain

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

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

One aspect as reported herein is a multispecific antibody comprising:

a) a full-length antibody that specifically binds to a first antigen and is composed 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 further antigens (i.e. the second and/or third and/or fourth and/or fifth antigen, preferably, specifically bind to one further antigen, i.e. the second antigen),

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

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

In one embodiment, one or two identical single chain Fab fragments binding 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 binding 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 binding 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 binding to a second antigen are fused to the full length antibody via 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 binding to a second antigen are fused to the full length antibody via 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 binding to a second antigen are fused to the full length antibody via 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 to 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 to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker at the N-terminus of its VH domain,

c) a second polypeptide consisting of:

ca) 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 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 via a peptide linker,

and

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 to the second antigen,

and

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 an interchain disulfide bridge by introducing a disulfide bond between the following positions:

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 numbering 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., Nuclear Medicine & Biology, Vol. 25, (1998) 387-393; or Schmidt, M., et al., Oncogene (1999) 181711-1721. In one embodiment, the optional disulfide bond between the variable domains of the polypeptides of b) and c) is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the optional disulfide bond between the variable domains of the polypeptides of b) and c) is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (always numbering according to the EU index of Kabat). In one embodiment, it is preferred that the trivalent bispecific antibody does not have the optional disulfide-stabilized 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 to a first antigen, and

b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to 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 that specifically bind to one or two other antigens (i.e., the third and/or fourth antigen) are fused to the C- or N-terminus of the light or heavy chain of a) and/or b) via a peptide linker,

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

The antibody in a) does not comprise the modifications reported in b), and the heavy and light chains in a) are separated chains.

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 a scFv fragment and a scFab fragment.

In one embodiment, the antigen binding peptide is a 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 comprises two identical antigen-binding peptides in c) that specifically bind to a third antigen. In a preferred embodiment, the two identical antigen-binding peptides are fused to the C-termini 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 two antigen-binding peptides in c) that specifically bind to a third and a fourth antigen. In one embodiment, the two antigen-binding peptides are fused to the C-termini of the heavy chains of a) and b) via the same peptide connector. 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, which specifically bind to a first antigen (and comprise two Fab fragments),

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

and

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 with each other, and/or the constant domains CL and CH1 are replaced with each other,

or

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

and

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

or

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

and

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

or

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

or

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

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

In one embodiment said further Fab fragments are both fused via a peptide linker at the C-terminus of the heavy chain of a) or at the N-terminus of the heavy chain of a).

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

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 the two Fab fragments of a), or in the two Fab fragments of b), the variable domains VL and VH are replaced with one another,

and/or

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

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

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

and/or

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

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

i) In the two Fab fragments of a), the constant domains CL and CH1 are replaced with each other.

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

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

and/or

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

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

i) In the two Fab fragments of b), the constant domains CL and CH1 are replaced with each other.

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

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

b) two light chains of the first antibody described in a),

c) a (modified) heavy chain of a second antibody, which 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 to the C-terminus of the heavy chain via a peptide linker, and

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

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

One aspect as reported herein is a bispecific antibody comprising:

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

b) a heavy chain and a light chain of a second full-length antibody that specifically binds to 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,

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

The antibody in a) does not comprise the modifications reported in b), and the heavy and light chains are separated chains.

One aspect as reported herein is a bispecific antibody comprising:

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

b) an Fv fragment which specifically binds to a second antigen and comprises a VH2 domain and a ^VL2 domain, wherein the two domains are linked to each other by a disulfide bridge,

wherein only one of the VH ² domain and the VL ² domain is fused via a peptide linker to the heavy chain or light chain of a full-length antibody that specifically binds to a first antigen,

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

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

In one embodiment, the other of the VH ² domain and the VL ² domain is not fused via a peptide linker to the heavy chain or light chain of a full length antibody that specifically binds to a first antigen.

In all aspects as 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 which requires 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 CH3-modification to support heterodimerization have been described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291, which are included herein by reference. Typically, in methods known in the art, the CH3 domain of a first heavy chain and the CH3 domain of a second heavy chain are modified in a complementary manner such that a heavy chain comprising one modified CH3 domain no longer homodimerizes with another heavy chain of the same structure (e.g., a CH3-modified first heavy chain may no longer homodimerize with another CH3-modified first heavy chain; and a CH3-modified second heavy chain may no longer homodimerize with another CH3-modified second heavy chain). Thus, a heavy chain comprising one modified CH3 domain is forced to heterodimerize with another heavy chain comprising a complementary modified CH3 domain. In this embodiment of the invention, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are modified in a complementary manner by amino acid substitution, thereby forcing the first and second heavy chains to heterodimerize, while the first and second heavy chains are no longer able to homodimerize (e.g., due to steric reasons).

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

The CH3 domains of the multispecific antibodies reported herein can be altered using the "knob-into-hole" technique, which is described in detail, for example, in WO 96/027011, Ridgway, J.B., et al., Protein Eng. 9 (1996) 617-621; and Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are 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) can be a "knob" and the other a "hole." Introduction of a disulfide bridge further stabilizes the heterodimer (Merchant, A.M., et al., Nature Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) and increases yield.

In a preferred embodiment, the bispecific antibodies reported herein comprise a T366W mutation in the CH3 domain of the "knobs chain" and T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat-EU index). Additional interchain disulfide bridges between the CH3 domains can also be used (Merchant, A.M., et al., Nature Biotech. 16 (1998) 677-681), for example, by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and an E356C mutation or an S354C mutation into the CH3 domain of the "hole chain." Thus, in another preferred embodiment, the multispecific antibody as 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 as 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 an interchain disulfide bridge) (numbering according to the Kabat EU index).

However, other knobs-into-hole technologies as described in EP 1 870 459 A1 may alternatively or additionally be applied. In one embodiment, the multispecific antibody as reported herein comprises the R409D and K370E mutations in the CH3 domain of the "knobs chain" and the D399K and E357K mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index).

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

In one embodiment, the multispecific antibody as 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 as 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 additionally comprises R409D and K370E mutations in the CH3 domain of the “knobs 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 "knobs-into-holes technology", other technologies for modifying the CH3 domain of the heavy chain of multispecific antibodies to force heterodimerization are known in the art. These technologies, in particular those described in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954 and WO 2013/096291, are contemplated herein as alternatives to the "knobs-into-holes technology" for use in combination with the multispecific antibodies reported herein.

In one embodiment of the multispecific antibodies as reported herein, heterodimerization of the first and the second heavy chain of said multispecific antibody is supported using the method described in EP 1 870 459. This method is based on the introduction of oppositely charged amino acids at specific amino acid positions in the CH3/CH3-domain-interface between the two heavy chains, i.e. the first and the second heavy chain.

Therefore, this embodiment relates to the multispecific antibodies as reported herein, wherein in the tertiary structure of the antibody, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface located between the CH3 domains of the respective antibodies, wherein the amino acid sequence of each of the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain respectively comprises a set of amino acids located within the interface in the tertiary structure of the antibody, wherein the first amino acid from the set of amino acids within the interface located in the CH3 domain of one heavy chain is substituted with a positively charged amino acid, and the second amino acid from the set of amino acids within the interface located 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 "CH3 (+/-)-engineered multispecific antibodies" (wherein the abbreviation "+/-" indicates that the oppositely charged amino acids are introduced into the respective CH3 domains).

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

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

In one embodiment of the multispecific antibody as reported herein, heterodimerization of the first and second heavy chains of the multispecific antibody is supported using the method described in WO2013/157953. In one embodiment of the multispecific antibody as 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 (numbered according to the Kabat EU index). In another embodiment of the multispecific antibody as 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 (numbered according to the Kabat EU index).

In one embodiment of the multispecific antibody as 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). Additionally, the CH3 domain of the other heavy chain comprises at least one of the following substitutions: the amino acid Y at position 349 is substituted with E, the amino acid Y at position 349 is substituted with D, and the amino acid L at position 368 is substituted with E (numbering according to the Kabat EU index). In one embodiment, the amino acid L at position 368 is substituted with E (numbering according to the Kabat EU index).

In one embodiment of the multispecific antibody reported herein, the method described in WO2012/058768 is used to support heterodimerization of the first heavy chain and the 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 L at position 351 is replaced with Y and the amino acid Y at position 407 is replaced with A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is replaced with A, and the amino acid K at position 409 is replaced with F (numbered according to the Kabat EU index). In another embodiment, in addition to the aforementioned replacements, in the CH3 domain of the other heavy chain, at least one of the amino acids at the following positions is replaced: position 411 (originally T), 399 (originally D), 400 (originally S), 405 (originally F), 390 (originally N) and 392 (originally K) (numbered according to the Kabat EU index). Preferred replacements are:

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

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

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

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

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

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

In another embodiment of the multispecific antibodies as 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 Kabat EU index). In another embodiment of the multispecific antibodies 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 of the aforementioned embodiments, in the CH3 domain of the other heavy chain, the amino acid K at position 392 is substituted with E, the amino acid T at position 411 is substituted with E, the amino acid D at position 399 is substituted with R and the amino acid S at position 400 is substituted with R (numbering according to the Kabat EU index).

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

In one embodiment of the multispecific antibody as reported herein, heterodimerization of the first and second heavy chains of the multispecific antibody is supported using the method described in WO 2011/090762. WO 2011/090762 relates to amino acid modifications performed using the "knobs-into-holes" technique. In one embodiment of the CH3 (KiH)-engineered multispecific antibody as 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 the CH3 (KiH)-engineered multispecific antibody as 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 as reported herein, which is of IgG2 isotype, the method described in WO2011/090762 is used to support heterodimerization of the first and second heavy chains of said multispecific antibody.

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

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

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

In one embodiment of all aspects and embodiments as 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 as reported herein the antibody is a bivalent or trivalent antibody. In one embodiment the antibody is a bivalent antibody.

In one embodiment of all aspects as reported herein, the multispecific antibody has the constant domain structure of an IgG type antibody. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG1 subclass or of human IgG1 subclass with the mutations L234A and L235A. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG2 subclass. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG3 subclass. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG4 subclass or of human IgG4 subclass with the additional mutation S228P. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG1 subclass or of human IgG4 subclass. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is a human IgG1 subclass with mutations L234A and L235A (according to the Kabat EU index numbering). In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is a human IgG1 subclass with mutations L234A and L235A (according to the Kabat EU index numbering). In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is a human IgG4 subclass with mutations S228P and L235E (according to the Kabat EU index numbering). In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is a human IgG4 subclass with mutations S228P, L235E and P329G (according to the Kabat EU index numbering).

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

In another aspect, the anti-PDGF-B antibody according to any of the above embodiments may incorporate any of the features described in Sections 1-5 below, alone or in combination.

1. Antibody affinity

In certain embodiments, the antibodies provided herein have a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM (eg, 10 ⁻⁸ M or less, eg, 10 ⁻⁸ M-10 ⁻¹⁰ M, eg, 10 ⁻⁹ M-10 ⁻¹⁰ M).

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

When using surface plasmon resonance assays, alternatively, KD values can be measured as follows: an assay using BIAcore® or BIAcore® (BIAcore, Inc., Piscataway, NJ) is performed at 25° C. with an immobilized antigen CM5 chip at ˜10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg/mL (˜0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μL/min to obtain approximately 10 response units (RU) of coupled protein. Following the injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS with 0.05% polysorbate 20 (TWEEN-20 ^™ ) surfactant (PBST) were injected at a flow rate of approximately 25 μL/min at 25° C. Association rates ( _ka ) and dissociation rates ( _kd ) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (Evaluation software version 3.2). The equilibrium dissociation constant (KD) was calculated as the ratio _kd / _ka (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). If the on-rate exceeds 10 ⁶ M ⁻¹ s ⁻¹ as determined by surface plasmon resonance as described above, the off-rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25° C. in the presence of increasing antigen concentrations as measured with a spectrometer, such as a stop-flow spectrophotometer (Aviv Instruments) or an 8000-series SLM-AMINCO ^™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

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') ₂ , Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson, PJ et al., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, for example, Plueckthun, A., in: The Pharmacology of Monoclonal Antibodies, Vol. 113, ed. Rosenburg and Moore, Springer-Verlag, New York (1994), pp. 269-315; see also WO 93/16185; US 5,571,894 and US 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising residues that bind to a salvage receptor binding epitope and having increased in vivo half-life, see US 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 0 404 097; WO 1993/01161; Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134).

Single-domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US 6,248,516).

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 (eg, E. coli or phage).

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described in, for example, US4,816,567; and Morrison, S.L. et al., Proc.Natl.Acad.Sci.USA 81 (1984) 6851-6855). In one embodiment, a 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, a chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity for humans while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies include one or more variable domains: wherein HVR, such as CDR (or a portion thereof), is derived from a non-human antibody, and FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed in, for example, Almagro, J.C. and Fransson, J., Front.Biosci.13 (2008) 1619-1633, and further described in, for example, Riechmann, I. et al., Nature332 (1988) 323-329; Queen, C. et al., Proc.Natl.Acad.Sci.USA 86 (1989) 10029-10033; US5,821,337, US7,527,791, US6,982,321, and US 7,087,409; Kashmiri, S.V. et al., Methods 36 (2005) 25-34 (describing specificity determining region (SDR) grafting); Padlan, E.A., Mol. Immunol. 28 (1991) 489-498 (describing "surfacing"); Dall'Acqua, W.F. et al., Methods 36 (2005) 43-60 (describing "FR shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the "guided selection" approach for FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims, M.J. et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L.G. et al., J. Immunol. 151 (1993) 2623-2632); 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) 10678-10684 and Rosok, M.J. et al., J. Biol. Chem. 271 (19969 22611-22618).

4. Multispecific Antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, for example, bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity to 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, bispecific antibodies can bind to two different epitopes of PDGF-B. Bispecific antibodies can also be used to locate 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 chain-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 "knobs-into-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for the preparation of antibody Fc-heterodimer molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, S.A. et al., J. Immunol. 148 (1992) 1547-1553); using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (sFv) dimers (see, e.g., Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described in, e.g., 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).

The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs," which contain antigen binding sites that bind PDGF-B as well as another, different antigen (see, eg, US 2008/0069820).

The antibodies or fragments herein also include the 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 the antibody. By introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis, amino acid sequence variants of the antibody can be prepared. Such modifications include, for example, deleting residues and/or inserting residues and/or replacing residues from the amino acid sequence of the antibody. Any combination of deletions, insertions, and replacements can be prepared to obtain the final construct, provided that the final construct has the desired characteristics, for example, antigen binding.

a) Substitution, insertion and deletion variants

In certain embodiments, antibody variants with one or more amino acid replacements are provided. The target site for replacement mutagenesis includes HVR and FR. In the table below, conservative replacements are shown under the heading of "preferred replacements". In Table 1, more substantial changes are provided under the heading of "exemplary replacements", and reference amino acid side chain categories are further described below. Amino acid replacements can be introduced into the target antibody and the product can be screened for desired activity (e.g., retained/improved antigen binding, reduced immunogenicity or improved ADCC or CDC).

surface

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 affect chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

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

Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots" (i.e., residues encoded by codons that undergo mutation at 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 the resulting variant VH or VL tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, for example, in Hoogenboom, H.R. et al. in Methods in Molecular Biology. Biology (Molecular Biology Methods) 178 (2002) 1-37. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then established. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed 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 often targeted in particular.

In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the antigen-binding ability of the antibody. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may be, for example, outside the antigen-contacting residues of 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.

A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham, B.C. and Wells, J.A., Science 244 (1989) 1081-1085. In this method, a residue or target residue group (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. Other substitutions can 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 can be used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-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 the antibody with an enzyme (e.g., an enzyme for 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 antibodies are glycosylated. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

In the case where the antibody comprises an Fc region, the carbohydrates attached thereto can be changed. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, which are typically attached to Asn297 of the CH2 domain of the Fc region via an N-bond (see, e.g., Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32). Oligosaccharides can include various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose connected to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

In one embodiment, an antibody variant having a carbohydrate structure is provided, wherein the carbohydrate structure lacks fucose connected to the Fc region (directly or indirectly). 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 in the following manner: relative to the sum of all sugar structures (e.g., complex structures, hybrid structures, and high mannose structures) connected to Asn297 as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008/077546), the average amount of fucose at Asn297 in the sugar chain is calculated. Asn297 represents an asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 can also be located upstream or downstream ±3 amino acids of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, US 2003/0157108; US 2004/0093621. Examples of publications relating 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) 1239-1249; Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells defective in protein fucosylation (Ripka, J. et al., Arch, Biochem. Biophys. 249 (1986) 533-545; US 2003/0157108; and WO 2004/056312, particularly Example 11), and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).

Antibody variants can also be provided with bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants can have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; US 6,602,684; and US 2005/0123546. Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants can have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and 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 can be comprised of a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells used to mediate ADCC (i.e., NK cells) express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in US Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad, Sci. USA 82 (1985) 1499-1502); US Pat. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays can be employed (see, e.g., ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo, e.g., in an animal model, such as that described in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity (see, for example, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402). To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int. Immunol. 18 (2006: 1759-1769)).

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

Certain antibody variants with improved or decreased binding to FcRs have been described (see, e.g., US 6,737,056; WO 2004/056312, and Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

In certain embodiments, the antibody variant comprises an Fc region with 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, changes 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 US 6,194,551, WO 99/51642, and Idusogie, E.E. et al., J. Immunol. 164 (2000) 4178-4184.

Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer, R.L. et al., J. Immunol. 117 (1976) 587-593, and Kim, J.K. et al., J. Immunol. 24 (1994) 2429-2434), are described in US 2005/0014934. These antibodies comprise an Fc region with one or more substitutions 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, e.g., substitution of Fc region residue 434 ( US 7,371,826 ).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351 for other examples of Fc region variants.

d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to establish a cysteine-engineered antibody, for example, a "thio MAb", wherein one or more residues of the antibody are replaced with cysteine residues. In a specific embodiment, the replaced residues appear at accessible sites of the antibody. By replacing those residues with cysteine, reactive sulfhydryl groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to produce immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine-engineered antibodies can be produced as described in, for example, US 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous structural parts known in the art and easily obtained. Suitable structural parts 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers 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. Due to its stability in water, polyethylene glycol propionaldehyde can have the advantages of manufacturing aspect. The polymer can have any molecular weight and can 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 used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used in therapy for a specific condition, etc.

In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety is provided, wherein the non-proteinaceous moiety can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam, N.W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can have any wavelength and includes, but is not limited to, a wavelength that does not harm normal cells but heats the non-proteinaceous moiety to a temperature that kills cells in the vicinity of the antibody-non-proteinaceous moiety.

B. Recombinant Methods and Compositions

Antibodies can be produced using recombinant methods and compositions (e.g., as described in US 4,816,567). In one embodiment, an isolated nucleic acid is provided that encodes an anti-PDGF-B antibody described herein. The nucleic acid can 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 acid are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) the following vectors: (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector and a second vector, wherein the first vector comprises a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and the second vector comprises 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., a Y0, NS0, Sp20 cell). In one embodiment, a method for preparing 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 an anti-PDGF-B antibody, 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 acid can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be prepared in bacteria, especially when glycosylation and Fc effector functions are not required. Regarding the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., in: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be separated 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 fungi 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) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that 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., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES ^™ technology for producing antibodies in transgenic plants)).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293 cells, which are described, for example, in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, which are described, for example, in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma 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, which are described, for example, in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. 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) 4216-4220); and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

C. Determination

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

D. Immunoconjugates

The present 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 radioactive isotopes.

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 (see US 5,208,020, US 5,416,064 and EP 0 425 235 B1); auristatins such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see US 5,635,483, US 5,780,588, and US 7,498,298); dolastatin; calicheamicin or its derivatives (see US 5,712,374, US 5,714,586, US 5,739,116, US 5,767,285, US 5,770,701, US 5,770,710, US 5,773,001, and US 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); anthracyclines such as daunomycin or 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., Bioconjug. Chem. 16 (2005) 717-721; Nagy, A., et al., Proc. Natl. Acad. Sci. USA 97 (2000) 829-834; Dubowchik, G.M., et al., Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, H.D., et al., J. Med. Chem. 45 (2002) 4336-4343; and US 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but 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 proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis toxin, scutellaria baicalensis toxin, scutellaria serrata ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes.

In another embodiment, the immunoconjugate comprises an antibody as described herein that is conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu. When a radioconjugate is used for detection, it can include radioactive atoms for scintigraphy research, such as TC ^99m or I ¹²³ , or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 (reappearing), iodine-131, sodium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of the antibody and cytotoxic agent can be prepared using 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), 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 bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta, E.S. et al., Science 238 (1987) 1098-1104. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetate (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94/11026. The linker can be a "cleavable linker" that promotes the release of the cytotoxic drug in the cell. For example, an acid-sensitive linker, a peptidase-sensitive linker, a photosensitive linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari, R.V et al., Cancer Res. 52 (1992) 127-131; US 5,208,020).

The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with commercially available cross-linkers (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. As used herein, the term "detecting" includes quantitative or qualitative detection.

In one embodiment, an anti-PDGF-B antibody for use in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of PDGF-B in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-PDGF-B antibody as described herein under conditions that allow the anti-PDGF-B antibody to bind to PDGF-B, and detecting whether a complex is formed between the anti-PDGF-B antibody and PDGF-B. Such methods can be in vitro or in vivo methods. In one embodiment, an anti-PDGF-B antibody is used to select subjects who are eligible for a therapy using 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, directly detectable labels or moieties (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, for example, by enzymatic reactions or molecular interactions (such as enzymes or ligands). Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases (e.g., firefly luciferase and bacterial luciferase ( US 4,737,456 )), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase) coupled to enzymes that utilize hydrogen peroxide to oxidize the dye precursor (such as HR, lactoperoxidase, or microoxidase), biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical Preparations

Pharmaceutical formulations of anti-PDGF-B antibodies as described herein can be prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (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 octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, 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 (vinyl pyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; 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 nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, 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, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006/044908, the latter formulations including a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an anti-ANG2 antibody or an anti-VEGF antibody. Such active ingredients are suitably present in combination in amounts effective for their intended purpose.

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

Sustained-release preparations 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, eg, films, or microcapsules.

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

G. Methods of Treatment and Compositions

Any of the anti-PDGF-B antibodies provided herein can be used in therapeutic methods.

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

In another aspect, the present invention provides the use of an anti-PDGF-B antibody in the production or preparation of a medicament. In one embodiment, the medicament is used to treat an ocular vascular disease, preferably macular degeneration. In another embodiment, the medicament is used in a method for treating an ocular vascular disease, preferably macular degeneration, comprising administering an effective amount of the medicament to an individual suffering from an ocular vascular disease, preferably macular degeneration. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, for example, as described below. In another embodiment, the medicament is used to inhibit angiogenesis. In another embodiment, the medicament is used in a method for inhibiting angiogenesis in an individual, comprising administering an effective amount of the medicament to the individual, thereby inhibiting angiogenesis. The "individual" according to any of the above embodiments may be a human.

In another aspect, the present invention provides a method for treating an ocular vascular disease, preferably macular degeneration. In one embodiment, the method comprises administering an effective amount of an anti-PDGF-B antibody to an individual suffering from such an ocular vascular disease, preferably macular degeneration. 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. According to any of the above embodiments, the "individual" can be a human.

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

The antibodies of the present invention can be used alone or in combination with other agents in therapy. For example, the antibodies of the present 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-ANG2 antibody.

Such combination therapies as noted above include combined administration (wherein 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 can occur before, simultaneously with, and/or after 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 of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days.

Antibodies of the present invention (and any other therapeutic agent) can be administered in any suitable manner, including parenteral, intrapulmonary and intranasal administration, and, if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing can be by any appropriate approach, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-lived or long-term. Various dosing time schemes include, but are not limited to, single or multiple administrations at different time points, push injections, and pulse infusions are contemplated herein.

The antibodies of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the medicament, the method of administration, the time scheme of administration, and other factors known to medical practitioners. The antibodies do not need to be used together with one or more medicaments currently used to prevent or treat the condition under discussion. The effective amount of such other medicaments depends on the amount of the antibody present in the formulation, the type of condition or treatment, and other factors discussed above. These are generally used in the same dosage and with the administration routes described herein, or with about 1 to 99% of the dosage described herein, or with any dosage and any route determined to be suitable empirically/clinically.

For the prevention or treatment of disease, the appropriate dosage of the antibody of the present 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 progression of the disease, whether the antibody is administered for prevention or treatment purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is administered to the patient appropriately at one time or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg/kg to 15 mg/kg (e.g., 0.5 mg/kg-10 mg/kg) can be the initial candidate dose for administration to the patient, e.g., no matter by one or more separate administrations, or by continuous infusion. A typical daily dose may be in the range of about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the disease, the treatment typically continues until desired disease symptom inhibition occurs. An exemplary dosage of the antibody is in the range of about 0.05 mg/kg to about 10 mg/kg. Thus, a dosage of one or more about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) can be administered to the patient. Such dosage can be administered intermittently (e.g., weekly or every three weeks) (e.g., so that the patient receives about two to about twenty, or for example, about six, doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of the treatment can be easily monitored by conventional techniques and assays.

It will be understood that any of the above formulations or treatment methods can be achieved using an immunoconjugate of the invention instead of or in addition to an anti-PDGF-B antibody.

III. Products

In another aspect of the invention, an article of manufacture is provided that contains materials for treating, preventing, and/or diagnosing the conditions described above. The article of manufacture comprises a container and a label or package insert attached to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, and the like. The container can 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 for treating, preventing, and/or diagnosing the condition and can have a sterile access port (for example, the container can be an IV solution bag or a vial with a stopper pierceable by a hypodermic 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 used to treat a selected condition. In addition, the article of manufacture may comprise: (a) a first container containing a composition, wherein the composition comprises an antibody of the invention; and (b) a second container containing a composition, wherein the composition comprises another cytotoxic therapeutic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may also comprise a package insert indicating that the composition can be used to treat a specific 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 understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in combination with an anti-PDGF-B antibody.

IV. Specific Implementation Methods

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

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

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

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

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

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

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

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

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

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

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 human IgG1 subclass or of human IgG4 subclass.

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

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

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

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 to human PDGF-B but not to human PDGF-C.

18. The antibody of any one of embodiments 1-4, wherein the antibody specifically binds to 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:

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

b) a second light chain and a second heavy chain of an antibody that specifically binds to 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,

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

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

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) (according to Kabat numbering) (in a preferred embodiment, it is 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) (according to the Kabat EU index numbering),

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) (according to Kabat numbering) (in a preferred embodiment, it is 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) (according to the Kabat EU index numbering).

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

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

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

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

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

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

b) a second light chain and a second heavy chain of an antibody that specifically binds to 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,

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:

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

b) a second light chain and a second heavy chain of an antibody that specifically binds to 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,

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:

a) a full-length antibody that specifically binds to a first antigen and is composed 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 further antigens (i.e. the second and/or third and/or fourth and/or fifth antigen, preferably, specifically bind to one further antigen, i.e. the second antigen),

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

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:

a) a full-length antibody that specifically binds to 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 to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker at the N-terminus of its VH domain,

c) a second polypeptide consisting of:

ca) 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 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 via a peptide linker,

and

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 to the second antigen,

and

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 the following positions:

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 numbering according to the Kabat EU index).

31. The antibody of any one of embodiments 1-19, wherein the antibody 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 to a first antigen, and

b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to 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 that specifically bind to one or two other antigens (i.e., the third and/or fourth antigen) are fused to the C- or N-terminus of the light or heavy chain of a) and/or b) via a peptide linker,

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

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

a) two light chains and two heavy chains of an antibody, which specifically bind to a first antigen (and comprise two Fab fragments),

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

and

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 with each other, and/or the constant domains CL and CH1 are replaced with each other,

or

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

and

In the two Fab fragments in b), the variable domains VL and VH are replaced with each other,

or the constant domains CL and CH1 are replaced with each other,

or

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

and

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

or

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

or

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

And in the two Fab fragments of b), the variable domains VL and VH are replaced with 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:

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

b) two light chains of the first antibody described in a),

c) a (modified) heavy chain of a second antibody, which 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 said second antibody is fused to the C-terminus of said heavy chain via a peptide linker, and

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

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:

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

b) a heavy chain and a light chain of a second full-length antibody that specifically binds to 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,

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:

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

b) an Fv fragment, which specifically binds to a second antigen and comprises a VH ² domain and a VL ² domain, wherein the two domains are linked to each other by a disulfide bridge,

wherein only one of the VH ² domain and the VL ² domain is fused via a peptide linker to the heavy chain or light chain of a full-length antibody that specifically binds to a first antigen,

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

in

i) the first Fc-region polypeptide is selected from the group consisting of:

- human IgG1 Fc-region polypeptide,

- human IgG2 Fc-region polypeptide,

- human IgG3 Fc-region polypeptide,

- 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 with mutations S354C, T366S, L368A, Y407V

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

- a 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,

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

- a 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,

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

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

- a 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,

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

- a 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 mutation N392D,

and

ii) the second Fc-region polypeptide is selected from the group consisting of:

- human IgG1 Fc-region polypeptide,

- human IgG2 Fc-region polypeptide,

- human IgG3 Fc-region polypeptide,

- 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 the mutations D399K, D356K and/or E357K, and

- human IgG2, IgG3 or IgG4 having the 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

in

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 the mutations S228P, L235E, P329G, S354C, T366W, and the second Fc-region polypeptide is a human IgG4 Fc-region polypeptide having the 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) both the first and the second Fc-region polypeptide are of human IgG1 or human IgG4 subclass (derived from human origin) and comprise in the first Fc-region polypeptide one or two mutations selected from the group consisting of: i) I253A, H310A and H435A, or ii) the group consisting of H310A, H433A and Y436A, or iii) the group consisting of L251D, L314D and L432D (numbering according to the Kabat EU index numbering system) and in the second Fc-region polypeptide comprises one or two mutations selected from the group consisting of the mutations L251D, I253A, H310A, L314D, L432D, H433A, H435A and Y436A (numbering according to the Kabat EU index numbering system) EU index numbering system numbering) such that all mutations in the first and second Fc-region polypeptides when taken together result in the variant (human) IgG class Fc-region comprising the following mutations: i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D,

or

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

or

c) both the first and the second Fc-region polypeptide are of human IgG1 or human IgG4 subclass (i.e., are derived from human origin) and comprise the mutations I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D in the first as well as in the second Fc-region polypeptide (numbering according to the Kabat EU index numbering system), or comprise the mutation combination I253A/H310A/H435A in the first Fc-region polypeptide and the mutation combination H310A/H433A/Y436A in the second Fc-region polypeptide (numbering 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 class Fc-region polypeptide, and the second variant Fc-region polypeptide is derived from a second parent IgG class Fc-region polypeptide, wherein the first parent IgG class Fc-region polypeptide and the second parent IgG class Fc-region polypeptide are the same or different, 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 in which the first parent IgG species Fc-region polypeptide differs from the second parent IgG species Fc-region polypeptide, and

c) the IgG class 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 class Fc-region comprising the first parent IgG class Fc-region polypeptide in a) and the second parent IgG class Fc-region polypeptide in a),

wherein the first Fc-region polypeptide or the second Fc-region polypeptide or both Fc-region polypeptides independently 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 (hole-chain), and the second Fc-region polypeptide comprises the mutations S354C and T366W (knob-chain), and wherein the first Fc-region polypeptide (hole-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 via one or more disulfide bridges,

And wherein the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide both bind to protein A or both do not bind to protein A (numbering according to 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 the antibody of any one of embodiments 1-45 for treating ocular diseases, in particular ocular vascular diseases.

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

50. The antibody according to any one of embodiments 1-45, for use in treating 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 the antibody of any one of embodiments 1-45 in the preparation of a medicament for treating ocular vascular diseases.

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

60. A method for preparing 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 according to any one of embodiments 1 to 45,

b) culturing the cell so as to express the antibody, and

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

Example

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

Example 1

immunity

For immunization of mice (NMRI mice) and rabbits (human Ig locus transgenic rabbits), a time schedule based on RIMMS ("Rapid IMmunization, Multiple Sites") was 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 NUNC Maxisorb plates (100 μl/well) at 2.5 μg/ml (mouse) or 1.0 μg/ml (rabbit) in PBS, and then: the plates were blocked with 200 μl/well of 2% Crotein C in PBS; serial dilutions of antisera in PBS containing 0.5% Crotein C were applied in duplicate at 100 μl/well; mouse serum was detected with HRP-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch) diluted 1:16,000 in PBS containing 0.5% Crotein C, or rabbit serum was detected with biotinylated goat anti-human kappa antibody (Southern Biotech) diluted 1:5,000 and HRP-conjugated streptavidin diluted 1:8,000 in PBS containing 0.5% Crotein C, 100 μl/well. For all steps, the plates were incubated at 37°C for 1 hour. Between steps, the plates were washed three times with PBS containing 0.05% Tween 20. The signal was visualized by adding 100 μl/well soluble BM Blue POD substrate (Roche Diagnostics GmbH, Mannheim, Germany) and terminated by adding 100 μl/well 1 M HCl. The absorbance was read at 450 nm with 690 nm as reference. The titer was defined as the antiserum dilution that resulted in half the maximal signal.

Example 3

Rabbit-derived B cell clones

Isolation of rabbit peripheral blood mononuclear cells (PBMC)

Blood samples were collected from three immunized rabbits. Whole blood containing EDTA was diluted twice with 1x PBS (PAA, Pasching, Austria) and then subjected to density centrifugation using mammalian lymphocytes (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the manufacturer's instructions. PBMCs were washed twice with 1x PBS.

EL-4B5 medium

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

Flat coating

Sterile cell culture 6-well plates were coated with PBGF-BB (2 μg/ml) or a mixture of PDGF-AA and PDGF-CC proteins (1 μg/ml PDGF-AA and PDGF-CC) in carbonate buffer (0.1 M sodium bicarbonate, 34 mM disodium hydrogen carbonate, pH 9.55) at 4° C. overnight. Prior to use, the plates were washed three times with sterile PBS.

Depletion of macrophages/monocytes

PBMCs from half of the blood samples were plated on sterile 6-well plates (cell culture grade) to deplete macrophages and monocytes by nonspecific adhesion.

The remaining 50% of PBMCs were plated on plates pre-coated with a mixture of PDGF-AA and PDGF-CC proteins, thereby eliminating the binding of B cells to these proteins and removing macrophages and monocytes in one step.

Each well was filled with up to 4 ml of culture medium and up to ⁶ x 106 PBMCs from immunized rabbits and allowed to bind for 1 hour in an incubator at 37°C.

The cells in the supernatant (peripheral blood lymphocytes (PBL)) were used for the antigen panning step.

Enrichment of B cells on PDGF BB protein

Six-well tissue culture plates coated with PDGF-BB protein were seeded with up to 6 x 10 PBLs in ⁴ ml of culture medium and allowed to bind for 1 hour at 37°C in an incubator. Non-adherent cells were carefully removed by washing the wells 1-2 times with 1xPBS. The remaining adherent cells were detached by trypsinization for 10 minutes at 37°C in an incubator. Trypsinization was terminated with EL-4B5 culture medium. Cells were kept on ice prior to immunofluorescence staining.

Immunofluorescence staining and flow cytometry

Single cell sorting was performed using anti-IgG FITC (AbD Serotec, Düsseldorf, Germany). For surface staining, cells from the depletion and enrichment steps were incubated with anti-IgG antibodies conjugated to FITC in PBS and incubated in the dark at 4 ° C for 45 minutes. After staining, PBMCs were washed twice with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Before FACS analysis, propidium iodide (BD Pharmingen, San Diego, CA, USA) was added at a concentration of 5 μg/ml to distinguish between dead and live cells.

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

B-cell culture

Briefly, single sorted rabbit B-cells were cultured in 96-well plates with 200 μl/well of EL-4B5 medium containing Pansorbin cells (1:100,000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymocyte supernatant (MicroCoat, Bernried, Germany), and γ-irradiated murine EL-4B5 thymoma cells (2.5×10 ⁴ cells/well) at 37° C. in an incubator for 7 days. The supernatant of the B-cell culture was removed for screening, and the remaining cells were immediately collected and frozen at −80° C. in 100 μl of RLT buffer (Qiagen, Hilden, Germany).

Example 4

Hybridoma production

Cell culture

Mouse myeloma cell line P3x63-Ag8.653 was used as a fusion partner to generate mouse-mouse hybridomas. Cells were thawed approximately 14 days before fusion and cultured in the presence of 8-azaguanine. Every 3-4 days, cells were split and adjusted to a concentration of 1-2 x 10 ⁵ cells/mL.

Cell fusion

Material:

Mouse hybridoma medium (RPMI 1640 (PAN), FBS ultra-low-IgG, 2 mM L-glutamine, 1 mM 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°C)

Before fusion, myeloma cells were centrifuged briefly (250 rpm, 7 min.). The cell pellet was resuspended in culture medium. For one spleen, approximately 1-5*10 ⁷ cells were required.

For cell fusion, a lymphocyte:myeloma cell ratio of approximately 5:1 should be used. Resuspend P3x63-Ag8.653 cells in 50 ml of RPMI 1640 medium and centrifuge (250 rpm, 7 min.). Remove the supernatant. Then add the cells to the spleen cells.

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

PEG solution (37° C.) was added dropwise to the cells.

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

Example 5

Hybridoma culture

Suspend approximately 5 x ¹⁰ cells in 50 ml of Hyclone medium. Incubate the culture mixture for 96 hours. Then, add 75 ml of Hyclone medium. Continue culturing for 7 days. If viability drops below 40%, filter the cell suspension through a 0.22 μm filter and use the filtrate for purification.

Example 6

B-cell clones

PCR amplification of the V-domain

Total RNA was prepared from B-cell lysates (resuspended in RLT buffer - Qiagen - Cat. No. 79216) using the NucleoSpin 8/96 RNA Kit (Macherey &Nagel; 740709.4, 740698) according to the manufacturer's protocol. RNA was eluted with 60 μl of RNase-free water. cDNA was generated using 6 μl of RNA by reverse transcriptase reaction using Superscript III First Strand Synthesis Supermix (Invitrogen 18080-400) and oligo dT primers 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 μl of cDNA and AccuPrime supermix (Invitrogen 12344-040) in a final volume of 50 μl. Primers rbHC.up and rbHC.do were used for the heavy chain of wild-type rabbit B-cells, rbLC.up and rbLC.do were used for the light chain, and BcPCR_FHLC_leader.fw and BcPCR_huCkappa.rev were used for the light chain of transgenic rabbit B-cells. All forward primers were specific for the signal peptide (VH and VL respectively), while the reverse primers were specific for the constant region (VH and VL respectively). PCR conditions for RbVH+RbVL were as follows: 94°C hot start for 5 minutes; 35 cycles of 94°C for 20 seconds, 70°C for 20 seconds, 68°C for 45 seconds, and a final extension of 68°C for 7 minutes. PCR conditions for HuVL were as follows: hot start at 94°C for 5 minutes; 40 cycles of 94°C for 20 seconds, 52°C for 20 seconds, 68°C for 45 seconds, and a final extension at 68°C for 7 minutes.

8 μl of the 50 μl PCR solution was loaded onto a 48 E-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 μl 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 rabbit monoclonal bivalent antibodies, PCR products encoding VH or VL were cloned as cDNA into expression vectors by the overhang cloning method (RS Haun et al., BioTechniques 13 (1992) 515-518; MZ Li et al., Nature Methods 4 (2007) 251-256). The expression vector contained an expression cassette consisting of a 5′ CMV promoter containing intron A and a 3′ BGH polyadenylation sequence. In addition to the expression cassette, the plasmid also contained a pUC-derived replication origin for plasmid amplification in E. coli and a β-lactamase gene conferring ampicillin resistance. Three variants of the basic plasmid were used: one plasmid contained a rabbit IgG constant region designed to accommodate the VH region; two plasmids contained either a rabbit or human κLC constant region accommodating the VL region.

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

The purified PCR product was incubated with T4 DNA polymerase, which generates single-stranded overhangs. The reaction was terminated by the addition of dCTP.

In the next step, plasmid and insert are combined and incubated with recA, which induces site-specific restructuring. The recombinant plasmid is transformed into Escherichia coli. Next day, the bacterium colony of picking growth is used to detect correct recombinant plasmid 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 recombinantly express the selected candidates as monoclonal monovalent antibodies, the rabbit constant regions of all VH chains were converted to human constant regions encompassing the knob-mutated CH3 segments. For the VL chains derived from rabbit wild-type B cells, the rabbit Cκ constant region was converted to human. Immunoglobulin heavy and light chain variable regions were amplified using 4 μl of cDNA from the selected candidates with AccuPrime Supermix (Invitrogen 12344-040) in a final volume of 50 μl. The forward primer was specific for the signal peptide, and the reverse primer was specific for the CDR3-J region, which had a 20 bp overlap sequence (at the 3' end) with human constant region homology (VH and VL, respectively). PCR conditions for VH and VL chain amplification were as follows: 94°C hot start for 5 minutes; 35 cycles of 94°C for 20 seconds, 68°C for 20 seconds, 68°C for 45 seconds, and a final extension at 68°C for 7 minutes.

PCR products encoding VH or VL were cloned as cDNA into expression vectors using the overhang cloning method (RS Haun et al., BioTechniques 13 (1992) 515-518; MZ Li et al., Nature Methods 4 (2007) 251-256). The expression vector contained an expression cassette consisting of a 5′ CMV promoter containing intron A and a 3′ BGH polyadenylation sequence. In addition to the expression cassette, the plasmid also contained a pUC-derived replication origin for plasmid amplification in E. coli and a β-lactamase gene conferring ampicillin resistance. Two variants of the basic plasmid were used: one plasmid contained a human IgG constant region designed to accommodate the newly amplified VH chain, and the second plasmid contained a human κLC constant region that accommodated the VL chain.

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

The purified PCR product was incubated with T4 DNA polymerase, which generates single-stranded overhangs. The reaction was terminated by the addition of dCTP.

In the next step, plasmid and insert are combined and incubated with recA, which induces site-specific restructuring. The recombinant plasmid is transformed into Escherichia coli. Next day, the bacterium colony of picking growth is used to detect correct recombinant plasmid by plasmid preparation, restriction analysis and DNA sequencing.

Example 9

Human PDGF-BB binding ELISA

Binding analysis was performed using an enzyme-linked immunosorbent assay (ELISA)-based technique. The antigen, human PDGF-BB (Cell Signaling, Cat. No. 8921BF), was immobilized at a concentration of 125 ng/mL (25 μL in PBS, 0.5% BSA, and 0.05% Tween) on a 384-well microtiter plate (Thermo Scientific, Cat. No. 464718). Each of the following steps was followed by three routine washes (90 μL PBS, with dispersion and aspiration): 1) Blocking step: saturation of the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detection antibody at a dilution of 1:3000 (ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or, alternatively, ECL anti-mouse IgG-POD; NA9310V for murine antibodies). Optical density was determined at 370 nm 20-30 minutes after addition of the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). _EC50 was calculated using a four-parameter logistic model using GraphPad Prism 6.0 software.

Example 10

Cynomolgus monkey PDGF-BB binding ELISA

Binding analysis was performed using an enzyme-linked immunosorbent assay (ELISA)-based technique. The antigen, human PDGF-BB, was immobilized at a concentration of 125 ng/mL (25 μL in PBS, 0.5% BSA, and 0.05% Tween) on a 384-well microtiter plate (Thermo Scientific, Cat. No. 464718). Each of the following steps was followed by three routine washes (90 μL PBS, 0.5% BSA, 0.05% Tween, performed by dispersing and aspiration): 1) Blocking step: saturation of the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detection antibody at a dilution of 1:3000 (ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or, alternatively, ECL anti-mouse IgG-POD; NA9310V for murine antibodies). Optical density was determined at 370 nm 20-30 minutes after addition of the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). _EC50 was calculated using a four-parameter logistic model using GraphPad Prism 6.0 software.

Example 11

Mouse PDGF-BB binding ELISA

Binding analysis was performed using an enzyme-linked immunosorbent assay (ELISA)-based technique. Antigen mouse PDGF-BB (Peprotech 315-18) was immobilized on a 384-well microtiter plate (Thermo Scientific, Cat. No. 464718) at a concentration of 125 ng/mL (25 μL in PBS, 0.5% BSA, and 0.05% Tween). Each of the following steps was followed by three routine washes (90 μL PBS, 0.5% BSA, 0.05% Tween, performed by dispersion and aspiration): 1) Blocking step: saturation of the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detection antibody, dilution = 1:3000 (ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or, alternatively, ECL anti-mouse IgG-POD; NA 9310V for murine antibodies). Optical density was determined at 370 nm 20-30 minutes after addition of the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). _EC50 was calculated using a four-parameter logistic model using GraphPad Prism 6.0 software.

Example 12

Rat PDGF-BB binding ELISA

Binding analysis was performed using an enzyme-linked immunosorbent assay (ELISA)-based technique. Antigen rat PDGF-BB (R&D, 520-BB) was immobilized on a 384-well microtiter plate (Thermo Scientific, Cat. No. 464718) at a concentration of 125 ng/mL (25 μL in PBS, 0.5% BSA, and 0.05% Tween). Each of the following steps was followed by three routine washes (90 μL PBS, 0.5% BSA, 0.05% Tween, performed by dispersion and aspiration): 1) Blocking step: saturation of the unbound surface (1 hour, 2% BSA); 2) Increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) Detection antibody, dilution = 1:3000 (ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or, alternatively, ECL anti-mouse IgG-POD; NA9310V for murine antibodies). Optical density was determined at 370 nm 20-30 minutes after addition of the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). _EC50 was calculated using a four-parameter logistic model using GraphPad Prism 6.0 software.

Example 13

Human PDGF-AA binding ELISA

Binding analysis was performed using an enzyme-linked immunosorbent assay (ELISA)-based technique. Antigen human PDGF-AA (Peprotech, Cat. No. AF-100-13A) was immobilized at a concentration of 125 ng/mL (25 μL in PBS, 0.5% BSA, and 0.05% Tween) on a 384-well microtiter plate (Thermo Scientific, Cat. No. 464718). Each of the following steps was followed by three routine washes (90 μL PBS, 0.5% BSA, 0.05% Tween, performed by dispersion and aspiration): 1) Blocking step: saturation of the unbound surface (1 hour, 2% BSA); 2) Increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) Detection antibody, dilution = 1:3000 (ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or, alternatively, ECL anti-mouse IgG-POD; NA9310V for murine antibodies). Optical density was determined at 370 nm 20-30 minutes after addition of the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). _EC50 was calculated using a four-parameter logistic model using GraphPad Prism 6.0 software.

Example 14

Human PDGF-CC binding ELISA

Binding analysis was performed using an enzyme-linked immunosorbent assay (ELISA)-based technique. Antigen human PDGF-CC (Peprotech AF-100-00C) was immobilized on a 384-well microtiter plate (Thermo Scientific, Cat. No. 464718) at a concentration of 125 ng/mL (25 μL in PBS, 0.5% BSA, and 0.05% Tween). Each of the following steps was followed by three routine washes (90 μL PBS, 0.5% BSA, 0.05% Tween, performed by dispersion and aspiration): 1) Blocking step: saturation of the unbound surface (1 hour, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1 hour; 3) detection antibody, dilution = 1:3000 (ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or, alternatively, ECL anti-mouse IgG-POD; NA 9310V for murine antibodies). Optical density was determined at 370 nm 20-30 minutes after addition of the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). _EC50 was calculated using a four-parameter logistic model using GraphPad Prism 6.0 software.

Example 15

Protein-Protein Interaction Inhibition Assay: Human PDGF-B: Human PDGF-BB Receptor

The protein-protein interaction inhibition analysis of human PDGF-BB and human PDGF-BB receptor was performed using enzyme-linked immunosorbent assay (ELISA)-based technology. Human Fc-tagged PDGF-BB receptor protein (RnD, Cat.No.385-PR-100) was immobilized on 384-well microtiter plates (Thermo Scientific Cat.No.464718) at a concentration of 750 μg/mL (25 μL in PBS, 0.5% BSA and 0.05% Tween). Each of the following steps was followed by three routine washes (90 μL PBS, with dispersion and aspiration): 1) a blocking step (1 hour, 2% BSA) to saturate the unbound surface; 2) incubation of 15 μL of increasing concentrations of anti-PDGF-BB antibody with 15 μL of 75 nM biotinylated human PDGF-BB (Cell Signaling, Cat. No. 8921BF) in a 30 μL volume for 1 hour; 3) detection using peroxidase-labeled streptavidin (Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 11089153001). Optical density was determined at 370 nm 20-30 minutes after addition of the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). _IC50 values were calculated using a four-parameter logistic model using GraphPad Prism 6.0 software.

Example 16

Antibody purification from mouse hybridomas

The hybridoma supernatant containing the antibody was filtered and purified by two chromatography steps. Using HiTrap Protein G (GE Healthcare) balanced with PBS (1 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 137 mM NaCl, 2.7 mM KCl) (pH 7.4), the antibody was captured by affinity chromatography. Unbound protein was removed by washing with equilibrium buffer, and the antibody was recovered with 25 mM citric acid buffer (pH 3.0) and immediately neutralized to pH 6.0 with 1 M Tris-base (pH 9.0) after elution. Size exclusion chromatography on Superdex 200 ^TM (GE Healthcare) was used as the second purification step. The size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The solution containing the antibody was concentrated and stored at -80 ° C using an Ultrafree-CL centrifugal filter component equipped with a Biomax-SK membrane (Millipore, Billerica, MA).

The hybridoma supernatant comprising antibody is filtered and purified by two chromatography steps.Supernatant is mixed with 50%v/v 2M glycine, pH 8.6,600mM NaCl, and captured by affinity chromatography using the HiTrapMabSelectSuRe (GE Healthcare) balanced with 1M glycine, pH 8.6,300mM NaCl.Unbound protein is removed by cleaning with equilibrium buffer, and the antibody is reclaimed with 100mM citrate buffer (pH 2.8), and immediately neutralized to pH 6.0 with 1M Tris-base (pH 8.5) after elution.The size exclusion chromatography used on Superdex 200 ^TM (GE Healthcare) is used as the second purification step.The size exclusion chromatography is carried out in 20mM histidine buffer, 0.14M NaCl, pH 6.0. The antibody-containing solution was concentrated using Ultrafree-CL centrifugal filter units equipped with Biomax-SK membrane (Millipore, Billerica, MA) and stored at -80°C.

Example 17

Surface plasmon resonance spectroscopy of human PDGF-BB binding

Binding analysis was performed using a BIAcore B4000 system (GE Healthcare) using surface plasmon resonance spectroscopy-based techniques. 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 μg/mL (in PBS, 0.1% BSA, 0.05% Tween) using amine coupling chemistry. Increasing concentrations of anti-PDGF-BB antibody were applied in 10 mM HEPES pH 7.2, 150 mM NaCl. Apparent _k and _k were calculated using read times of 180 seconds for association and 600 seconds for dissociation. Apparent _K (avidity) was calculated using BIAcore T200 v2.0 fitting software.

Example 18

Surface Plasmon Resonance Spectroscopy Determination of PDGF-BB Binding in Cynomolgus Monkeys

Binding analysis was performed using a BIAcore B4000 system (GE Healthcare) using surface plasmon resonance spectroscopy-based techniques. 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 μg/mL (in PBS, 0.1% BSA, 0.05% Tween) using amine coupling chemistry. Increasing concentrations of anti-PDGF-BB antibody were applied in 10 mM HEPES pH 7.2, 150 mM NaCl. Apparent _k and _k were calculated using a read time of 180 seconds for association and 600 seconds for dissociation. Apparent _K (avidity) was calculated using BIAcore T200 v2.0 fitting software.

Example 19

Transfection and transient expression of humanized antibodies in HEK cells

Antibodies were transiently expressed in suspension-adapted HEK293F (FreeStyle 293-F cells; Invitrogen) cells using Transfection Reagent 293-free (Novagen).

After thawing in 125 ml shake flasks (incubated/shaken at 37°C, 7% _CO2 , 85% humidity, 135 rpm), cells had been passaged at least four times by dilution (volume 30 ml).

The cells were propagated to 3 x ¹⁰ cells/ml in a 250 ml volume. Three days later, the cells were split and newly seeded into 250 ml volumes in 1 liter shake flasks at a density of 7 x ¹⁰ cells/ml. Transfection was performed 24 hours after the cell density reached approximately 1.4-2.0 x ¹⁰ cells/ml.

Prior to transfection, 250 μg of plasmid DNA (122 μg of light chain and 128 μg of heavy chain) was diluted in a final volume of 10 ml with preheated (water bath; 37°C) Opti-MEM (Gibco). The solution was gently mixed and incubated at room temperature for no more than 5 minutes. Then, 333.3 μl of 293-free transfection agent was added to the DNA-OptiMEM solution. Gently mix and incubate at room temperature for 15-20 minutes. The entire volume of the mixture was added to a 1 L shake flask with a 250 ml HEK cell culture volume.

Incubate/shake at 37°C, 7% _CO2 , 85% humidity, 135 rpm for 6 or 7 days.

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

Example 20

Purification of antibodies from HEK supernatant

The culture supernatant containing the antibody was filtered and purified by two chromatography steps. The antibody was captured by affinity chromatography using a HiTrap MabSelectSuRe (GE Healthcare) balanced with PBS (1 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 137 mM NaCl, 2.7 mM KCl) (pH 7.4). Unbound protein was removed by washing with equilibration buffer, and the antibody was recovered with 100 mM citrate buffer (pH 2.8) and immediately neutralized to pH 6.0 with 1 M Tris-base (pH 9.0) after elution. Size exclusion chromatography on a Superdex 200 ^TM (GE Healthcare) was used as a second purification step. The size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The antibody-containing solution was concentrated using Ultrafree-CL centrifugal filter units equipped with Biomax-SK membrane (Millipore, Billerica, MA) and stored at -80°C.

Example 21

Analysis of antibody preparations

The protein concentration of the antibody preparations was determined by measuring the optical density (OD) at 280 nm 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 a LabChip GX II (PerkinElmer) with a Protein Express Chip and HT Protein Express Reagents Kit.

The aggregate content of the antibody preparations was determined by high performance SEC using a TSK-GEL QC-PAK GFC 300 with 2x PBS, pH 7.4 as running buffer or by high performance SEC using a BioSuite High Resolution SEC, 5 μm analytical size exclusion column (Waters GmbH) with 200 mM _{K 2} HPO ₄ /KH ₂ PO ₄ , 250 mM KCl, pH 7.0 as running buffer.

Example 22

Preparation and analysis of Fab fragments from antibodies:

5 mg of antibody (approximately 1 mg/ml in 20 mM histidine, 140 mM NaCl, pH 6.0) was incubated with 90 μl of L-cysteine solution (Merck Millipore; 250 mM in 20 mM histidine, 140 mM NaCl, pH 6.0) and 12 μl of papain (Roche Life Science; 3.2 U/mg antibody) at 37°C for 120 minutes. After cleavage, intact IgG and Fc fragments were removed using affinity chromatography on a HiTrap MabSelectSuRe (GE Healthcare) equilibrated with PBS (1 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 137 mM NaCl, 2.7 mM KCl) (pH 7.4). Subsequently, the flow-through of the MabSelectSuRe chromatography was further purified using size exclusion chromatography on a Superdex 200 ^™ (GE Healthcare) as a second purification step. The size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The solution containing the Fab fragments was concentrated using Ultrafree-CL centrifugal filter units equipped with Biomax-SK membrane (Millipore, Billerica, MA) and stored at -80°C.

The protein concentration of the Fab-fragments was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on 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 a reducing agent (5 mM 1.4-dithiothreitol) and staining with Simply Blue Safe Stain (Invitrogen).

The aggregate content of the Fab preparations was determined by high performance SEC using a Superdex 200 10/300 GL analytical grade size exclusion column (GE Healthcare) with 2x PBS, pH 7.4 as running buffer.

Example 23

3T3 cell proliferation ELISA

To determine the proliferation inhibition, a BrdU colorimetric assay was used according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany, #11 647 229 001). In this assay, 5 ng/ml human PDGF-BB and the antibody were used.

Example 24

Phospho-PDGF-R β (Tyr751) sandwich ELISA

10,000 3T3 cells/well were cultured in DMEM medium supplemented with 10% newborn calf serum for 24 hours. The cells were then cultured in Hunger's medium (DMEM medium with 0.5% newborn calf serum) for 24 hours.

Before addition, the antibodies (1 μg/ml) were pre-incubated for 2 hours in a medium containing 5 ng/ml PDGF-BB. 3T3 cells were cultured in Hunger's medium with 5 ng/ml human PDGF-BB and the antibodies for 10 minutes.

Prior to lysis, cells were washed four times with PBS. Cell lysis was performed in 100 μl of lysis buffer.

The lysate was incubated overnight at 4°C in the wells of a 96-well plate coated with rabbit anti-PDGF-Rβ antibody. The wells were then washed four times with PBS. After a one-hour incubation with mouse anti-phospho-PDGF receptor β antibody, the wells were washed four times with wash buffer. For detection, the wells were incubated with 100 μl of TMB substrate solution for 30 minutes. The reaction was terminated by adding 100 μl of stop solution. Absorbance was measured at 450 nm.

Example 25

Cell migration assay

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

process

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

Plate preparation

Upper chamber: (50 μl culture medium + 100 μl cell suspension)

Coat both sides with 20 μg/ml fibronectin (Sigma n° F0895-2mg) in PBS:

40 μl of fibronectin solution was added to the sensor side (bottom side) of each well and the upper chamber was incubated in a fume hood for 30 minutes. Carefully aspirate the fibronectin solution from the sensor side to avoid touching the electrodes. The UC was flipped so that the well side was facing up and the sensor side was facing down. 50 μl of fibronectin solution was added to the well and incubated at room temperature (RT) in a fume hood for 30 minutes. Carefully aspirate the solution from the inside of the well. 50 μl of CSC serum-free culture medium without growth factors was added to the well.

Lower chamber: 160 μl/well of your sample to be tested (1X concentrated)

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

Cell preparation

The cells were detached with cell dissociation buffer and resuspended at 1.5x10 ⁵ cells/ml in CellSystems CSC serum-free medium without growth factors (n°SF-4ZR-500-S); the CIM-plate 16 was removed from the RTCA analyzer and 15,000 primary human retinal pericytes ((ACBRI 183 CellSystems), immortalized with hTERT) in basal medium (Cell Systems CSC serum-free medium without growth factors n°SF-4ZR-500-S) were added to the UC/well/100 μl; the plate was returned to the RTCA analyzer and the 10-20 hour measurement was immediately started.

Example 26

Cell proliferation assay

CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, G3580).

Primary human retinal pericytes (ACBRI 183 Cell Systems, immortalized with hTERT), 2500 cells/well/100 μl

culture medium

Growth medium = CSC complete medium, culture boost (ACBRI n°4Z=-500)

Assay medium = CSC serum medium without growth factors (ACBRI n° SF-4ZR-500-S)

Balbc3T3, 5000 cells/well/100 μl

culture medium

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

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

Proliferation assay

Cells were seeded in growth medium 24-48 hours before assay. When cells were 80-90% confluent, cells were collected with trypsin solution. Cells were resuspended in growth medium at 0.25×10 ⁵ cells/ml (or 0.5×10 ⁵ cells/ml). 100 μl of cell suspension was added to each well of a 96-well plate. Incubated at 37°C for 24 hours. Growth medium was removed and 100 μl of assay medium was added to each well. Incubated for 24 hours. 100 μl of standard or sample (2X concentrated, diluted in assay medium) was added to each well. Incubated at 37°C with 5% CO ₂ for 72 hours. 40 μl of dye solution was added to the cells and incubated at 37°C. Read at 490 nm at different times (1h to 8h).

Example 27

Anti-PDGF-BB antibody kinetic screening

Using BIACORE T200 instrument (GE Healthcare), the combination of anti-PDGF-BB antibody and human PDGF-BB was studied by surface plasmon resonance. Using the amine coupling kit supplied by GE Healthcare, about 20 resonance units (RU) of recombinant human PDGF-BB (5 μg/ml; ordering code 220-BB; R&D Systems) were coupled to a Series S C1 chip (GE Healthcare BR-1005-35) at pH 4.0. The running buffer used for fixation was HBS-N pH 7.4 (10 mM HEPES, 150 mM NaCl, pH 7.4, GE Healthcare). For the following kinetic characterizations, the running and dilution buffer was HBS-P pH 7.4 (10 mM HEPES, 150 mM NaCl, 0.05% surfactant P20, pH 7.4, GE Healthcare). The flow cell was set to 25 ° C - and the sample block was set to 12 ° C - and sensitized twice with running buffer.

The association was measured by injecting anti-PDGF-BB antibodies at 30 nM and 3 nM concentrations in solution at a flow rate of 100 μl/min for 30 seconds. The dissociation phase was monitored until 600 seconds and initiated by switching from the sample solution to the running buffer. The surface was regenerated by washing with two consecutive 60-second injections of 0.85% H ₃ PO ₄ (phosphoric acid) solution at a flow rate of 5 μl/min. Differences in the bulk refractive index were corrected by subtracting the response obtained from a blank surface. The blank injection was also subtracted (= double referencing). For the calculation of K _D and other kinetic parameters, the Langmuir 1:1 model was used.

Example 28

Kinetic characterization of anti-PDGF-BB Fab

Using BIACORE T200 instrument (GE Healthcare), the combination of anti-PDGF-BB Fab sample and human PDGF-BB was studied by surface plasmon resonance. Using the amine coupling kit supplied by GE Healthcare, about 50 resonance units (RU) of recombinant human PDGF-BB (0.5 μg/ml; ordering code 220-BB; R&D Systems) were coupled to a Series S CM3 chip (GE Healthcare BR-1005-36) at pH 4.0. The running buffer used for fixing was HBS-N pH 7.4 (10 mM HEPES, 150 mM NaCl, pH 7.4, GE Healthcare). For the following kinetic characterizations, the running and dilution buffer was HBS-P pH 7.4 (10 mM HEPES, 150 mM NaCl, 0.05% surfactant P20, pH 7.4, GE Healthcare). The flow cell was set to 25 ° C - and the sample block was set to 12 ° C - and sensitized twice with running buffer.

The association was measured by injecting different concentrations of anti-PDGF-BB Fab in solution (starting at 300 nM and serially diluted 1:3) for 180 seconds at a flow rate of 30 μl/min. The dissociation phase was monitored until 900 seconds and initiated by switching from the sample solution to the running buffer. The surface was regenerated by washing with 0.85% H ₃ PO ₄ (phosphoric acid) solution for 60 seconds at a flow rate of 5 μl/min. The bulk refractive index difference was corrected by subtracting the response obtained from a blank surface. The blank injection was also subtracted (= double reference). For the calculation of KD and other kinetic parameters, the Langmuir 1:1 model was used.

Example 29

Chemical degradation test

The samples were divided into three aliquots and rebuffered in 20 mM His/His*HCl, 140 mM NaCl, pH 6.0 or in PBS and stored at 40°C (His/NaCl) or 37°C (PBS). Control samples were stored at -80°C.

At the end of the incubation period, samples were analyzed for relative activity concentration (BIAcore), aggregation (SEC), and fragmentation (capillary electrophoresis or SDS-PAGE) and compared to untreated controls.

Example 30

Thermal stability

Samples were prepared at a concentration of 1 mg/mL in 20 mM histidine/histidine hydrochloride, 140 mM NaCl, pH 6.0, transferred by centrifugation through a 0.4 μm filter plate into an optical 384-well plate, and covered with paraffin oil. Hydrodynamic radii were measured repeatedly by dynamic light scattering on a DynaPro plate reader (Wyatt) while samples were heated from 25°C to 80°C at a rate of 0.05°C/min.

Alternatively, samples were transferred to 10 μL microcuvette arrays and static light scattering data as well as fluorescence data when excited with a 266 nm laser were recorded using an Optim 1000 instrument (Avacta Inc.) while they were heated from 25 to 90° C. at a rate of 0.1° C./min.

The aggregation onset temperature is defined as the temperature at which the hydrodynamic radius (DLS) or scattered light intensity (Optim 1000) starts to increase.

The melting temperature is defined as the inflection point in a 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 illustrations and examples should not be construed as limiting the scope of the invention. The functional contents of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Example 31

Preparation and purification of bispecific antibodies

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

After thawing in a 125 ml shake flask (incubated/shaken at 37° C., 7% CO 2 , 85% humidity, 135 rpm), the cells were passaged by dilution every third or fourth day for at least four passages (volume 30 ml). Cells were expanded by inoculating them in 250 ml of culture medium at a cell density of 3×10 ⁵ cells/ml. Three days later, the cells were split and newly inoculated in 500 ml of culture medium at a density of 2× ^{10 5} cells/ml. Four days later, the cells were split and newly inoculated in 1 liter of culture medium at a density of 7×10 5 cells/ml (incubated/shaken at 37° C., 7% CO 2 , 85% humidity, 110 rpm). After 24 hours, transfection was performed at a cell density of approximately 1.4-2.0×10 ⁶ cells/ml.

Prior to transfection, 1000 μg of plasmid DNA (2 x 250 μg of light chain encoding plasmid DNA and 2 x 250 μg of heavy chain encoding plasmid DNA) was diluted in a final volume of 40 ml of preheated (water bath; 37°C) Opti-MEM (Gibco). The solution was gently mixed and incubated at room temperature for no more than 5 minutes. Then, 1333 μl of 293-free transfection agent 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. The cells were further cultured for 7 days at 37°C, 7% CO2, 85% humidity with shaking at 110 rpm.

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

The culture supernatant containing the antibody was filtered and purified by at least two chromatography steps. The antibody was captured by affinity chromatography using a CaptureSelect prepacked column IgG-CH1 (life technologies, #494320005) equilibrated with PBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl) (pH 7.4). Unbound protein was removed by washing with the equilibration buffer, and the antibody was recovered with 25 mM citric acid buffer (pH 3.0) and neutralized to pH 6.0 with 1 M Tris-base (pH 9.0) immediately after elution.

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

Anti-PDGF-B/ANG2 antibody (clone 0144-0004) was further purified using hydrophobic interaction chromatography (HIC). Ammonium sulfate was added to the antibody solution to a final concentration of 1 M. The solution was applied to a Butyl Sepharose 4 Fast Flow (GE Healthcare) column equilibrated with 1 M ammonium sulfate, 35 mM acetate, pH 5.6. The antibody was eluted with a linear gradient (0-100%) using 35 mM acetate, pH 5.6. Antibody-containing fractions were pooled and applied to size exclusion chromatography.

The protein concentration of the antibody preparations was determined by measuring the optical density (OD) at 280 nm 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 a LabChip GX II (PerkinElmer) with a protein expression array and HT protein expression kit.

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

Antibody 0144 is a CrossMab antibody comprising the ANG2 binding sites of SEQ ID NO: 117 (VH) and SEQ ID NO: 118 (VL) and the PDGF-B binding sites of SEQ ID NO: 92 (VH) and SEQ ID NO: 97 (VL).

Antibody 0117 is a CrossMab antibody comprising the VEGF binding sites of SEQ ID NO: 119 (VH) and SEQ ID NO: 120 (VL) and the PDGF-B binding sites of SEQ ID NO: 92 (VH) and SEQ ID NO: 97 (VL).

Antibody 0145 is a CrossMab antibody comprising the ANG2 binding sites of SEQ ID NO: 119 (VH) and SEQ ID NO: 120 (VL) and the PDGF-B binding sites of SEQ ID NO: 101 (VH) and SEQ ID NO: 106 (VL).

Antibody 0146 is a CrossMab antibody comprising the ANG2 binding sites of SEQ ID NO: 117 (VH) and SEQ ID NO: 118 (VL) and the PDGF-B binding sites of SEQ ID NO: 121 (VH) and SEQ ID NO: 122 (VL).

Example 32

Bispecific Antibody Kinetic Characterization

PDGF-BB

Binding of the bispecific anti-PDGF-BB/ANG2 antibody to human PDGF-BB was investigated by surface plasmon resonance using a BIACORE T200 instrument (GE Healthcare). Approximately 50 resonance units (RU) of recombinant human PDGF-BB (0.5 μg/ml; ordering code 220-BB; R&D Systems) were coupled to a Series S CM3 chip (GE Healthcare BR-1005-36) at pH 4.0 using an amine coupling kit supplied by GE Healthcare. The running buffer used for immobilization was HBS-N pH 7.4 (10 mM HEPES, 150 mM NaCl, pH 7.4, GE Healthcare). For the kinetic characterization described below, the running and dilution buffer was HBS-P pH 7.4 (10 mM HEPES, 150 mM NaCl, 0.05% surfactant P20, pH 7.4, GE Healthcare). The flow cell was set at 25°C and the sample block was set at 12°C and primed twice with running buffer.

The association was measured by injecting different concentrations of the bispecific antibody in solution (starting at 300 nM and serially diluted 1:3) for 180 seconds at a flow rate of 30 μl/min. The dissociation phase was monitored until 900 seconds and initiated by switching from the sample solution to the running buffer. The surface was regenerated by washing with a 0.85% H ₃ PO ₄ (phosphoric acid) solution for 60 seconds at a flow rate of 5 μl/min. The bulk refractive index differences were corrected by subtracting the response obtained from a blank surface. The blank injection (= double reference) was also subtracted. For the calculation of KD and other kinetic parameters, the Langmuir 1:1 model was used.

ANG2

Binding of the bispecific antibody to the human ANG2-RBD-mouse Fc-region fusion was studied by surface plasmon resonance using a BIACORE T200 instrument (GE Healthcare). Approximately 4000 RU of anti-mouse Fc-region antibody (10 μg/ml anti-mouse (Fc) antibody) was coupled to a Series SCM5 chip (GE Healthcare BR-1005-30) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. HBS-N (10 mM HEPES, 150 mM NaCl, pH 7.4, GE Healthcare) was used as the running buffer during the immobilization step. For the kinetic characterization described below, the sample and running buffer was HBS-P (10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% surfactant P20; GE Healthcare). The flow cell was set at 25°C and the sample block at 12°C and primed twice with running buffer prior to kinetic characterization.

The human ANG2-RBD-murine Fc-region fusion was captured by injecting a 1 μg/ml solution for 30 seconds at a flow rate of 5 μl/min. Association was measured by injecting the bispecific antibody at different concentrations (starting at 300 nM and serially diluted 1:3) in solution at a flow rate of 90 μl/min for 90 seconds. The dissociation phase was monitored until 600 seconds and initiated by switching from the sample solution to the running buffer. All surfaces were regenerated by washing with a 3 M MgCl solution for 60 seconds at a flow rate of 5 μl/min. Bulk refractive index differences were corrected by subtracting the response obtained from the anti-mouse IgG antibody (Fc) surface. A blank injection (double reference) was also subtracted. To calculate KD and other kinetic parameters, a Langmuir 1:1 model was used.

VEGF

The binding of the bispecific antibodies to human VEGF isoform 121 was studied by surface plasmon resonance using a BIACORE T200 instrument (GE Healthcare). The anti-hexa-histidine antibody was coupled to a CM5 chip (GE Healthcare BR-1005-30) using an amine coupling kit supplied by GE Healthcare according to the manufacturer's instructions. HBS-N (10 mM HEPES, 150 mM NaCl pH 7.4, GE Healthcare) was used as the running buffer during the immobilization step. For the following kinetic characterizations, the sample and running buffer were HBS-P (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20; GE Healthcare). The flow cell was set to 25° C. and the sample block was set to 12° C. and primed twice with running buffer prior to kinetic characterization.

The human VEGF isoform 121 containing the histidine tag was captured by injecting the solution at a flow rate of 5 μl/min for 30 seconds. The association was measured by injecting the bispecific antibody at different concentrations in solution (starting at 300 nM and serially diluted 1:3) at a flow rate of 90 μl/min for 90 seconds. The dissociation phase was monitored until 600 seconds and initiated by switching from the sample solution to the running buffer. All surfaces were regenerated by washing with a 3M MgCl2 solution for 60 seconds at a flow rate of 5 μl/min. The bulk refractive index differences were corrected by subtracting the reaction obtained from the anti-hexa-histidine antibody surface. The blank injection (= double reference) was also subtracted. For the calculation of KD and other kinetic parameters, the Langmuir 1:1 model was used.

Sequence Listing

<110> Hoffmann-La Roche GmbH

<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

<210> 1

<211> 116

<212> PRT

<213> House mouse (Mus musculus)

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

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Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Tyr Thr Phe Ser Ser Tyr

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

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

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

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

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

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Thr Val Ser Ser

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

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

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

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

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

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

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

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

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

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Tyr Thr Phe Ser Ser Tyr

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

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

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Lys Val Arg Val Thr Ile Thr Ala Asp Thr 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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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Tyr Thr Phe Ser Ser 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 Asn Glu Lys Phe

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

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Tyr Thr Phe Ser Ser Tyr

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Trp Ile Glu Trp Val Arg Gln Arg 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 Thr 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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<400> 23

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

1 5 10 15

Gly

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> PDGFB-0044 HVR-H3-003

<400> 24

Thr Thr Gln Asp Phe Asp Ser

1 5

<210> 25

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> PDGFB-0044 VH-004

<400> 25

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Tyr Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser 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> 26

<211> 7

<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

1 5 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 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 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 Ile Thr 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 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> 57

<211> 5

<212> PRT

<213> Homo sapiens

<400> 57

Ser Phe Trp Met Thr

1 5

<210> 58

<211> 6

<212> PRT

<213> Homo sapiens

<400> 58

Ser Ala Gly Gly Gly Ile

1 5

<210> 59

<211> 17

<212> PRT

<213> Homo sapiens

<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> Homo sapiens

<400> 60

Ser Gly Gly Asp Ile Tyr Ser Asp Phe Asp Tyr

1 5 10

<210> 61

<211> 107

<212> PRT

<213> Homo sapiens

<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> Homo sapiens

<400> 62

Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 63

<211> 7

<212> PRT

<213> Homo sapiens

<400> 63

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 64

<211> 9

<212> PRT

<213> Homo sapiens

<400> 64

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210> 65

<211> 120

<212> PRT

<213> Homo sapiens

<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> Homo sapiens

<400> 66

Ser Phe Trp Met Thr

1 5

<210> 67

<211> 6

<212> PRT

<213> Homo sapiens

<400> 67

Ser Ala Gly Gly Gly Ile

1 5

<210> 68

<211> 17

<212> PRT

<213> Homo sapiens

<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> Homo sapiens

<400> 69

Ser Gly Gly Asp Ile Tyr Ser Asp Phe Asp Tyr

1 5 10

<210> 70

<211> 107

<212> PRT

<213> Homo sapiens

<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> Homo sapiens

<400> 71

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 72

<211> 7

<212> PRT

<213> Homo sapiens

<400> 72

Thr Thr Ser Ser Leu Gln Ser

1 5

<210> 73

<211> 9

<212> PRT

<213> Homo sapiens

<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 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 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> 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

65 70 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 5 10 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> Homo sapiens

<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 Leu Ile 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 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 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 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

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 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

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 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> 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 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> 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 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> 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

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> 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 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> 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 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> 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 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> 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 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 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

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> 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 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> 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 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> 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

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> 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 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> 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

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> 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

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> 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

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> 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

245 250 255

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> 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

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> 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 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> 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 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr 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 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

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 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> 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

385 390 395 400

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> 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

115 120 125

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> 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

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> 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

145 150 155 160

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> 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 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> 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

115 120 125

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> 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 Pro Val 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 Tyr Cys 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

355 360 365

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

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> 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 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 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 (9)

1. An antibody that specifically binds to human PDGF-B, wherein the antibody comprises a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO:92 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO:97. 2. The antibody according to claim 1, wherein the antibody is a bispecific antibody. 3. The antibody according to claim 2, wherein the antibody is a bivalent antibody. 4. The antibody according to claim 2, wherein the antibody specifically binds to human PDGF-B and VEGF. 5. The antibody according to claim 2, wherein the antibody specifically binds to human PDGF-B and ANG2. 6. The antibody according to any one of claims 1-5, wherein the antibody is a human IgG1 subclass or a human IgG4 subclass. 7. A pharmaceutical preparation comprising the antibody according to any one of claims 1-6. 8. The pharmaceutical formulation of claim 7, further comprising a pharmaceutically acceptable carrier. 9. The pharmaceutical formulation according to claim 7 or 8, further comprising an additional therapeutic agent selected from anti-ANG2 antibody and anti-VEGF antibody.