VEGF antibodies

Abstract

The present invention relates to an antibody against VEGF (vascular endothelial growth factor)-A165b. The invention also relates to pharmaceutical compositions, kits and methods for use in therapy.

Description

Technical Field

[0001] The present invention relates to VEGF-A 165 b and targeting of monoclonal antibodies that bind VEGF-A 165 b, such as monoclonal antibodies (mAbs). Background Art

[0002] Peripheral vascular disease is a major cause of amputation and a very common cause of death from cardiovascular disease. In obesity and diabetes patients, due to enhanced monocyte-derived Wnt5a signaling, an anti-angiogenic form of VEGF is increased, resulting in a lack of new blood vessel formation (Kikuchi et al. 2014, Nat Med).

[0003] The growth of new blood vessels (angiogenesis) required for all tumor growth is stimulated by the expression of vascular endothelial growth factor (VEGF). VEGF is upregulated in all known solid tumors, but also in atherosclerosis, diabetic retinopathy, arthritis, and many other conditions. Conventional VEGF isoforms are widely described as pro-angiogenic cytokines. A splice variant of VEGF-A called VEGF-A 165 b is expressed as a protein in normal cells and tissues and circulates in human plasma (see Figure 34 ). VEGF-A 165 b binds to VEGF receptor 2 with the same affinity as VEGF-A 165 a, but does not activate the receptor or stimulate the same downstream signaling pathways. In addition, it attenuates VEGF-A 165 a-mediated phosphorylation and signaling of VEGF receptor 2 in cultured cells. VEGF-A 165 b is not angiogenic, and it inhibits VEGF-A 165 a-mediated angiogenesis in rabbit cornea, rat mesentery, mouse mammary gland, dorsal chamber and ovary, as well as in proliferative oxygen-induced retinopathy, choroidal neovascularization, and cancer growth. Tumors expressing VEGF-A 165 a grow significantly slower than tumors expressing VEGF-A 165 a, indicating that the switch in splice variant expression from VEGF-A 165 a to VEGF-A 165 b can inhibit tumor growth.

[0004] It has also been previously found that anti-VEGF-A 165The b murine monoclonal antibody stimulates angiogenesis in a hindlimb ischemia model of genetically obese (ob / ob) or mice fed a high-fat, high-sucrose diet for 12 weeks (Kikuchi et al. 2014. Nat Med.; 20(12):1464-1471, which is incorporated herein by reference), and the anti-VEGF-A 165 The b murine monoclonal antibody stimulates angiogenesis in an impaired hindlimb ischemia model of mice with eNOS genetic deficiency or myoglobin overexpression (Kuppuswamy et al., Cells 2022, 11, 2676). However, antibodies that are therapeutically effective, preferably effective for humans, are needed. SUMMARY OF THE INVENTION

[0005] An object of the present invention is to provide a therapeutic monoclonal antibody against VEGF-A 165 b.

[0006] A first aspect of the present invention relates to an antibody or an antigen-binding fragment thereof that is specific for a splice variant of vascular endothelial growth factor (VEGF), wherein the splice variant is VEGF-A 165 b or any VEGF sequence containing the sequence encoded by exon 8b of the VEGF gene (such as VEGF-Ax and VEGF 189 b).

[0007] In some embodiments, the antibody is a full antibody. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fv fragments and Fab-like fragments (e.g., Fab fragments, Fab' fragments, and F(ab)2 fragments). "Fv fragment" includes single-chain Fv, disulfide-bonded Fv, and domain antibody. "Fab-like fragment" includes Fab fragment, Fab' fragment, and F(ab)2 fragment.

[0008] In some embodiments, the antibody or its antigen-binding fragment is recombinant. In some embodiments, the antibody or its antigen-binding fragment is monoclonal. In some embodiments, the antibody or its antigen-binding fragment is polyclonal. In some embodiments, the antibody or its antigen-binding fragment is murine. In some embodiments, the antibody or its antigen-binding fragment is chimeric. In some embodiments, the antibody or its antigen-binding fragment is human or humanized.

[0009] In some embodiments, the antibody or its antigen-binding fragment comprises or consists of the following complementarity-determining regions (CDRs) as determined by the IMGT assay:

[0010] a. V H CDR1: GFDFSRYW (SEQ ID NO:1);

[0011] b.V H CDR2: IHPYSSTI (SEQ ID NO:2);

[0012] c.V H CDR3: ARAFAY (SEQ ID NO:3);

[0013] d.V L CDR1: QSLLDSDGKTY (SEQ ID NO:4);

[0014] e.V L CDR2: LVS (SEQ ID NO:5); and / or

[0015] f.V L CDR3: WQGTHFPYT (SEQ ID NO:6).

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following CDRs, as determined by the Kabat assay:

[0017] a.V H CDR1: RYWMSW (SEQ ID NO:7);

[0018] b.V H CDR2: EIHPYSSTINYTPSVKD (SEQ ID NO:8);

[0019] c.V H CDR3: AFAY (SEQ ID NO:9);

[0020] d.V L CDR1: R SSQSLLDSDGKTYLN (SEQ ID NO:10) or K SSQSLLDSDGKTYLN (SEQ ID NO:11);

[0021] e.V L CDR2: LVSKLDS (SEQ ID NO:12); and / or

[0022] f.V L CDR3: WQGTHFPYT (SEQ ID NO:13).

[0023] In some embodiments, the V L CDR1 determined by the Kabat assay is *SSQSLLDSDGKTYLN (SEQ IDNO:14), where * is R or K.

[0024] In some embodiments, the CDR sequences are determined by the Kabat method. In some embodiments, the CDR sequences are determined by the IMGT method.

[0025] In some embodiments, the antibody or its antigen-binding fragment comprises or consists of the following variable heavy chains:

[0026] a. EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTI

[0027] NYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA (SEQ ID NO:15; also referred to herein as the V H chain) of the parental murine mAb clone "58 / 8 / 31 / 1 / 13";

[0028] b. EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA

[0029] (SEQ ID NO:15); also referred to herein as "VH0");

[0030] c. EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTI

[0031] NYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO:16; also referred to herein as "VH1");

[0032] d. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFIISRDNAKNSVYLQLNSLRAEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO:17; also referred to herein as "VH2");

[0033] e. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWISEIHPYSSTI

[0034] NYTPSVKDRFTISRDNAKNSLYLQMNSLRDEDTALYYCARAFAYWGQGTLVTVSS (SEQ ID NO:18; also referred to herein as "VH3");

[0035] f. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSS (SEQ

[0036] ID NO:19; also referred to herein as "VH4"); or

[0037] g. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSA (SEQ ID NO:20; also referred to herein as "VH5").

[0038] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable heavy chain sequence, where substitutions or deletions at specific positions are indicated in parentheses and deletions are marked as "X":

[0039] ● EVKLLESGGGLVQPGGS(L / P)KLSCAASGFDFSRYW(M / R)SWVRQAP

[0040] GK(G / E)LEWIGEIHPYSSTINYTPSVKDK(F / L)IISRD(N / S)AKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA (SEQ ID NO:21)

[0041] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus heavy chain sequence, where the most common amino acid is indicated at each position (SEQ ID NO:22) (the variable domain is highlighted in bold and the CDRs are double underlined):

[0042] ●

[0043]

[0044] TTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF (SEQ ID NO:22) ；

[0045] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following common variable heavy chain sequences or consists of the same, where the most common amino acids are indicated at each position (SEQ ID NO: 15): The following common variable light chain sequences or consists of the same, where the most common amino acids are indicated at each position (SEQ ID NO: 31):

[0046] ●

[0047]

[0048] (SEQ ID NO:15).

[0049] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following variable light chain or consists of the following variable light chain:

[0050] a. DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO:23; also referred to herein as the V L chain of parental murine mAb clone "58 / 8 / 31 / 1 / 13");

[0051] b. DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO:23; also referred to herein as "VL0");

[0052] c. DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO:24; also referred to herein as "VL1");

[0053] d. DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWELQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKR (SEQ ID NO:25; also referred to herein as "VL2");

[0054] e. DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYCWQGTHFPYTFGGGTKVEVKR (SEQ ID NO:26; also referred to herein as "VL3");

[0055] f. DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGKTYLNWLQQRPGQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR (SEQ ID NO:27; also referred to herein as "VL4"); or

[0056] g. DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO:28; also referred to herein as "VL5").

[0057] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable light chain sequence, where substitutions or deletions are at specific positions, indicated in parentheses, and deletions are marked as "X":

[0058] ● D(I / V)VMTQ(T / S)PL(T / S)(L / S)(S / P)VT(I / L / P)GQPASISC(K / R)SSQSLLDSDGKTYLNW(L / F / E / Y)(L / Q)Q(R / K)PGQ(S / P)P(K / R / Q)(R / L)LIYLVSKLDSGVPDRF(T / S)GSG(S / A)GTDFTLKISRVE(A / P)ED(L / V)GVYYCWQGTHFPYTFG(G / Q)GTK(V / L)E(I / V)KR (SEQID NO:29)

[0059] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable chain sequences, where the most common amino acids are indicated at each position (SEQ ID NO: 30) (the variable domains are highlighted in bold and the CDRs are double-underlined as determined by IMGT):

[0060] ●

[0061]

[0062] In some embodiments, the antibody or antigen-binding fragment thereof comprises Figure 1 Figure 2

[0063] ●

[0064]

[0065] In some embodiments, the variable heavy chain sequence and / or variable light chain sequence further comprises a signal peptide sequence, such as MGWTLVFLFLLSVTAGVHS (SEQ ID NO: 32).

[0066] Thus, an antibody or antigen-binding fragment thereof can be formed by combining any VH specified above with any VL specified above. For example, VH4 or VH5 can be combined with the following VL2 (which may optionally contain a linker, such as (G4S)3, between the two domains):

[0067] VH4-VL2 (SEQ ID NO: 33)

[0068] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSS[linker]DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWELQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKRVH5-VL2 (SEQ ID NO: 34)

[0069] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSA[Linker]DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWELQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKR

[0070] It should be understood that any one of the VH domains can be combined with any one of the VL domains. In some embodiments, VH0 is combined with VL0, VL1, VL2, VL3, VL4, or VL5. In some embodiments, VH1 is combined with VL0, VL1, VL2, VL3, VL4, or VL5. In some embodiments, VH2 is combined with VL0, VL1, VL2, VL3, VL4, or VL5. In some embodiments, VH3 is combined with VL0, VL1, VL2, VL3, VL4, or VL5. In some embodiments, VH4 is combined with VL0, VL1, VL2, VL3, VL4, or VL5. In some embodiments, VH5 is combined with VL0, VL1, VL2, VL3, VL4, or VL5.

[0071] In some embodiments, the antibody or its antigen-binding fragment further comprises a constant domain. For example, the constant domain can correspond to the following sequence:

[0072] Heavy chain constant domain:

[0073] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:35)

[0074] Light chain constant region:

[0075] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:36)

[0076] When VH# or VL# further includes the corresponding heavy or light chain constant region, it is respectively referred to as HC# or LC#. For example, taking VH4 and including the heavy chain constant region gives HC4, and taking VL2 and including the light chain constant region gives LC2. Thus, for this example, combining the heavy chain domain and the light chain domain gives HC4-LC2 (also referred to herein as "HC4LC2" or "HC4 LC2", and all are interchangeable).

[0077] In some embodiments, the antibody or its antigen-binding fragment comprises or consists of the VH4-VL2 CDR sequences, and / or comprises or consists of the VH4-VL2 VH and / or VL sequences, optionally further comprising the heavy chain constant region and / or the light chain constant region specified herein (i.e., the antibody or its antigen-binding fragment may comprise or consist of the HC4-LC2 sequence). In some embodiments, the antibody or its antigen-binding fragment comprises or consists of the VH5-VL2 CDR sequences, and / or comprises or consists of the VH5-VL2 VH and / or VL sequences, optionally further comprising the heavy chain constant region and / or the light chain constant region specified herein (i.e., the antibody or its antigen-binding fragment may comprise or consist of the HC5-LC2 sequence).

[0078] In some embodiments, the antibody or its antigen-binding fragment is not specific for any other VEGF splice variant that does not contain exon 8b. An example of a VEGF splice variant that does not contain exon 8b is VEGF-A 165 a. Thus, in some embodiments, the antibody or its antigen-binding fragment is not specific for VEGF-A 165 a.

[0079] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a moiety. The moiety can be an amino acid sequence motif, optionally contained within the heavy chain constant domain. The moiety can be used to increase the in vivo half-life of the antibody or antigen-binding fragment thereof. The moiety can be selected from the group of post-translational modifications consisting of polyethylene glycol (PEG), glycosylation, fatty acids, and dextran. The moiety can be a gene fusion protein. For example, the gene fusion protein can be human serum albumin. Additionally or alternatively, the gene fusion protein can be a cytokine, which can form an immunocytokine. In some embodiments, the moiety is a half-life extension motif, optionally, wherein the half-life extension motif is contained within the Fc domain and not within the variable domain.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof is pegylated.

[0081] In some embodiments, the moiety is a cytotoxic moiety. For example, the cytotoxic moiety can comprise or consist of a radioisotope, such as a radioisotope selected from the group consisting of astatine-211, bismuth-212, bismuth-213, iodine-131, yttrium-90, lutetium-177, samarium-153, and palladium-109. Alternatively or additionally, the cytotoxic moiety can comprise or consist of a toxin, such as but not limited to saporin or calicheamicin. Alternatively or additionally, the cytotoxic moiety can comprise or consist of a chemotherapeutic agent such as an antimetabolite.

[0082] In some embodiments, the moiety is a detectable moiety. For example, the detectable moiety can comprise or consist of a radioisotope, such as a radioisotope selected from the group consisting of: technetium-99m; indium-111; gallium-67; gallium-68; arsenic-72; zirconium-89; iodine-124; thallium-201. Alternatively or additionally, the detectable moiety can comprise or consist of a paramagnetic isotope, such as a paramagnetic isotope selected from the group consisting of: gadolinium-157; manganese-55, dysprosium-162, chromium-52; iron-56.

[0083] A second aspect of the invention relates to a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to the first aspect of the invention, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0084] In some embodiments, the pharmaceutical composition is suitable for a particular route of delivery. For example, the delivery can be subcutaneous, intravenous, intramuscular, intracranial, or intraocular delivery.

[0085] A third aspect of the present invention relates to a kit comprising an antibody or an antigen-binding fragment thereof according to the first or second aspect of the present invention. In some embodiments, the kit further comprises instructions for use. Any one or more parts of the kit may be stored in vials in a form that requires reconstitution and / or mixing with other components before use. Any one or more parts of the kit may be in lyophilized form.

[0086] A fourth aspect of the present invention relates to an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention for use as a medicament, or a pharmaceutical composition according to the second aspect of the present invention.

[0087] A fifth aspect of the present invention relates to the use of an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or a pharmaceutical composition according to the second aspect of the present invention, in the manufacture of a medicament for the treatment of the diseases, syndromes or conditions specified herein.

[0088] A sixth aspect of the present invention relates to an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or a pharmaceutical composition according to the second aspect of the present invention, for use in the treatment or prevention of the diseases, syndromes or conditions specified herein.

[0089] A seventh aspect of the present invention relates to a method of treatment or diagnosis, wherein the method comprises administering an effective amount of an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or a pharmaceutical composition according to the second aspect of the present invention.

[0090] An eighth aspect of the present invention relates to a method of diagnosis, wherein the method comprises subjecting a sample to an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or a pharmaceutical composition according to the second aspect of the present invention. In some embodiments, the method is an in vitro or ex vivo method. In some embodiments, the sample is an isolated sample, such as a sample isolated from a subject. In some embodiments, the sample is selected from the group consisting of body fluids, cells, cell populations, tissues, organs, plasma and serum.

[0091] In some embodiments, the diseases, syndromes or conditions are selected from the group consisting of:

[0092] - VEGF-related diseases (preferably VEGF-A, even more preferably VEGF-A 165 b);

[0093] - ischemia (peripheral, intestinal / mesenteric, coronary / heart, brain / cerebral, retinal, limb, or renal);

[0094] - peripheral arterial disease (PAD);

[0095] - atherosclerosis;

[0096] - Conditions related to diabetes (such as diabetic retinopathy, diabetic nephropathy, painful diabetic neuropathy, diabetic neuropathy);

[0097] - Sclerosis (e.g., systemic sclerosis / scleroderma);

[0098] - Raynaud's syndrome;

[0099] - Arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, osteoarthritis);

[0100] - Ischemia-related skin conditions (such as cyanosis and gangrene);

[0101] - Retinal ischemic diseases (such as rhegmatogenous retinal detachment and proliferative vitreoretinopathy, retinal ischemia, central vein occlusion, branch vein occlusion, non-proliferative diabetic retinopathy);

[0102] - Lung diseases related to angiogenesis alterations (e.g., asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease);

[0103] - Inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease);

[0104] - Neuron ischemic conditions (e.g., chronic pain, peripheral neuropathy, traumatic neuropathy, chemotherapy-induced peripheral neuropathy);

[0105] - Stroke;

[0106] - Preeclampsia;

[0107] - Hypertension;

[0108] - Obesity;

[0109] - Hair loss;

[0110] - Renal failure (e.g., IgA nephropathy, hereditary kidney diseases such as Denys-Drash Syndrome or Frasier Syndrome, chronic kidney disease, acute kidney disease, glomerular nephropathy);

[0111] - Angiogenesis / vasculogenesis (including tumor-related angiogenesis and VEGF-A 165 a-mediated angiogenesis);

[0112] - Cancer (e.g., solid tumors);

[0113] - Deep vein thrombosis (DVT);

[0114] - Refractory angina; and

[0115] - Myocardial infarction (MI) or post-MI conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 3 : PCR using several combinations of Ig variable region primers.

[0117] Figure 4 : Sequence alignment of the heavy chain using the Clustal W 2.1 multiple sequence alignment tool (www.expasy.ch).

[0118] Figure 5 : 2D representation of the V region of the heavy chain CDR loops or Collier de Perles (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501). Amino acids are represented by single-letter abbreviations. CDRs specified using the IMGT numbering system are restricted by the amino acids shown in squares (anchoring positions), which belong to the neighboring FR-IMGT. According to the IMGT unique numbering, the shaded circles correspond to missing positions. Arrows indicate the direction of the β-strands and their different names in the 3D structure (from the IMGT receptor repertoire, http: / / imgt.cines.fr).

[0119] Figure 6 : Sequence alignment of the light chain using the Clustal W 2.1 multiple sequence alignment tool (www.expasy.ch).

[0120] Lane No. : 2D representation of the V region of the light chain CDR loops or Collier de Perles (Lefranc, M.-P. eta / ., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501). Amino acids are represented by single-letter abbreviations. CDRs specified using the IMGT numbering system are restricted by the amino acids shown in squares (anchoring positions), which belong to the neighboring FR-IMGT. According to the IMGT unique numbering, the shaded circles correspond to missing positions. Arrows indicate the direction of the β-strands and their different names in the 3D structure (from the IMGT receptor repertoire, http: / / imgt.cines.fr).

[0121] Sample : 4% to 20% denaturing, reducing (A) and non-reducing (B) SDS-PAGE analysis of Ab 1126HC0 LC0 to HC1 LC5#250719. Molecular weight markers are shown in kilodaltons. The lanes are as follows:

[0122] Batch Amount (μg) Condition* PageRule (Thermo Fisher) NA 1 NA R / NR Ab 1126 HC0 LC0 R / NR 2 Ab 1126 HC1 LC1 250719 2 R / NR 3 Ab 1126 HC1 LC2 250719 2 R / NR 4 Ab 1126 HC1 LC3 250719 2 R / NR 5 Ab 1126 HC1 LC4 250719 2 R / NR 6 Ab 1126 HC1 LC5 250719 2 R / NR 7 Ab 1126 HC1 LC5 250719 2 R / NR

[0123] *R = Reducing; NR = Non-reducing.

[0124] Figure 7 : 4% to 20% denaturing, reducing (A) and non-reducing (B) SDS-PAGE analysis of Ab 1126HC2 LC1 to HC2 LC5 #250719 and #080819. Molecular weight markers are shown in kilodaltons. The lanes are as follows:

[0125] Lane No. Sample Batch Amount (μg) Condition* 1 PageRule (Thermo Fisher) NA NA R / NR 2 Ab 1126 HC2 LC1 250719 2 R / NR 3 Ab 1126 HC2 LC2 250719 2 R / NR 4 Ab 1126 HC2 LC3 080819 2 R / NR 5 Ab 1126 HC2 LC4 250719 2 R / NR 6 Ab 1126 HC2 LC5 250719 2 R / NR

[0126] *R = Reducing; NR = Non-reducing.

[0127] Figure 8 : 4% to 20% denaturing, reducing (A) and non-reducing (B) SDS-PAGE analysis of Ab 1126HC3 LC1 to HC3 LC5 #250719. Molecular weight markers are shown in kilodaltons. The lanes are as follows:

[0128] Lane No. Sample Batch Amount (μg) Condition* 1 PageRule (Thermo Fisher) NA NA R / NR 2 Ab 1126 HC3 LC1 250719 2 R / NR 3 Ab 1126 HC3 LC2 250719 2 R / NR 4 Ab 1126 HC3 LC3 250719 2 R / NR 5 Ab 1126 HC3 LC4 250719 2 R / NR 6 Ab 1126 HC3 LC5 250719 2 R / NR

[0129] *R = Reducing; NR = Non-reducing.

[0130] Figure 9 : 4% to 20% denaturing, reducing (A) and non-reducing (B) SDS-PAGE analysis of Ab 1126HC4 LC1 to HC4 LC5 #250719. Molecular weight markers are shown in kilodaltons. The lanes are as follows:

[0131] Lane No. Sample Batch Amount (μg) Condition* 1 PageRule (Thermo Fisher) NA NA R / NR 2 Ab 1126 HC4 LC1 250719 2 R / NR 3 Ab 1126 HC4 LC2 250719 2 R / NR 4 Ab 1126 HC4 LC3 250719 2 R / NR 5 Ab 1126 HC4 LC4 250719 2 R / NR 6 Ab 1126 HC4 LC5 250719 2 R / NR

[0132] *R = Reducing; NR = Non-reducing.

[0133] Figure 10 : 4% to 20% denaturing, reducing (A) and non-reducing (B) SDS-PAGE analysis of Ab 1126HC5 LC1 to HC5 LC5 #250719. Molecular weight markers are shown in kilodaltons. The lanes are as follows:

[0134] Lane No. Sample Batch Amount (μg) Condition* 1 PageRule (Thermo Fisher) NA NA R / NR 2 Ab 1126 HC5 LC1 250719 2 R / NR 3 Ab 1126 HC5 LC2 250719 2 R / NR 4 Ab 1126 HC5 LC3 250719 2 R / NR 5 Ab 1126 HC5 LC4 250719 2 R / NR 6 Ab 1126 HC5 LC5 250719 2 R / NR

[0135] *R = Reducing; NR = Non-reducing.

[0136] Figure 11: Characterization of IgG:antigen interaction. Referencing the calibrated BLI binding curves (black), monitored on the surface of non-covalently immobilized antibody (variant IDs shown below the sensograms) at 25 °C in electrophoresis buffer for various antigen concentrations. The apparent dissociation rate constant (k d ) and the association rate constant (k a ) were determined by globally fitting a 1:1 binding model to the sensograms using the software provided with the instrument. Table 2 summarizes the global fitting results.

[0137] Figure 12 : 4% to 20% denaturing, reducing, and non-reducing SDS-PAGE analysis of Ab 1126HC0 LC0 lot 011020. Molecular weight markers are shown in kilodaltons. The lanes are as follows:

[0138] Lane No. Sample Batch Amount (μg) Condition 1 PageRule (Thermo Fisher) NA NA Reducing 2 Ab 1126 HC0 LC0 011020 2 Reducing 3 Blank NA NA NA 4 Ab 1126 HC0 LC0 011020 2 Non-reducing

[0139] Figure 13 : 4% to 20% denaturing, reducing, and non-reducing SDS-PAGE analysis of Ab 1126HC4 LC2 lot 061020. Molecular weight markers are shown in kilodaltons. The lanes are as follows:

[0140] Lane No. Sample Batch Amount (μg) Condition 1 PageRule (Thermo Fisher) NA NA Reducing 2 Ab 1126 HC4 LC2 061020 2 Reducibility 3 Blank NA NA NA 4 Ab 1126 HC4 LC2 061020 2 Non - reducibility

[0141] Figure 14 : 4% to 20% denaturing, reducing, and non-reducing SDS-PAGE analysis of Ab 1126HC5 LC2 lot 061020. Molecular weight markers are shown in kilodaltons. The lanes are as follows:

[0142] Lane number Sample Batch Amount (μg) Condition 1 PageRule (Thermo Fisher) NA NA Reducibility 2 Ab 1126 HC5 LC2 061020 2 Reducibility 3 Blank NA NA NA 4 Ab 1126 HC5 LC2 061020 2 Non - reducibility

[0143] Figure 15 : Analytical gel filtration elution curve observed for Ab 1126HC0 LC0. Sample Ab 1126HC0LC0 lot 011020 was analyzed and a chromatogram was obtained. The chromatogram is shown and the integration results are shown in the corresponding table. The peak corresponding to the monomer fraction (based on the retention volume of the control protein [see Figure 18 and Figure 19 ) is >98%. SEC analysis of Ab 1126 variant HC0 LC0 was performed using a Superdex 200 Increase 10 / 300GL column (see Figure 18 ). Acetone control samples were run before and after the sample and were identical within experimental error (see Figure 19 ).

[0144] Figure 16: Analytical gel filtration elution profiles observed for Ab 1126HC4 LC2. Sample Ab 1126HC4 LC2 lot 061020 was analyzed and chromatograms were obtained. The chromatograms are shown and the integration results are shown in the corresponding table. The peak corresponding to the monomer fraction (based on the retention volume of the control protein [see Figure 18 and Figure 19 ) is >98%. SEC analysis of the Ab 1126 variant HC4 LC2 was performed using a Superdex 200 Increase 10 / 300GL column (see Figure 18 ). Acetone control samples were run before and after the samples and were identical within experimental error (see Figure 19 ).

[0145] Figure 17 : Analytical gel filtration elution profiles observed for Ab 1126HC5 LC2. Sample Ab 1126HC5 LC2 lot 061020 was analyzed and chromatograms were obtained. The chromatograms are shown and the integration results are shown in the corresponding table. The peak corresponding to the monomer fraction (based on the retention volume of the control protein [see Figure 18 and Figure 19 ) is >98%. SEC analysis of the Ab 1126 variant HC5 LC2 was performed using a Superdex 200 Increase 10 / 300GL column (see Figure 18 ). Acetone control samples were run before and after the samples and were identical within experimental error (see Figure 19 ).

[0146] Figure 18 : HMW calibration kit. Calibration data for Superdex 200 Increase 10 / 300GL.

[0147]

[0148]

[0149] Figure 19 : SEC analysis of acetone control using Superdex 200 Increase 10 / 300GL column. SEC analysis of acetone control samples was performed before and after sample analysis.

[0150] Figure 20 : Fv domain model of humanized VH1 / VL1 predicted by Abodybuilder. Potential sequence defects are circled (N-glycosylation; Met oxidation; Trp oxidation; Asp isomerization). The same potential sequence defects are present in all humanized variants.

[0151] Figure 21 : (A) Anti-VEGF-A, anti-VEGF 165 b, VEGF 165 b and rh-Fc-VEGFR2 pictograms. (B) Anti-VEGF 165 b (HC4 LC2) inhibition of VEGF 165 b binding to the fc-VEGF receptor 2.

[0152] Figure 22 : In the presence of VEGF 165 a, chimeric anti-VEGF 165 b (HC0 LC0) and humanized anti-VEGF 165 b (HC4LC2 bevacizumab) and murine anti-VEGF 165 b antibody (mα165b) cell migration assay.

[0153] Figure 23 : In the presence of VEGF 165 a, for mα165b, chimeric anti-VEGF 165 b (HC0 LC0) and humanized anti-VEGF 165 b (HC4 LC2 bevacizumab (Vexobicizumab)) and previously published murine anti-VEGF 165 b antibody (MRVL56 / 1, AbCam) cell migration assay. B. IC50 for each of the antibodies (Vex = HC4 LC2).

[0154] Figure 24 : (A) Pictorial representation of cell migration assay of inhibition of endothelial cell migration mediated by human monocytes in diabetic PAD. (B) Results of the cell migration assay.

[0155] Figure 25 : Schematic of the murine experiment described in Example 4.

[0156] Figure 26 : Murine analysis of angiogenesis in hindlimb ischemia. Mice in the example were fed a high-fat high-sucrose (HFHS) diet for 12 weeks, then blood flow imaging was performed by laser speckle in mice before (pre-operative) and after (post-operative) femoral artery ligation and on subsequent days.

[0157] Figure 27 : Graphical quantification of murine analysis of blood flow in hindlimb ischemia. Speckle intensity was calculated as blood flow in the ipsilateral (ischemic) relative to the contralateral side. Ratio of blood flow relative to pre-operative. ** = p < 0.05, two-way ANOVA compared to IgG on the same day.

[0158] Figure 28: (A) Example of the gastrocnemius muscle of a mouse treated with IgG of visosumab 28 days after ischemia; (B) Angiogenesis was calculated as ipsilateral (ischemic) versus contralateral IB4+ve vessels; (C) Arteriogenesis was calculated as ipsilateral (ischemic) versus contralateral smooth muscle actin (SMA)+ve vessels. ** = p < 0.01, *** = p < 0.001, unpaired t-test compared to IgG.

[0159] Figure 29 : Schematic diagram of the rat experiment described in Example 4.

[0160] Figure 30 : Rat analysis of angiogenesis in hindlimb ischemia. Examples of paw blood flow imaging by laser speckle in rats before (pre-operative) and after (post-operative) femoral artery ligation and on subsequent days. Speckle intensity was calculated as ipsilateral (ischemic) versus contralateral blood flow.

[0161] Figure 31 : Graphical quantification of rat analysis of hindlimb ischemia blood flow, * = p < 0.05 compared to IgG on the same day, # = p < 0.05 compared to pre-operative, = P < 0.001 compared to pre-operative, two-way ANOVA.

[0162] Figure 32: (A) Example of the gastrocnemius muscle of a STZ rat treated with IgG of visosumab 28 days after ischemia; (B) Angiogenesis was calculated as ipsilateral (ischemic) versus contralateral IB4+ve vessels; (C) Arteriogenesis was calculated as ipsilateral (ischemic) versus contralateral smooth muscle actin (SMA)+ve vessels. ** = p < 0.01, *** = p < 0.001, unpaired t-test compared to IgG.

[0163] Figure 33: A. Human monocytes inhibit the migration of endothelial cells through a porous membrane. 1 μg / ml HC4 LC2 significantly reverses this inhibition of the migration of human umbilical vein cells. N = 3, cells from individual subjects. One-way ANOVA. B. Monocytes from 7 patients with peripheral vascular disease inhibit migration towards 165 VEGF-A

[0164] Figure 34 : A single gene can lead to opposite VEGF isoforms - pro-angiogenic VEGF-A 165 a and anti-angiogenic VEGF-A 165 b.

[0165] Figure 35 : Key to displaying antibodies derived from parental hybridomas.

[0166] Figure 36 : To determine the neutralizing effect of the antibody, VEGFR2 was bound to an ELISA plate, and anti-VEGF-A 165 b antibody was mixed with rhVEGF-A 165 b at increasing VEGF-A 165 b antibody concentrations. Both humanized (HC4LC2) and chimeric (HC0LC0) anti-VEGF 165 b were able to significantly reduce the affinity of VEGF 165 b for the VEGF receptor 2 (VEGFR2), to an extent slightly lower than the positive control of G6-31 (a pan-VEGF antibody that binds to the VEGF-A receptor binding site).

[0167] Figure 37 : In the presence of VEGF 165 a, cell migration assays of humanized anti-VEGF 165 b (HC5 LC2) and mouse anti-VEGF 165 b antibody (mα165b is referred to as "56 / 8" in the figure). Detailed implementation

[0168] All publications, patents, and patent applications cited herein, whether above or below, are hereby incorporated by reference in their entirety.

[0169] It should be understood that the different applications of the disclosed antibodies, uses, methods, pharmaceutical compositions, and kits can be customized according to specific needs in the art. It should also be understood that the terms used herein are only for describing specific embodiments of the present invention and are not intended to be limiting.

[0170] Furthermore, as used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a, an" and "the" include plural referents. Thus, for example, reference to "an antibody" includes "antibodies", reference to "an antigen" includes two or more such antigens, reference to "a subject" includes two or more such subjects, and so on.

[0171] The present invention relates to antibodies or antigen-binding fragments thereof that are specific for splice variants of vascular endothelial growth factor (VEGF). Many splice variants of VEGF are known. Preferably, the splice variant is VEGF-A 165 b or contains isoforms of the VEGF gene such as VEGF-Ax, isoform 15 of VEGF-A, and VEGF 189Any VEGF sequence of the sequence encoded by exon 8b of (b). The meaning of "splice variant" is that the gene encoding VEGF may be alternatively spliced, resulting in different isoforms or variants that are translated into alternative VEGF molecules.

[0172] Advantageously, the described anti-VEGF-A 165 b antibody is effective in reversing the anti-angiogenic effects of monocytes from patients with peripheral arterial disease both in vitro and in vivo. It is desired to generate a set of antibodies with an affinity <10 nM that can block VEGF-A 165 b-mediated inhibition of human VEGF-A 165 a-mediated angiogenesis in endothelial cells in culture. The present invention includes chimeric antibodies (mouse variable domains VH and VL and human heavy and light chain constant domains HC0LC0) and humanized versions of the VEGF-A 165 b antibody. The chimeric antibody has an affinity of 300 pM, and the humanized versions have affinities of 600 pM (2 clones - HC4LC2 and HC5LC2), 900 pM (HC4 LC1), 5.0 nM (HC1 LC1), 2.1 nM (HC1LC2), 4.4 nM (HC2 LC1), 3.6 nM (HC2LC2), 1.5 nM (HC3 LC1), 1.17 nM (HC3 LC2), and 3.2 nM (HC5 LC1). Three of these antibodies have been tested in cell assays and shown to have significantly higher potency (IC 50 >1 μg / ml) than the parental mouse antibody (IC 50 HC0 LC0 0.44 μg / ml, HC4 LC2 0.137 μg / ml, HC5 LC2 0.114 μg / ml). This represents a significant advancement in targeting antibodies to VEGF-A 165 b and provides potential new therapies for diseases associated with poor vasculature such as ischemia.

[0173] In some embodiments, the affinity of the antibody or its antigen-binding fragment is from 100 pM to 1000 pM, such as from 200 pM to 800 pM, preferably from 300 pM to 600 pM. In some embodiments, the affinity is about 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 1.17 nM, 1.5 nM, 2.1 nM, 3.2 nM, 3.6 nM, 4.4 nM, 5 nM, or higher. In terms of affinity, "higher" means an increase in affinity. For example, 1 pM is a higher (i.e., increased, improved, better, greater) affinity than 1 nM.

[0174] An antibody or an antigen-binding fragment thereof may be specific for VEGF-A 165 b. The antibody or an antigen-binding fragment thereof may not bind or may not significantly bind VEGF-A 165 a. Preferably, the antibody or an antigen-binding fragment thereof binds VEGF-A 165 b and does not bind or does not significantly bind VEGF-A 165 a or any other splice variant of VEGF that uses the proximal splice site in exon 8 encoding CDKPRR. The antibody may bind VEGF-A 165 b with an affinity that is at least 50x or 100x greater than that for VEGF-A 165 a or any other exon 8 proximal splice variant of VEGF. In some embodiments, if the splice variant excludes exon 8b, the antibody or an antigen-binding fragment thereof does not bind.

[0175] In some embodiments, the antibody or an antigen-binding fragment thereof may have an affinity / dissociation constant (k 165 ) for binding to VEGF-A D b that is less than 1 nanomolar. The antibody or an antigen-binding fragment thereof may have an affinity / dissociation constant (k 165 ) for binding to VEGF-A D b that is less than about 0.8 nM. In another embodiment, the antibody or an antigen-binding fragment thereof may have an affinity / dissociation constant (k 165 ) for binding to VEGF-A D b that is about 0.6 nM (600 pM) or less. The antibody or an antigen-binding fragment thereof may have an affinity / dissociation constant (k 165 ) for binding to VEGF-A D b that is between about 0.3 nM and about 0.8 nM. The antibody or an antigen-binding fragment thereof may have an affinity / dissociation constant (k 165 ) for binding to VEGF-A D b that is between about 0.5 nM and about 0.7 nM. The antibody or an antigen-binding fragment thereof may have an affinity / dissociation constant (k 165 ) for binding to VEGF-A D ) that is between about 0.55 nM and about 0.65 nM.

[0176] In some embodiments, the antibody or an antigen-binding fragment thereof has one or more of the following properties:

[0177] a. Reduces the affinity of VEGF-A 165 b for binding to vascular endothelial growth factor receptor 2 (VEGFR2);

[0178] b. Blocks VEGF-A 165b-mediated inhibition of cell migration, such as when tested in vitro, where the antibody or its antigen-binding fragment effectively inhibits cell migration, comparable to or better than parental murine monoclonal and chimeric antibodies used as controls;

[0179] c. Reversal of diabetes-induced inhibition of endothelial cell migration mediated by human monocytes in peripheral arterial disease (PAD) (e.g., as measured as described in Example 4, compared to a control).

[0180] The present invention advantageously provides an antibody or its antigen-binding fragment that has significant high specificity for VEGF-A 165 b and is humanized for efficacy and safety in human therapy.

[0181] In a preferred embodiment, the VEGF-A 165 b target of the antibody or its antigen-binding fragment is human VEGF-A 165 b. The sequence of VEGF-A 165 b may include the C-terminal sequence of TCRSLTRKD (SEQ ID NO: 37). The antibody or its antigen-binding fragment may have specific affinity for a peptide comprising or consisting of the sequence TCRSLTRKD (SEQ ID NO: 37), optionally conjugated to keyhole limpet hemocyanin (KLH). The sequence TCRSLTRKD (SEQ ID NO: 37) corresponds to the last 9 amino acids of human VEGF165b (which was used as an immunogen to generate the antibody).

[0182] VEGF-A variants that fully or partially contain the TCRSLTRKD sequence include VEGFA_human isoform 15 of vascular endothelial growth factor A and the precursor of vascular endothelial growth factor A isoform VEGF-Ax, as follows:

[0183] > vascular endothelial growth factor A sp|P15692-15|VEGFA_human isoform 15 OS=Homo sapiens OX=9606 GN=VEGFA (SEQ ID NO: 159)

[0184] MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRSLTRKD>NP_001303939.1 Vascular endothelial growth factor A isoform VEGF-Ax precursor [Homo sapiens] (SEQ ID NO:160)

[0185] MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQ

[0186] EYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHN

[0187] KCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKP

[0188] RRSAGQEEGASLRVSGTRSLTRKD

[0189] As used herein, the term "antibody" includes whole antibodies and any antigen-binding portion thereof (i.e., "antigen-binding fragment") or single chains. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or an antigen-binding portion thereof. Each heavy chain comprises a heavy-chain variable region (abbreviated herein as VH) and a heavy-chain constant region. Each light chain comprises a light-chain variable region (abbreviated herein as VL) and a light-chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0190] The heavy chain can be of any isotype, including IgG (IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM, and IgE. The light chains include κ chains and λ chains.

[0191] Thus, in some embodiments, the variable regions (VH and VL) of the antibodies of the invention can further comprise (e.g., be attached to) an antibody constant region or a portion thereof. For example, the variable region of an antibody can be attached at its C-terminus to an antibody light-chain constant domain, including the human Cκ or Cλ chain. Similarly, the variable region of an antibody can be attached at its C-terminus to all or a portion of an immunoglobulin heavy chain from any antibody isotype, such as IgG, IgA, IgE, and IgM, and any one of the isotype subclasses, particularly IgG1, IgG2, and IgG4.

[0192] Also relevant are antibodies and antigen-binding fragments thereof that have been "isolated" so as to be present in a physical environment different from that in which they might exist in nature, or antibodies and antigen-binding fragments thereof that have been modified so as to be different in amino acid sequence from a naturally occurring antibody.

[0193] According to the present invention, the term "isolated" refers to the state in which the specific antibody or antigen-binding fragment thereof or nucleic acid encoding the same of the present invention is preferably present. Antibodies and their antigen-binding fragments and nucleic acids will generally be free or substantially free of substances naturally associated therewith, such as other polypeptides (e.g., host cell proteins) or nucleic acids, which are present in their natural environment or, when prepared by recombinant DNA techniques carried out in vitro or in vivo, in the environment in which they are prepared (e.g., cell culture). Specific monoclonal antibodies and nucleic acids can be formulated with diluents or adjuvants or excipients and still be considered isolated for practical purposes - for example, if used to coat microtiter plates for immunoassays, the components will generally be mixed with gelatin or other carriers, or when used for diagnosis or therapy, will be mixed with pharmaceutically acceptable carriers, diluents, and / or excipients. Specific binding antibodies or their antigen-binding fragments can be glycosylated, either naturally or through a heterologous eukaryotic cell system, or they can be non-glycosylated (e.g., if produced by expression in prokaryotic cells or mammalian cells lacking specific glycosylation pathways, or if the antibody or antigen-binding fragment has been specifically engineered to remove glycosylation sites).

[0194] The antibody or its antigen-binding portion can be a polyclonal antibody or a monoclonal antibody. The antibody or its antigen-binding portion can be produced by any suitable method. For example, suitable methods for producing monoclonal antibodies are disclosed in "Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Application", SGR Hurrell (CRC Press, 1982). Recombinant techniques can also be used.

[0195] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen such as VEGF-A 165 b. It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, and dAb fragments. Single-chain antibodies such as scFv, which are referred to as nanobodies, as well as heavy-chain antibodies such as VHH and camelid antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of these fragments can be screened in the same manner as for intact (i.e., full-length) antibodies.

[0196] The antibodies for use in the methods of the present invention can be human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable regions where both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto a human framework sequence - such antibodies are commonly referred to as chimeric or humanized.

[0197] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues, commonly referred to as the input residues, are typically taken from the variable domain of the input. Humanization can be effected essentially by replacing the human complementarity determining regions with the corresponding rodent complementarity determining regions as described (see, e.g., Jones et al., 1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536l; US 4,816,567, the disclosures of which are incorporated herein by reference). In practice, a humanized antibody can be a typical human antibody in which some complementarity determining region residues and possibly some framework residues are replaced with residues from a similar site in a rodent (e.g., mouse) antibody. For example, a chimeric antibody can be an antibody in which the VH and VL of a rodent antibody are fused to human IgG constant regions (CH and CL or CK). On the other hand, a humanized antibody is an antibody in which the CDRs from a rodent antibody replace the CDRs in a human antibody framework that has a high degree of sequence identity / homology with the parental rodent antibody. This process is referred to as CDR grafting. It should be understood that such antibodies are not limited to similar sites in rodent antibodies. Instead, similar sites can be from other non-human parental antibody species, including, for example, rabbits, camels, monkeys, etc.

[0198] Human antibodies for use in the methods of the invention are generally human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic non-human animals (e.g., transgenic mice) having a genome comprising a human heavy chain transgene and a light chain transgene that are fused to immortalized cells. Human antibodies can also be prepared by in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus. The term “human antibody derivative” refers to any modified form of a human antibody, such as a conjugate of an antibody with another agent or antibody.

[0199] Alternatively, an antibody or antigen-binding portion thereof according to the invention can be a humanized antibody.

[0200] The term “humanized” refers to antibody molecules that are typically prepared using recombinant techniques and that have antigen-binding sites derived from immunoglobulins of non-human species and remaining immunoglobulin structures based on the structure and / or sequence of human immunoglobulins.

[0201] In one embodiment, the antigen-binding site can comprise a complete non-human antibody variable domain fused to a human constant domain, in which case the antibody is “chimeric”. Chimeric antibodies are discussed by Neuberger et al. (1998, 8 th International Biotechnology Symposium Part 2, 792-799).

[0202] Alternatively, in a fully humanized antibody (described herein as “humanized”), only the complementarity-determining regions (CDRs) of such variable domains are grafted into appropriate human framework regions of human variable domains. The framework residues of such humanized molecules can be wild-type (e.g., fully human), or they can be modified to contain one or more amino acid substitutions not found in human antibodies whose sequences have served as the basis for humanization. Humanization reduces or eliminates the likelihood that a “humanized antibody” will be immunogenic in a human individual, but the possibility of an immune response to the foreign variable domain remains (LoBuglio, A.F. et al. (1989) “Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224).

[0203] Those skilled in the art will understand that for human therapy or diagnosis, human antibodies or humanized antibodies are preferably used. Humanized forms of non-human (e.g., murine) antibodies are genetically engineered chimeric antibodies or antibody fragments that preferably have the fewest amino acid residues derived from the non-human antibody. Humanized antibodies include such antibodies in which the complementarity-determining regions of a human antibody (the acceptor antibody) are replaced with residues of the complementarity-determining regions from a non-human species (the donor antibody) having the desired function, such as a mouse, rat, or rabbit. In some cases, the Fv framework residues of the human antibody are replaced with corresponding non-human residues. Humanized antibodies may also contain residues that are neither present in the acceptor antibody nor in the input complementarity-determining regions or framework sequences. In general, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the complementarity-determining regions correspond to those of the non-human antibody and all or substantially all of the framework regions correspond to those of the relevant human consensus sequence. A humanized antibody preferably also includes at least a portion of the antibody constant region, such as the Fc region, typically derived from a human antibody (see, e.g., Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

[0204] Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues, often referred to as input residues, are typically taken from the input variable domain. Humanization can generally be performed as described by replacing human complementarity-determining regions with corresponding rodent complementarity-determining regions (see, e.g., Jones et al., 1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536l; US 4,816,567). In practice, a humanized antibody can be a typical human antibody in which some complementarity-determining region residues and possibly some framework residues are replaced with residues from similar sites in a rodent antibody.

[0205] A variety of techniques known in the art can also be used to identify human antibodies, including identification from phage display libraries (see, for example, Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581; Cole et al., 1985, Monoclonal antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner et al., 1991. J. Immunol. 147:86-95).

[0206] Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable regions to make them as close as possible to the human form. The variable regions of both the heavy and light chains are known to contain three complementarity-determining regions (CDRs) that vary in response to the antigen under discussion and determine binding affinity, flanked by four framework regions (FRs) that are relatively conserved in a given species and are thought to provide the scaffold for the CDRs. When preparing non-human antibodies against a specific antigen, the variable regions can be "engineered" or "humanized" by transplanting the CDRs derived from the non-human antibody into the FRs present in the human antibody to be modified. The application of this method to various antibodies has been reported by the following references: Sato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science 239:1534-1536; Kettleborough, C.A. et al. (1991) "Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation," Protein Engineering 4:773-3783; Maeda, H. et al. (1991) "Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity," Human Antibodies Hybridoma 2:124-134; Gorman, S.D. et al. (1991) "Reshaping A Therapeutic CD4 Antibody," Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185; Tempest, P.R.et al. (1991) "Reshaping AHuman Monoclonal Antibody To Inhibit Human Respiratory Syncytial VirusInfection in vivo," Bio / Technology 9:266-271; Co, M.S. et al. (1991) "HumanizedAntibodies For Antiviral Therapy," Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873; Carter, P. et al. (1992) "Humanization Of An Anti-p185her2 Antibody For HumanCancer Therapy," Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; and Co, M.S. et al. (1992) "Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen," J. Immunol. 148:1149-1154.

[0207] In some embodiments, the humanized antibody retains all six CDR sequences (e.g., a humanized murine antibody that contains all six CDRs from a murine antibody). In other embodiments, the humanized antibody has one or more (one, two, three, four, five, six) CDRs that are altered relative to the original antibody, which are also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody. The ability to humanize antibodies is well known (see, e.g., U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).

[0208] Any antibody mentioned herein can be provided in isolated form or, optionally, in connection (directly or indirectly) with another moiety. The other moiety can be a therapeutic molecule, such as a cytotoxic moiety, an antibiotic, or a drug.

[0209] Therapeutic molecules can be directly attached (e.g., by chemical conjugation) to the antibodies or antigen-binding fragments of the present invention. Methods for conjugating molecules thereto are known in the art. For example, carbodiimide conjugation (Bauminger and Wilchek (1980) Methods Enzymol. 70, 151-159) can be used to conjugate a variety of agents, including doxorubicin, to antibodies or peptides. The water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), is particularly suitable for conjugating functional moieties to binding moieties.

[0210] Other methods for conjugating a moiety to an antibody can also be used. For example, periodate oxidation followed by reductive alkylation with an appropriate reactant can be used, as can glutaraldehyde crosslinking. However, it is well known that regardless of the method chosen to produce the conjugates of the present invention, it must be determined that the antibody retains its targeting ability and the functional moiety retains its relevant function.

[0211] The cytotoxic moiety can be directly and / or indirectly cytotoxic. "Direct cytotoxicity" means that the moiety is itself cytotoxic. "Indirect cytotoxicity" means that the moiety, although not itself cytotoxic, can induce cytotoxicity, for example, by its action on another molecule or by further action on it. The cytotoxic moiety can be cytotoxic only when inside the cell and preferably not cytotoxic when outside the cell.

[0212] The antibody or its antigen-binding portion can be linked to a cytotoxic moiety that is a directly cytotoxic chemotherapeutic agent. Optionally, the cytotoxic moiety is a directly cytotoxic polypeptide. Cytotoxic chemotherapeutic agents are well known in the art. In the context of the present invention, an antibody or its antigen-binding portion having a cytotoxic chemotherapeutic agent is used for use in combination with different chemotherapies. In some embodiments, the antibody or its antigen-binding portion is not conjugated to a cytotoxic chemotherapeutic agent.

[0213] Cytotoxic chemotherapeutic agents, whether conjugated to an antibody or an antigen-binding portion thereof or used alone as chemotherapy, such as anticancer agents, include: alkylating agents, including nitrogen mustards such as methyl bis(chloroethyl)amine (HN2), cyclophosphamide, ifosfamide, phenylalanine mustard (L-sarcolysin), and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozocin (streptozotocin); and triazenes such as dacarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); antimetabolites, including folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), and cytarabine (cytosine arabinoside); and purine analogs and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin). Natural products, including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin D (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biologic response modifiers such as interferon alpha. Other agents, including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenediones such as mitoxantrone and anthracyclomycin; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); and adrenocortical inhibitors such as mitotane (o,p'-DDD) and aminoglutethimide; paclitaxel and analogs / derivatives; and hormone agonists / antagonists such as flutamide and tamoxifen.

[0214] The cytotoxic moiety can be a cytotoxic peptide or polypeptide moiety that causes cell death. Cytotoxic peptides and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor, and the like. Methods for conjugating them to targeting moieties such as antibodies are also known in the art. Other ribosome-inactivating proteins are described as cytotoxic agents in WO 96 / 06641. Pseudomonas exotoxin can also be used as a cytotoxic polypeptide. Certain cytokines such as TNFα and IL-2 can also be used as cytotoxic agents.

[0215] Certain radioactive atoms may also be cytotoxic if delivered in sufficient doses. Thus, the cytotoxic moiety may comprise a radioactive atom that, in use, delivers a sufficient amount of radioactivity to the target site to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-111, rhenium-186, rhenium-188, or yttrium-90, or any other isotope that emits sufficient energy to disrupt adjacent cells, organelles, or nucleic acids. Preferably, the isotope and density of the radioactive atom in the agent of the present invention are such that a dose of more than 4000 cGy (preferably at least 6000 cGy, 8000 cGy, or 10000 cGy) is delivered to the target site, and preferably to the cells and their organelles, particularly the cell nucleus, at the target site.

[0216] The radioactive atom can be attached to an antibody, antigen-binding fragment, variant, fusion, or derivative thereof in a known manner. For example, EDTA or another chelator can be attached to the binding moiety and used to attach 111In or 90Y. Tyrosine residues can be directly labeled with 125I or 131I.

[0217] The cytotoxic moiety can be a suitable indirect cytotoxic polypeptide. An indirect cytotoxic polypeptide can be a polypeptide having enzymatic activity and capable of converting a non-toxic and / or relatively non-toxic prodrug into a cytotoxic drug. For antibodies, this type of system is commonly referred to as ADEPT (antibody-directed enzyme prodrug therapy). The system requires the antibody to localize the enzyme moiety to the desired site in the patient, and after a period of time allowing the enzyme to localize at the site, a prodrug that is a substrate for the enzyme is administered, and the final product of the catalysis is a cytotoxic compound. The aim of the method is to maximize the drug concentration at the desired site and minimize the drug concentration in normal tissues. The cytotoxic moiety can be capable of converting a non-cytotoxic prodrug into a cytotoxic drug.

[0218] The enzyme and prodrug using the system targeting an enzyme as described herein can be any of those previously proposed. The cytotoxic substance can be any existing anticancer drug, such as an alkylating agent; an agent that intercalates into DNA; an agent that inhibits any key enzyme such as dihydrofolate reductase, thymidylate synthase, ribonucleotide reductase, nucleoside kinase, or topoisomerase; or an agent that causes cell death by interacting with any other cellular component. Etoposide is an example of a topoisomerase inhibitor.

[0219] The reported prodrug systems include those listed in Table A.

[0220] Table A: Reported prodrug systems.

[0221]

[0222] Suitable enzymes for forming part of the enzyme moiety include: exopeptidases such as carboxypeptidases G, G1 and G2 (for glutamated nitrogen mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs) and aminopeptidases (for 2-α-aminoacyl MTC prodrugs); endopeptidases such as, for example, thrombolysin (for thrombin prodrugs); hydrolases such as phosphatases (e.g., alkaline phosphatase) or sulfatases (e.g., arylsulfatase) (for phosphorylated or sulfated prodrugs); amidases such as penicillin amidase and arylacylamidase; lactamases such as β-lactamase; glycosidases such as β-glucuronidase (for β-glucuronide anthracyclines), α-galactosidase (for amygdalin) and β-galactosidase (for β-galactoanthracyclines); deaminases such as cytosine deaminase (for 5FC); kinases such as urokinase and thymidine kinase (for ganciclovir); reductases such as nitroreductase (for CB1954 and analogues), azoreductase (for azobenzene mustard) and DT-diaphorase (for CB1954); oxidases such as glucose oxidase (for glucose), xanthine oxidase (for xanthine) and lactoperoxidase; DL-racemases, catalytic antibodies and cyclodextrins.

[0223] Preferably, the prodrug is relatively non-toxic compared to the cytotoxic drug. Generally, in a suitable in vitro cytotoxicity test, its toxicity is less than 10%, preferably less than 1%.

[0224] The moiety that is capable of converting the prodrug to the cytotoxic drug is active when separated from the remainder of the agent of the invention, but is only active when (a) it is combined with the remainder of the agent of the invention, and (b) the agent of the invention is attached to, adjacent to, or internalized in the target cell.

[0225] When the moieties are both polypeptides, the two moieties can be linked together by any of the conventional polypeptide cross-linking methods. For example, an antibody or its antigen-binding portion can be enriched in thiol groups, and the other moiety can be reacted with a bifunctional agent capable of reacting with those thiol groups, such as N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Amide and thioether bonds (e.g., obtained with m-maleimidobenzoyl-N-hydroxysuccinimide ester) are generally more stable in vivo than disulfide bonds.

[0226] The cytotoxic moiety can be a radiosensitizer. Radiosensitizers include fluoropyrimidines, thymidine analogs, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3-aminobenzodiamide, etanidazole, pimonidazole, and misonidazole. Additionally, delivering genes into cells can radiosensitize them, such as the delivery of the p53 gene or cyclin D. The additional moiety can be a moiety that becomes cytotoxic or releases a cytotoxic moiety after radiation. For example, the boron-10 isotope releases cytotoxic alpha particles when appropriately irradiated. Similarly, the cytotoxic moiety can be a moiety for photodynamic therapy, such as a photosensitizer.

[0227] "Therapeutically effective amount", "effective amount", or "therapeutically effective" means that a given substance is administered to a subject suffering from the disorder in an amount sufficient to cure, alleviate, or partially inhibit one or more of the condition or its symptoms. Such therapeutic treatment can result in a reduction in the severity of the symptoms of the disease or an increase in the frequency or duration of asymptomatic periods. The effective amount for a given purpose and a given agent will depend on the severity of the disease or injury and the weight and general condition of the subject. This can be a predetermined amount of the active antibody calculated to produce the desired therapeutic effect in relation to the required additives and diluents (i.e., carriers or administration vehicles). Additionally, it is intended to be an amount sufficient to reduce or prevent clinically significant deficiencies in the activity, function, and responses of the host. Alternatively, a therapeutically effective amount is sufficient to improve a clinically significant condition of the host. As understood by those skilled in the art, the amount of the compound can vary depending on its specific activity. A suitable dose can contain a predetermined amount of the active composition calculated to produce the desired therapeutic effect in relation to the required diluent. A therapeutically effective amount can be determined by a ordinarily skilled medical or veterinary worker based on patient characteristics such as age, weight, gender, condition, complications, other diseases, etc., as is well known in the art.

[0228] The optimal dose can be determined by a physician based on many parameters, including for example age, gender, weight, the severity of the condition being treated, the active ingredient being administered, and the route of administration. Generally, a serum concentration of the polypeptide and antibody that allows receptor saturation is desired. A concentration in excess of about 0.1 nM is usually sufficient. For example, a dose of 100 mg / kg of an antibody provides a serum concentration of about 20 nM for about eight days. As a rough guide, the dose of an antibody can be from 10 mg / kg to 300 mg / kg per week.

[0229] The dosage of the composition will depend on the nature of the monoclonal antibody, such as its binding affinity and in vivo plasma half-life, the concentration of the polypeptide in the formulation, the route of administration, the site and rate of dosing, the target organ, the clinical resistance of the patient involved, the pathological condition afflicting the patient, etc., all of which are within the skill of the physician. For example, a dosage of 300 μg of antibody per administration to each patient can be provided, however the dosage can range from about 10 μg to 6 g per dose. Different dosages are used in a series of successive inoculations; the practitioner can perform an initial inoculation and then boost with a relatively smaller dose of the antibody.

[0230] The term "subject" (which may be used interchangeably herein with "patient") includes any animal, including a human, in need of treatment with an antibody or antigen-binding portion thereof of the present invention. The subject or patient can be a mammal or a non-mammal. Preferably, the subject is a mammal, such as a horse, or a cow, or a sheep, or a pig, or a camel, or a dog, or a cat. Most preferably, the mammalian patient is a human.

[0231] In some embodiments, the antibody or antigen-binding portion thereof is formulated and / or adapted for delivery by a route selected from the group consisting of: intravenous; intramuscular; subcutaneous; intracranial; and intraocular. For example, the antibody or antigen-binding portion thereof can be formulated and / or adapted for intravenous (i.e., "i.v" or "IV") delivery.

[0232] In some embodiments, the antibody or antigen-binding portion thereof is delivered to a subject by a route selected from the group consisting of: intravenous; intramuscular; subcutaneous; intracranial; and intraocular. For example, the antibody or antigen-binding portion thereof can be delivered intravenously.

[0233] It is contemplated that injection will be the primary route of therapeutic administration of the composition, however delivery via a catheter or other surgical tube is also used. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal, intradermal, intramuscular, intracranial, and intraocular administration. The liquid formulation can be used after reconstitution from a powder or lyophilized formulation.

[0234] For intravenous injection or injection via other parenteral routes, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity, and stability. Those skilled in the relevant art can readily prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. As needed and as further described herein, preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included.

[0235] The composition can be administered locally or systemically.

[0236] Depending on the route of administration, the antibody or its antigen-binding portion may be coated with a material to protect the agent from acids and other natural conditions that may inactivate or denature the antibody or its antigen-binding portion. Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers for use in the pharmaceutical compositions of the invention include water, buffered water, and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Appropriate fluidity may be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. In many cases, it is preferred to include in the composition an isotonic agent such as sugar, polyols such as mannitol, sorbitol, or sodium chloride.

[0237] Methods and formulations for various routes of administration are well known in the art.

[0238] An antibody or its antigen-binding fragment can be defined by its binding affinity. The term "binding activity" is intended to refer to the tendency of an antibody molecule to bind or not bind to a target. Binding affinity can be quantified by determining the dissociation constant (Kd) of the antibody and its target. Similarly, the specificity of an antibody for its target can be defined according to the relative dissociation constant (Kd) of the antibody for its target, as compared to the dissociation constant of the antibody and another non-target molecule.

[0239] Typically, the Kd of an antibody relative to its target is at least 2-fold lower, preferably 5-fold lower, more preferably 10-fold lower than the Kd relative to other non-target molecules such as irrelevant substances or accompanying substances in the environment. More preferably, the Kd will be at least 50-fold lower, even more preferably at least 100-fold lower, such as at least 200-fold lower, and still more preferably at least 1000-fold lower.

[0240] The value of this dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those presented by, for example, Caceci et al. (Byte 9:340-362, 1984). For example, the Kd can be determined using a double filtration nitrocellulose filter binding assay, such as that disclosed by Wong and Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Other standard assays for assessing the binding ability of a ligand (such as an antibody) to a target are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding kinetics of an antibody (e.g., binding affinity) can also be evaluated by standard assays in the art, such as by surface plasmon resonance (SPR) analysis using a BIAcore TM (SPR) system or by biolayer interferometry (BLI) using an Octet system.

[0241] Competitive binding assays can be performed, in which the binding of an antibody to a target is compared to the binding of another known ligand of the target, such as another antibody, to the target. The concentration at which 50% inhibition occurs is called the Ki. Under ideal conditions, Ki is equivalent to Kd. The Ki value will never be less than Kd, so the measured value of Ki can be conveniently used to provide an upper limit for Kd.

[0242] The antibodies or antigen-binding fragments thereof described herein preferably are capable of binding their target with an affinity that is up to two-fold, 10-fold, 50-fold, 100-fold or greater than their affinity for binding another non-target molecule.

[0243] The antibody can be or can comprise a variant or fragment of one of the specific anti-VEGF-A 165 b antibodies disclosed herein, provided that the variant or fragment retains specificity for VEGF-A 165 b.

[0244] The fragment preferably is the antigen-binding portion of the antibody described herein. Fragments can be prepared by truncation, e.g., by removing one or more amino acids from the N- and / or C-terminus of the polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids can be removed in this manner from the N- and / or C-terminus. Fragments also can be generated by one or more internal deletions.

[0245] The variant can comprise one or more substitutions, deletions or additions relative to the sequence of the specific anti-VEGF-A 165 b antibody disclosed herein. The variant can comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from the specific sequence disclosed herein. A "deletion" variant can comprise the deletion of a single amino acid, a small group of amino acids such as 2, 3, 4 or 5 amino acids, or the deletion of a larger amino acid region, such as the deletion of a specific amino acid domain or other feature. A "substitution" variant preferably involves replacing one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid can be replaced with a replacement amino acid having similar properties, e.g., another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid.

[0246] In some embodiments, a variant comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 substitutions relative to any sequence disclosed herein. In some embodiments, a variant comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 deletions relative to any sequence disclosed herein. In some embodiments, the substitutions and / or deletions are in the variable heavy chain sequence. In some embodiments, the substitutions and / or deletions are in the variable light chain sequence. In some embodiments, the substitutions and / or deletions are in the variable heavy chain sequence and the variable light chain sequence. In some embodiments, the substitutions and / or deletions are not in the CDR sequences of the heavy chain and / or light chain sequences. For example, a variant may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 substitutions in SEQ ID NO: 19, 20, and / or 25. Alternatively or additionally, a variant may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 deletions in SEQ ID NO: 19, 20, and / or 25.

[0247] In some embodiments, a variant comprises or consists of a certain percentage sequence identity with a sequence disclosed herein (e.g., variable heavy chain sequence, variable light chain sequence, and / or CDR sequence). In some embodiments, a variant comprises or consists of 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% sequence identity with a sequence disclosed herein (e.g., variable heavy chain sequence, variable light chain sequence, and / or CDR sequence). In some embodiments, a variant comprises or consists of a certain percentage sequence identity with the variable heavy chain sequence and / or variable light chain sequence while retaining the same CDR sequences as the corresponding variable heavy chain sequence and / or variable light chain sequence, respectively.

[0248] In some embodiments, the antibody or its antigen-binding fragment may comprise a variable light chain sequence selected from SEQ ID NO: 15-22 or an amino acid sequence having at least 60% sequence identity therewith, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the antibody or its antigen-binding fragment may comprise a variable light chain sequence selected from SEQ ID NO: 23-31 or an amino acid sequence having at least 60% sequence identity therewith, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the antibody or its antigen-binding fragment may comprise a CDR sequence selected from SEQ ID NO: 1-6 or an amino acid sequence having at least 60% sequence identity therewith, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the antibody or its antigen-binding fragment may comprise a CDR sequence selected from SEQ ID NO: 7-13 or an amino acid sequence having at least 60% sequence identity therewith, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the antibody or its antigen-binding fragment may comprise the V L CDR1 sequence selected from SEQ ID NO: 14 or an amino acid sequence having at least 60% sequence identity therewith, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the V H CDR1 sequence comprises 1, 2, 3, 4, 5, or 6 substitutions or consists of the same. In some embodiments, the V H CDR2 sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions or consists of the same. In some embodiments, the V H CDR3 sequence comprises 1, 2, 3, or 4 substitutions or consists of the same. In some embodiments, the V L CDR1 sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions or consists of the same. In some embodiments, the V L CDR2 sequence comprises 1, 2, 3, 4, 5, 6, or 7 substitutions or consists of the same. In some embodiments, the V L CDR3 sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions or consists of the same.

[0249] In some embodiments, the antibody or antigen-binding fragment thereof comprises VH4-VL2 (SEQ ID NO:33) or an amino acid sequence having at least 60% sequence identity therewith, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises VH5-VL2 (SEQ ID NO:34) or an amino acid sequence having at least 60% sequence identity therewith, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.

[0250] In some embodiments, the antibody or antigen-binding fragment thereof may comprise a CDR selected from any one or more of the variable heavy chain sequences of SEQ ID NOs: 15-22. In some embodiments, the antibody or antigen-binding fragment thereof may comprise a CDR selected from any one or more of the variable light chain sequences of SEQ ID NOs: 23-31.

[0251] Some properties of the 20 major amino acids that can be used to select suitable substituents are as follows:

[0252]

[0253]

[0254] Preferred "variants" include those in which the amino acids present in the sequence are structural analogs of, rather than the naturally occurring amino acids. The amino acids used in the sequence may also be derivatized or modified, such as being labeled, provided that the function of the antibody is not significantly adversely affected.

[0255] Variants can be prepared during antibody synthesis or by post-production modification, or when the antibody is in recombinant form, using known site-directed mutagenesis techniques, or digestion and / or ligation of nucleic acids.

[0256] The invention also includes within its scope the polypeptides and polynucleotides described herein and sequences that are substantially identical thereto, e.g., having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto. In one embodiment, the CDR sequences of the sequences may be the same, but the variable regions outside the CDRs (e.g., any of the VH and / or VL sequences designated herein) may vary for a given variable sequence, e.g., having at least 70%, 80%, 85%, 90%, 95%, or 99% identity. In one embodiment, the CDR sequences of the sequences may be the same, but for a given framework sequence, the framework regions may vary, e.g., having at least 70%, 80%, 85%, 90%, 95%, or 99% identity. In another embodiment, the framework regions may vary by 1, 2, 3, 4, or 5 amino acid modifications such as substitutions, deletions, or insertions. The variant may retain its target binding function.

[0257] The percent identity between two amino acid sequences or two nucleic acid sequences is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps may be introduced in the first sequence for optimal alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. "Optimal alignment" is an alignment of two sequences that results in the highest percent identity. The percent identity is determined by comparing the number of identical amino acid residues or nucleotides within the sequences (i.e., percent identity = number of identical positions / total number of positions × 100).

[0258] Preferably, the amino acid sequence of the variant antibody has greater than 60%, or greater than 70%, e.g., 75% or 80%, preferably greater than 85%, e.g., greater than 90% or 95% amino acid identity to the VL or VH domain of the antibody disclosed herein. Depending on the size of the full-length polypeptide, this level of amino acid identity may be seen over the full length of the relevant SEQ ID NO sequence or over a portion of the sequence, e.g., over 20, 30, 50, 75, 100, 150, 200, or more amino acids.

[0259] Mathematical algorithms known to those of skill in the art can be used to determine the percentage of identity between two sequences. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990), as modified by Karlin and Altschul (1993). The NBLAST and XBLAST programs of Altschul et al. (1990) have incorporated this algorithm. The NBLAST program can be used to perform BLAST nucleotide searches with a score = 100 and wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. The XBLAST program can be used to perform BLAST protein searches with a score = 50 and wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997). Alternatively, PSI-Blast can be used to perform iterative searches to detect distant relationships between molecules (Id.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program can be used (e.g., XBLAST and NBLAST). See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller. The ALIGN program (version 2.0), which is part of the GCG sequence alignment software package, has incorporated this algorithm. Other sequence analysis algorithms in the art include ADVANCE and ADAM described in Torellis and Robotti (1994); and the FASTA algorithm described in Pearson and Lipman (1988). In FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0260] The antibodies and antigen-binding fragments thereof described herein can be modified by mutagenesis to remove one or more sites of N-linked glycosylation (HCDR2), Met oxidation (HCDR1), Trp oxidation, or Asp isomerization. Advantageously, modifying the antibodies and antigen-binding fragments thereof, e.g., to remove one or more sites of N-linked glycosylation (HCDR2), Met oxidation (HCDR1), Trp oxidation, or Asp isomerization, can avoid defects caused by such post-translational modifications that reduce antigen-binding affinity, lead to product heterogeneity and aggregation, or negatively impact their expression levels during production or manufacturing.

[0261] The residue numbering in this text can be determined by the Kabat numbering system. For example, the heavy chains HC4 or HC5 of the antibodies described herein may contain the mutations N59Q and / or T61A. Equivalent residues may be mutated in any of the heavy chains described herein. Additionally or alternatively, the light chains LC1 or LC2 of the antibodies described herein may contain one or more of the mutations selected from S32A, D33E, G34A, and D60E. Equivalent residues may be mutated in any of the light chains described herein.

[0262]

[0263]

[0264] In one embodiment, the antibody or antigen-binding fragment thereof described herein may be selected from one or more of the following heavy chains, where specific mutations in the heavy or light chains are indicated in parentheses after the relevant chain: HC4(N59Q)LC1, HC4(N59Q)LC2, HC5(N59Q)LC2, HC4(T61A)LC2, HC5(T61A)LC2, HC4 LC1(S32A), HC4LC2(S32A), HC5 LC2(S32A), HC4 LC1(D33E), HC4 LC2(D33E), HC5 LC2(D33E), HC4 LC1(G34A), HC4 LC2(G34A), HC5 LC2(G34A).

[0265] In some embodiments, the antibody or antigen-binding fragment thereof contains HC4(N59Q). In some embodiments, the antibody or antigen-binding fragment thereof contains HC5(N59Q). In some embodiments, the antibody or antigen-binding fragment thereof contains HC4(T61A). In some embodiments, the antibody or antigen-binding fragment thereof contains HC5(T61A). In some embodiments, the antibody or antigen-binding fragment thereof contains LC1(S32A). In some embodiments, the antibody or antigen-binding fragment thereof contains LC2(S32A). In some embodiments, the antibody or antigen-binding fragment thereof contains LC1(D33E). In some embodiments, the antibody or antigen-binding fragment thereof contains LC2(D33E). In some embodiments, the antibody or antigen-binding fragment thereof contains LC1(G34A). In some embodiments, the antibody or antigen-binding fragment thereof contains LC2(G34A). In some embodiments, the antibody or antigen-binding fragment thereof contains LC1(D60E). In some embodiments, the antibody or antigen-binding fragment thereof contains LC2(D60E). In some embodiments, the antibody or antigen-binding fragment thereof contains HC4(D270A). In some embodiments, the antibody or antigen-binding fragment thereof contains HC4(P329A). In some embodiments, the antibody or antigen-binding fragment thereof contains HC4(P331S).

[0266] In some embodiments, the antibody or antigen-binding fragment thereof comprises a combination of any of the foregoing mutations. For example, in some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) HC4 (N59Q) and LC1 (S32A); (ii) HC4 (N59Q) and LC1 (D33E); (iii) HC4 (N59Q) and LC1 (G34A); (iv) HC4 (N59Q) and LC1 (D60E); (v) HC4 (T61A) and LC1 (S32A); (vi) HC4 (T61A) and LC1 (D33E); (vii) HC4 (T61A) and LC1 (G34A); or (viii) HC4 (T61A) and LC1 (D60E). In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) HC5 (N59Q) and LC1 (S32A); (ii) HC5 (N59Q) and LC1 (D33E); (iii) HC5 (N59Q) and LC1 (G34A); (iv) HC5 (N59Q) and LC1 (D60E); (v) HC5 (T61A) and LC1 (S32A); (vi) HC5 (T61A) and LC1 (D33E); (vii) HC5 (T61A) and LC1 (G34A); or (viii) HC5 (T61A) and LC1 (D60E).

[0267] In some embodiments, a linker is present between the HC domain and the LC domain. For example, in some embodiments, the antibody or its antigen-binding fragment comprises a domain combination selected from the list consisting of: SEQ ID NO:129 + linker + SEQ ID NO:132; SEQ ID NO:130 + linker + SEQ ID NO:132; SEQ ID NO:104 + linker + SEQ ID NO:132; SEQ ID NO:105 + linker + SEQ ID NO:132; SEQ ID NO:106 + linker + SEQ ID NO:132; SEQ ID NO:107 + linker + SEQ ID NO:132; SEQ ID NO:129 + linker + SEQ ID NO:133; SEQ ID NO:130 + linker + SEQ ID NO:133; SEQ ID NO:104 + linker + SEQ ID NO:133; SEQ ID NO:105 + linker + SEQ ID NO:133; SEQ ID NO:106 + linker + SEQ ID NO:133; SEQ ID NO:107 + linker + SEQ ID NO:133; SEQ ID NO:129 + linker + SEQ ID NO:111; SEQ ID NO:130 + linker + SEQ ID NO:111; SEQ ID NO:104 + linker + SEQ ID NO:111; SEQ ID NO:105 + linker + SEQ ID NO:111; SEQ ID NO:106 + linker + SEQ ID NO:111; SEQ ID NO:107 + linker + SEQ ID NO:111; SEQ ID NO:129 + linker + SEQ ID NO:112; SEQ ID NO:130 + linker + SEQ ID NO:112; SEQ ID NO:104 + linker + SEQ ID NO:112; SEQ ID NO:105 + linker + SEQ ID NO:112; SEQ ID NO:106 + linker + SEQ ID NO:112; SEQ ID NO:107 + linker + SEQ ID NO:112; SEQ ID NO:129 + linker + SEQ ID NO:113; SEQ ID NO:130 + linker + SEQ ID NO:113; SEQ ID NO:104 + linker + SEQ ID NO:113; SEQ ID NO:105 + linker + SEQ ID NO:113; SEQ ID NO:106 + linker + SEQ ID NO:113;SEQ ID NO:107 + linker + SEQ ID NO:113; SEQ ID NO:129 + linker + SEQ ID NO:114; SEQ ID NO:130 + linker + SEQ ID NO:114; SEQ ID NO:104 + linker + SEQ ID NO:114; SEQ ID NO:105 + linker + SEQ IDNO:114; SEQ ID NO:106 + linker + SEQ ID NO:114; SEQ ID NO:107 + linker + SEQ ID NO:114; SEQ ID NO:129 + linker + SEQ ID NO:115; SEQ ID NO:130 + linker + SEQ ID NO:115; SEQ IDNO:104 + linker + SEQ ID NO:115; SEQ ID NO:105 + linker + SEQ ID NO:115; SEQ ID NO:106 + linker + SEQ ID NO:115; SEQ ID NO:107 + linker + SEQ ID NO:115; SEQ ID NO:129 + linker + SEQ ID NO:116; SEQ ID NO:130 + linker + SEQ ID NO:116; SEQ ID NO:104 + linker + SEQ IDNO:116; SEQ ID NO:105 + linker + SEQ ID NO:116; SEQ ID NO:106 + linker + SEQ ID NO:116; SEQ ID NO:107 + linker + SEQ ID NO:116; SEQ ID NO:129 + linker + SEQ ID NO:117; SEQ IDNO:130 + linker + SEQ ID NO:117; SEQ ID NO:104 + linker + SEQ ID NO:117; SEQ ID NO:105 + linker + SEQ ID NO:117; SEQ ID NO:106 + linker + SEQ ID NO:117; SEQ ID NO:107 + linker + SEQ ID NO:117; SEQ ID NO:129 + linker + SEQ ID NO:118; SEQ ID NO:130 + linker + SEQ IDNO:118; SEQ ID NO:104 + linker + SEQ ID NO:118; SEQ ID NO:105 + linker + SEQ ID NO:118; SEQ ID NO:106 + linker + SEQ ID NO:118; and SEQ ID NO:107 + linker + SEQ ID NO:118.;

[0268] In some embodiments, the heavy chain is selected from one of SEQ ID NO: 99-107. In some embodiments, the light chain is selected from one of SEQ ID NO: 108-118. Any combination of the heavy chain and the light chain can be used to generate an antibody or an antigen-binding fragment thereof.

[0269] Abrogating the Fc function of antibodies can be beneficial, for example, in situations where receptor agonists are used to crosslink receptors and induce signal transduction, or receptor antagonists are used to block receptor:ligand interactions to prevent signal transduction. Binding of Fcγ receptors on effector cells or binding of C1q may be undesirable because it can lead to the unwanted killing of biologically important cells expressing the receptor through ADCC or CDC. A single mutation of Leu235Glu in the CH2 domain of the Fc portion of human IgG1 was found to be sufficient to knock out binding to Fc receptors on U937 cells (Wines et al., 2000, The IgG fc contains distinct fc receptor (fcr) binding sites: the leukocyte receptors fcγri and fcγriia bind to a region in the fc distinct from that recognized by neonatal fcr and protein a. J Immunol, May 15, 2000, 164(10)5313-5318). After this initial mutation was discovered, the combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) was found to abrogate FcγRIIa binding (Lund et al., 1991, Human FcγRI and FcγRII interact with distinct but overlapping sites on human IgG, J. Immunol. 147:2657; Lund et al., 1992, Multiple binding sites on the CH2 domain of IgG for mouse FcγRII, Mol. Immunol. 29:53). These two mutations were later shown to abrogate detectable binding of IgG1 and IgG4 to FcγRI, IIa, and IIIa (Sarmay et al., 1992, Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fcγ receptor, Mol. Immunol. 29:633).Other mutations are known in the art, such as in Saunders K.O Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life 2019 Front. Immunol., 07 June 2019 Sec. Comparative Immunology.

[0270] Thus, in one embodiment, where the antibody or antigen-binding fragment thereof comprises an Fc portion, the antibody or antigen-binding fragment thereof according to the invention may comprise a LALA (SEQ ID NO: 121) modification in the CH2 domain of the Fc portion. The antibody according to the invention may comprise Leu234Ala and Leu235Ala modifications, or their equivalent residues (e.g., equivalent Leu residues that may not be numbered 234 or 235 respectively) in the CH2 domain of the Fc portion.

[0271] In one embodiment where the antibody or antigen-binding fragment thereof comprises an Fc portion, the antibody or antigen-binding fragment thereof according to the invention may comprise an Fc portion that is mutated to reduce the cytotoxic off-target effect on specialized epithelial cells in the eye and kidney due to the presence of surface-bound VEGF-A 165 b. For example, the Fc region may be selected from the group consisting of SEQ ID NO: 164 - 166. Exemplary HC4 sequences having such Fc mutations may be selected from the group consisting of SEQ ID NO: 161 - 163.

[0272] With respect to amino acid sequences, "sequence identity" means a sequence having the stated value when evaluated using ClustalW (Thompson et al., 1994, supra) with the following parameters:

[0273] Pairwise alignment parameters - method: exact, matrix: PAM, gap opening penalty: 10.00, gap extension penalty: 0.10;

[0274] Multiple alignment parameters - matrix: PAM, gap opening penalty: 10.00, delayed divergence %: 30, end gap penalty: on, gap separation distance: 0, negative matrix: no, gap extension penalty: 0.20, residue specific gap penalty: on, hydrophilic gap penalty: on, hydrophilic residues: GPSNDQEKR (SEQ ID NO: 120). Sequence identity at a particular residue is intended to include simply derived identical residues.

[0275] The anti-VEGF-A of the invention 165A b antibody can bind to the same epitope as the specific antibodies disclosed herein because such an antibody may mimic the action of the disclosed antibodies. Whether an antibody binds to the same epitope as another antibody can be determined by conventional methods. For example, the binding of each antibody to the target can be carried out using a competitive binding assay. Methods for performing competitive binding assays are well known in the art. For example, they may involve contacting the antibody and the target molecule under conditions where the antibody can bind to the target molecule. Then, the antibody / target complex can be contacted with a second (test) antibody, and the extent to which the test antibody is able to displace the first antibody from the antibody / target complex can be evaluated. Such evaluation can be carried out using any suitable technique, including, for example, surface plasmon resonance, ELISA, or flow cytometry techniques. The ability of the test antibody to inhibit the binding of the first antibody to the target indicates that the test antibody can compete with the first antibody for binding to the target, and thus the test antibody binds to the same epitope or region on the target as the first antibody, and thus can mimic the action of the first antibody.

[0276] The present invention also provides a kit comprising an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or a pharmaceutical composition according to the second aspect of the present invention. For example, the kit may comprise (a) a therapeutically effective amount of an antibody or an antigen-binding portion thereof that specifically binds to VEGF-A as described herein 165 b.

[0277] The kit of the present invention may additionally comprise one or more other reagents or instruments capable of performing any of the examples mentioned above. Such reagents or instruments include one or more of the following: a suitable buffer (aqueous solution) and a tool for administering the anti-VEGF-A 165 b antibody (such as a container or instrument containing a needle). The kit may include instructions for performing the combination therapy or method described herein.

[0278] The anti-VEGF-A 165 b antibody described herein or provided in the kit of the present invention can be provided as a pharmaceutical composition formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc., which are physiologically compatible and also compatible with the desired route of administration.

[0279] The pharmaceutical composition may include a pharmaceutically acceptable antioxidant. These compositions may also contain excipients and adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The presence of microorganisms can be prevented by the above sterilization procedures and by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be necessary to include isotonic agents such as sugars, sodium chloride, and the like in the composition. In addition, extended absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption such as aluminum monostearate and gelatin.

[0280] The pharmaceutical composition must generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the required amount of the active agent (e.g., antibody) into a suitable solvent having one or a combination of the above-listed ingredients as required, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active drug plus any additional required ingredients from a previously sterile-filtered solution. The pharmaceutical composition may contain additional active ingredients as well as those mentioned above.

[0281] Suitable pharmaceutically acceptable buffers, diluents, carriers, and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A.Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are hereby incorporated by reference herein).

[0282] The term "buffer" is intended to include an aqueous solution containing an acid-base mixture for the purpose of stabilizing the pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole acetate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

[0283] The term "diluent" is intended to include an aqueous or non-aqueous solution for the purpose of diluting the agent in a pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or an oil such as safflower oil, corn oil, peanut oil, cottonseed oil, olive oil or sesame oil.

[0284] The term "adjuvant" is intended to include any compound added to the formulation to increase the biological effect of the agent of the present invention. The adjuvant may be one or more of zinc, copper or silver salts with different anions, such as fluoride, chloride, bromide, iodide, cyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate and acetate of different acyl compositions. The adjuvant may also be a cationic polymer such as cationic cellulose ether, cationic cellulose ester, deacetylated hyaluronic acid, chitosan, cationic dendrimer, cationic synthetic polymer such as poly(vinylimidazole) and cationic polypeptides such as polyhistidine, polylysine, polyarginine and peptides containing these amino acids.

[0285] Excipients can be one or more of carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrin, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinylpyrrolidone, all of which have different molecular weights and are added to the composition, for example, for viscosity control, for achieving bioadhesion, or for protecting lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, monoglycerides, diglycerides, and triglycerides, ceramides, sphingolipids, and glycolipids, all of which have different acyl chain lengths and degrees of saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, and they are added to the composition for reasons similar to polymers. Examples of minerals are talc, magnesium oxide, zinc oxide, and titanium oxide, which are added to the composition to obtain benefits such as reducing fluid accumulation or favorable pigment properties.

[0286] The active antibody-based agent of the present invention can be formulated into any type of pharmaceutical composition suitable for its delivery in the art.

[0287] In one embodiment, the pharmaceutical composition of the present invention can be in the form of liposomes, in which, in addition to other pharmaceutically acceptable carriers, the agent is combined with an amphiphilic agent such as a lipid, which exists in aggregated forms such as micelles, insoluble monolayers, and liquid crystals. Lipids suitable for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysophosphatidylcholine, phospholipids, saponins, bile acids, etc. Suitable lipids also include the above-mentioned lipids modified with poly(ethylene glycol) in the polar head group for extending the blood circulation time. The preparation of such liposome formulations can be found, for example, in US 4,235,871.

[0288] The pharmaceutical composition of the present invention can also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA), or poly(caprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in microsphere production. The preparation of such microspheres can be found in US 5,851,451 and EP 0213 303.

[0289] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of nanoparticles, such as based on poly-γ-glutamic acid. Details of the preparation and use of such nanoparticles can be found in WO 2011 / 128642. Those skilled in the art will understand that one or more of the active ingredients in the combination therapy of the present invention can be formulated in separate nanoparticles, or two active ingredients can be formulated in the same nanoparticles.

[0290] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of a polymer gel, wherein polymers such as starch, cellulose ethers, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, polyvinyl imidazole, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinyl pyrrolidone are used to thicken the solution containing the medicament. The polymer may also contain gelatin or collagen.

[0291] Alternatively, the medicament can simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, or an oil (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil), gum tragacanth, and / or various buffers.

[0292] It should be understood that the pharmaceutical composition of the present invention can include ions and a defined pH value for enhancing the action of the active agent. Additionally, the composition can be subjected to conventional pharmaceutical operations, such as sterilization, and / or can contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.

[0293] According to another aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or a pharmaceutical composition according to the second aspect of the present invention, for treating or preventing ischemia in a subject.

[0294] According to another aspect of the present invention, there is provided the use of an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or a pharmaceutical composition according to the second aspect of the present invention, in the manufacture of a medicament for treating or preventing ischemia in a subject.

[0295] According to another aspect of the present invention, there is provided a method for treating or preventing ischemia, the method comprising administering to a subject in need thereof an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or a pharmaceutical composition according to the second aspect of the present invention.

[0296] Ischemia may be a result of peripheral artery disease (PAD) (also known as peripheral arterial disease). In one embodiment, the disease to be treated or prevented is a disease associated with ischemia. In one embodiment, the disease to be treated or prevented is peripheral artery disease (PAD) and / or a condition associated with PAD.

[0297] Ischemia may be selected from cardiac or coronary ischemia, intestinal ischemia, cerebral ischemia, limb ischemia, retinal ischemia, renal ischemia, and related skin diseases such as cyanosis and gangrene.

[0298] According to another aspect of the invention, there is provided a monoclonal antibody of the invention for treating or preventing a disease in which VEGF-A 165 b is elevated relative to VEGF-A 165 a, said diseases including but not limited to myocardial infarction (MI) including ST-elevation MI, angina, systemic sclerosis / scleroderma, Raynaud's syndrome, ulcerative colitis, Crohn's disease, inflammatory bowel disease, diabetic retinopathy, diabetic nephropathy, painful diabetic neuropathy, diabetic neuropathy, stroke, preeclampsia, hypertension, obesity, or hair loss.

[0299] According to another aspect of the invention, there is provided the use of an antibody or an antigen-binding fragment thereof according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention in the manufacture of a medicament for treating or preventing a disease in which VEGF-A 165 b is elevated relative to VEGF-A 165 a, said diseases including but not limited to myocardial infarction (MI), including ST-elevation MI, angina, systemic sclerosis / scleroderma, Raynaud's syndrome, ulcerative colitis, Crohn's disease, inflammatory bowel disease, diabetic retinopathy, diabetic nephropathy, painful diabetic neuropathy, diabetic neuropathy, stroke, preeclampsia, hypertension, obesity, or hair loss.

[0300] According to another aspect of the invention, there is provided a method of treating or preventing a disease in which VEGF-A 165 b is elevated relative to VEGF-A 165 a, said diseases including but not limited to myocardial infarction (MI), including ST-elevation MI, angina, systemic sclerosis / scleroderma, Raynaud's syndrome, ulcerative colitis, Crohn's disease, inflammatory bowel disease, diabetic retinopathy, diabetic nephropathy, painful diabetic neuropathy, diabetic neuropathy, stroke, preeclampsia, hypertension, obesity, or hair loss, said method comprising administering to a subject in need thereof an antibody or an antigen-binding fragment thereof according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention.

[0301] A convenient way to generate the antibodies or antigen-binding portions according to the present invention is to use nucleic acids in an expression system to express the nucleic acids encoding them.

[0302] Accordingly, in a further aspect, the present invention provides a nucleic acid comprising a sequence encoding an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or its heavy and / or light chains.

[0303] The present invention further provides an isolated nucleic acid encoding a specific monoclonal antibody of the present invention. The nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid encoding a specific antibody or antigen-binding portion of the present invention as defined above. Those skilled in the art will be able to determine substitutions, deletions, and / or additions to these nucleic acids, which will still provide an antibody or its antigen-binding portion according to the present invention.

[0304] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes, which comprise at least one nucleic acid as described above. The present invention also provides recombinant host cells comprising one or more of the above constructs. As mentioned above, the nucleic acid encoding the antibody or antigen-binding portion of the present invention forms one aspect of the present invention, as does the method for its production, which includes expression from its encoding nucleic acid. Expression can be conveniently achieved by culturing the recombinant host cells containing the nucleic acid under suitable conditions. After production by expression, any suitable technique can be used to isolate and / or purify the specific monoclonal antibody, which can then be used as needed.

[0305] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and insect cell (baculovirus) systems. Mammalian cell lines that can be used for expressing heterologous polypeptides in the art include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, and many others. Those skilled in the art can obtain expression in cultured eukaryotic cells as an option for generating specific monoclonal antibodies, see, for example, Frenzel A, Hust M, Schirrmann T. Expression of recombinant antibodies. Front Immunol. 2013 Jul 29;4:217. doi:10.3389 / fimmu.2013.00217. PMID:23908655; PMCID:PMC3725456.

[0306] A suitable vector can be selected or constructed, which contains suitable regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other suitable sequences. The vector can be a plasmid, virus, such as a phage or phagemid, as required. For further details, see, for example, Sambrook et al., (1989). Many known techniques and protocols for manipulating nucleic acids (e.g., in the preparation of nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression, and protein analysis) are described in detail in Ausubel et al., (1992).

[0307] Accordingly, in another aspect of the present invention, there is provided a cell comprising the aforementioned nucleic acid. In one embodiment, the cell is a mammalian cell, such as a CHO or HEK cell. An antibody or an antigen-binding fragment thereof can be recombinantly expressed in any one of the cells selected from CHO, NS0, Sp2 / 0, HEK293, and PER.C6.

[0308] Another aspect of the present invention provides a host cell containing the nucleic acid disclosed herein. In one embodiment, the nucleic acid of the present invention is integrated into the genome (e.g., chromosome) of the host cell. According to standard techniques, integration can be facilitated by including sequences that promote recombination with the genome.

[0309] In a further aspect, the present invention provides a method for preparing a specific antibody or antigen-binding fragment of the present invention, the method comprising expressing the nucleic acid under conditions that result in the expression of the antibody or antigen-binding fragment in a cell, and recovering the antibody or antigen-binding fragment.

[0310] The specific antibody or antigen-binding fragment according to the present invention can be used in a method for treating a human or animal body, such as a method for treating ischemia in a patient (preferably a human), the method comprising administering to the patient an effective amount of the specific antibody or antigen-binding fragment of the present invention.

[0311] Example

[0312] Example 1: Monoclonal sequencing

[0313] mRNA was extracted from the hybridoma cell pellet. Total RNA was extracted from the pellet using the Fusion Antibody Pie in-house RNA extraction protocol. cDNA was reverse transcribed from the RNA using an oligo(dT) primer. PCR reactions were set up using variable domain primers to amplify the VH and VL regions of the monoclonal antibody DNA. Agarose gel electrophoresis of the amplified PCR products was as Figure 1 shown.

[0314] The VH and VL products were cloned into the Thermo Fisher sequencing vector pCR TMIn 2.1, the first 10 cells were transformed with the cloned plasmid, and positive transformants were screened by PCR. The selected colonies were picked and subjected to DNA sequencing analysis on an ABI 3130xI genetic analyzer. The results can be seen in Figure 2 , and its sequence is replicated as follows.

[0315] Heavy chain

[0316] ● VH2.2 protein (SEQ ID NO:38)

[0317] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPPVYP

[0318] ● VH2.2 nucleic acid (SEQ ID NO:39)

[0319] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTTTATCCT

[0320] ● VH2.4 protein (SEQ ID NO:40)

[0321] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYP

[0322] ● VH2.4 Nucleic Acid (SEQ ID NO:41)

[0323] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTCTATCCC

[0324] ● VH2.5 Protein (SEQ ID NO:42)

[0325] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWRSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLA

[0326] ● VH2.5 Nucleic Acid (SEQ ID NO:43)

[0327] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGAGGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGTAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTCTATCCCTTGGCC

[0328] ● VH2.7 protein (SEQ ID NO:44)

[0329] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKLIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF

[0330] ● VH2.7 nucleic acid (SEQ ID NO:45)

[0331] GAGGTGAAGCTTCTCGAATCTGGAGGTGGTCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTAATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTTGTCAAAGGTTATTTC

[0332] ● VH2HB.1 protein (SEQ ID NO:46)

[0333] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKELEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPPVYPLA

[0334] ● VH2HB.1 nucleic acid (SEQ ID NO:47)

[0335] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGAGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTTTATCCATTGGCC

[0336] ● VH2HB.2 protein (SEQ ID NO:48)

[0337] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF

[0338] ● VH2HB.2 nucleic acid (SEQ ID NO:49)

[0339] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCCGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAAGGTTATTTC

[0340] ● VH2HB.3 protein (SEQ ID NO:48)

[0341] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF

[0342] ● VH2HB.3 nucleic acid (SEQ ID NO:50)

[0343] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTTGTCAAAGGTTATTTC

[0344] ● VH2HB.4 protein (SEQ ID NO:51)

[0345] EVKLLESGGGLVQPGGSPKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF

[0346] ● VH2HB.4 nucleic acid (SEQ ID NO:52)

[0347] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCCGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAAGGTTATTTC

[0348] ● VH2HB.5 protein (SEQ ID NO:53)

[0349] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPPVYPLV

[0350] ● VH2HB.5 nucleic acid (SEQ ID NO:54)

[0351] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTCTATCCCTTGGTC

[0352] ● VH2HB.6 protein (SEQ ID NO:48)

[0353] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF

[0354] ● VH2HB.6 nucleic acid (SEQ ID NO:55)

[0355] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGTTATTTC

[0356] ● VH4HB.1 protein (SEQ ID NO:56)

[0357] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDSAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF

[0358] ● VH4HB.1 nucleic acid (SEQ ID NO:57)

[0359] GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAGCGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTC

[0360] ● Consensus sequence (SEQ ID NO:58) (positions in parentheses indicate potential substitutions at those positions, X = blank)

[0361] EVKLLESGGGLVQPGGS(L / P)KLSCAASGFDFSRYW(M / R)SWVRQAPGK(G / E)LEWIGEIHPYSSTINYTPSVKDK(F / L)I ISRD(N / S)AKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPP(S / P)VYP(L / X)(A / V / X)(P / X)(G / X)(S / X)(A / X)(A / X)(Q / X)(T / X)(N / X)(S / X)(M / X)(V / X)(T / X)(L / X)(G / X)(C / X)(L / X)(V / X)(K / X)(G / X)(Y / X)(F / X)

[0362] ● Consensus sequence, with the most common amino acid indicated at each position (SEQ ID NO:48) (variable regions are highlighted in bold, sequencing primers are underlined, and CDRs are double underlined)

[0363]

[0364] The Complementary Determining Regions (CDRs) marked with double underlines are determined by the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-272 (1999)). The consensus amino acid sequence from VH sequencing is obtained from at least 5 amino acid sequences, as shown in the alignment in Figure 2 below. Figure 3 Figure 1 shows a graphical representation of the variable domain labeled with IMGT.

[0365] Light chain

[0366] ● VL2.1 protein (SEQ ID NO: 137)

[0367] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF

[0368] ● VL2.1 nucleic acid (SEQ ID NO: 149)

[0369] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCT

[0370] ● VL2.2 protein (SEQ ID NO: 138)

[0371] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVC

[0372] ● VL2.2 nucleic acid (SEQ ID NO:150)

[0373] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGC

[0374] ● VL2.3 protein (SEQ ID NO:139)

[0375] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK

[0376] ● VL2.3 nucleic acid (SEQ ID NO:151)

[0377] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCCGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAA

[0378] ● VL2.6 protein (SEQ ID NO:140)

[0379] DIVMTQTPLTLSVTIGQPASISCKSSQCLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR

[0380] ● VL2.6 nucleic acid (SEQ ID NO:152)

[0381] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGTGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACGCTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGA

[0382] ● VL2.7 protein (SEQ ID NO:141)

[0383] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR

[0384] ● VL2.7 nucleic acid (SEQ ID NO:153)

[0385] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGA

[0386] ● VL2.8 protein (SEQ ID NO:142)

[0387] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR

[0388] ● VL2.8 nucleic acid (SEQ ID NO:154)

[0389] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGA

[0390] ● VL2HB.2 protein (SEQ ID NO:143)

[0391] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK

[0392] ● VL2HB.2 nucleic acid (SEQ ID NO:155)

[0393] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAA

[0394] ● VL2HB.3 protein (SEQ ID NO:144)

[0395] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK

[0396] ● VL2HB.3 nucleic acid (SEQ ID NO:156)

[0397] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAA

[0398] ● VL2HB.4 protein (SEQ ID NO:145)

[0399] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR

[0400] ● VL2HB.4 nucleic acid (SEQ ID NO:157)

[0401] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGA

[0402] ● VL2HB.7 protein (SEQ ID NO:146)

[0403] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR

[0404] ● VL2HB.7 nucleic acid (SEQ ID NO:158)

[0405] GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGA

[0406] ● Consensus sequence (SEQ ID NO:147) (Positions in parentheses indicate potential alternatives at those positions)

[0407] D(I / V)VMTQ(T / S)PL(T / S)(L / S)(S / P)VT(I / L / P)GQPASISC(K / R)SSQSLLDSDGKTYLNW(L / F / E / Y)(L / Q)Q(R / K)PGQ(S / P)P(K / R / Q)(R / L)LIYLVSKLDSGVPDRF(T / S)GSG(S / A)GTDFTLKISRVE(A / P)ED(L / V)GVYYCWQGTHFPYTFG(G / Q)GTK(V / L)E(I / V)KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR

[0408] ● Consensus sequence, where the most common amino acids are indicated at each position (SEQ ID NO:148) (Variable regions are highlighted in bold, sequencing primers are underlined, and CDRs are double underlined)

[0409]

[0410] The CDRs indicated by double underlining are determined by the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-272 (1999)). The consensus amino acid sequence from VH sequencing is obtained from at least 5 amino acid sequences, as Figure 4 shown in the alignment in Figure 5 Figure 4 shows a graphical representation of the variable domain marked by IMGT.

[0411] Example 2: Transient expression, purification and affinity screening of the humanized anti-VEGF-A165b clone mα165b (also referred to herein as 56 / 8 / 31 / 1 / 16) variant (25+1) of Ab 1126

[0412] Summary

[0413] The humanized variant of Ab 1126 is a 150 kilodalton (kDa) antibody that contains two heavy chains and two light chains, complexed together via disulfide bonds. Mammalian expression vectors encoding each variant were transfected into CHO cells, and batch cultures of each variant were grown for up to seven days. The expressed antibodies were purified from the cell culture supernatant via affinity chromatography. The concentration and purity of the purified antibody products were determined. The results of these quality control experiments are detailed below.

[0414] Expression and purification of Ab 1126

[0415] DNA encoding the variant amino acid sequence of Ab 1126 was synthesized (see sequences below) and cloned into the mammalian transient expression plasmid pETE V2 (characteristics of the fusion antibody). The Ab1126 variant was expressed using a CHO-based transient expression system, and the resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. The Ab 1126 variant was purified from the cell culture supernatant via affinity chromatography (using state-of-the-art AKTA chromatography equipment). The purified antibody was dialyzed / buffer exchanged into phosphate-buffered saline solution. The purity of the antibody was determined to be >95% as judged by reducing and denaturing sodium dodecyl sulfate polyacrylamide gel Figures 6 to 10 ). The antibody concentration was determined by measuring the absorbance at 280 nm, and the standard extinction coefficient of the antibody 205,500 M -1 cm -1 (or 1.0 mg / ml = 1.37 A280 [assuming MW = 150,000 Da]) was used to calculate the antibody concentration. The details of the purified antibody products provided are summarized in Table 1; and Figure 35 summarizes the antibody sequences derived from the parental hybridoma.

[0416] Ab 1126 variant sequence (the signal peptides for expressing the heavy and light chains of the antibody are underlined and can be excluded from the heavy and light chain sequences):

[0417] >Ab 1126HC0 (SEQ ID NO:71, with signal peptide)

[0418] MGWTLVFLFLLSVTAGVHS EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0419] >Ab 1126HC0 (SEQ ID NO:125, without signal peptide)

[0420] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0421] >Ab 1126HC1(SEQ ID NO:72, with signal peptide)

[0422] MGWTLVFLFLLSVTAGVHS EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0423] >Ab 1126HC1 (SEQ ID NO:126, without signal peptide)

[0424] EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0425] >Ab 1126HC2 (SEQ ID NO:73, with signal peptide)

[0426] MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFIISRDNAKNSVYLQLNSLRAEDTAVYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0427] >Ab 1126HC2 (SEQ ID NO:127, without signal peptide)

[0428] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFIISRDNAKNSVYLQLNSLRAEDTAVYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0429] >Ab 1126HC3 (SEQ ID NO:74, with signal peptide)

[0430] MGWTLVFLFLLSVTAGVHS EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWISEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRDEDTALYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0431] >Ab 1126HC3 (SEQ ID NO:128, without signal peptide)

[0432] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWISEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRDEDTALYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0433] >Ab 1126HC4(SEQ ID NO:75, with signal peptide)

[0434] MGWTLVFLFLLSVTAGVHS EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0435] >Ab 1126HC4 (SEQ ID NO:129, without signal peptide)

[0436] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0437] >Ab 1126HC5 (SEQ ID NO:76, with signal peptide)

[0438] MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0439] >Ab 1126HC5(SEQ ID NO:130, without signal peptide)

[0440] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0441] >Ab 1126LC0 (SEQ ID NO:77, with signal peptide)

[0442] MVSSAQFLGLLLLCFQGTRC DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0443] >Ab 1126LC0 (SEQ ID NO:131, without signal peptide)

[0444] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0445] >Ab 1126LC1 (SEQ ID NO:78, with signal peptide)

[0446] MVSSAQFLGLLLLCFQGTRC DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0447] >Ab 1126LC1 (SEQ ID NO:132, without signal peptide)

[0448] DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0449] >Ab 1126LC2(SEQ ID NO:79, with signal peptide)

[0450] MVSSAQFLGLLLLCFQGTRC DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0451] >Ab 1126LC2(SEQ ID NO:133, without signal peptide)

[0452] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0453] >Ab 1126LC3(SEQ ID NO:80, with signal peptide)

[0454] MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYCWQGTHFPYTFGGGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0455] >Ab 1126LC3(SEQ ID NO:134, without signal peptide)

[0456] DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYCWQGTHFPYTFGGGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0457] >Ab 1126LC4(SEQ ID NO:81, with signal peptide))

[0458] MVSSAQFLGLLLLCFQGTRC DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGKTYLNWLQQRPGQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0459] >Ab 1126LC4(SEQ ID NO:135, without signal peptide)

[0460] DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGKTYLNWLQQRPGQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0461] >Ab 1126LC5(SEQ ID NO:82, with signal peptide))

[0462] MVSSAQFLGLLLLCFQGTRC DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0463] >Ab 1126LC5(SEQ ID NO:136, without signal peptide)DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0464] Table 1: Purification summary: Ab 1126 variants. Abbreviations are as follows: ND = not determined. *Purity was determined by analysis of (relative band intensity) of Coomassie blue-stained reducing and denaturing SDS-PAGE.

[0465] Sample Concentration (mg / ml) Volume (ml) Total (mg) Potential yield (mg / L) *Purity HC0 LC0 0.64 1.66 1.06 42.40 ≥95％ HC1 LC1 0.77 1.70 1.31 52.40 ≥95％ HC1 LC2 0.58 1.71 0.99 39.60 ≥95％ HC1 LC3 0.72 1.74 1.25 50.00 ≥95％ HC1 LC4 0.71 1.74 1.24 49.60 ≥95％ HC1 LC5 1.00 1.79 1.79 71.60 ≥95％ HC2 LC1 1.04 1.83 1.90 76.00 ≥95％ HC2 LC2 0.89 1.93 1.72 68.80 ≥95％ HC2 LC3 0.63 2.96 1.89 75.60 ≥95％ HC2 LC4 0.99 1.89 1.87 74.80 ≥95％ HC2 LC5 0.72 1.86 1.36 53.60 ≥95％ HC3 LC1 1.05 1.97 2.07 82.80 ≥95％ HC3 LC2 0.85 1.99 1.69 67.60 ≥95％ HC3 LC3 0.57 1.35 0.77 51.30 ≥95％ HC3 LC4 0.50 1.50 0.75 30.00 ≥95％ HC3 LC5 0.86 1.47 1.26 50.40 ≥95％ HC4 LC1 0.89 1.57 1.40 56.00 ≥95％ HC4 LC2 1.30 1.68 2.18 87.20 ≥95％ HC4 LC3 1.00 1.79 1.79 71.60 ≥95％ HC4 LC4 0.73 1.88 1.39 54.80 ≥95％ HC4 LC5 0.87 1.61 1.40 56.00 ≥95％ HC5 LC1 1.13 1.90 2.15 86.00 ≥95％ HC5 LC2 0.93 1.91 1.78 71.20 ≥95％ HC5 LC3 0.84 1.61 1.35 54.00 ≥95％ HC5 LC4 0.81 1.81 1.47 58.80 ≥95％ HC5 LC5 0.79 1.82 1.44 57.60 ≥95％

[0466] Kinetic analysis: Summary

[0467] The IgG antibody was immobilized on the biosensor using a suitable capture surface, and the binding of soluble antigen to the immobilized antibody was monitored by BLI (Octet). The resulting sensorgrams were analyzed using the software provided by the manufacturer (Fortebio).

[0468] Kinetic analysis: Materials and methods

[0469] Antigen: Human VEGF165b, Sino Biological, Catalog No. 29656-HNAB, Lot No. LC13JU1402

[0470] **Note** VEGF165b usually exists as a disulfide-linked homodimer

[0471] Details of the test antibody:

[0472] ● Antibody identifier = HCX LCX

[0473] ● Species = Humanized

[0474] ● Capture method = Anti-human IgG Fc (AHC)

[0475] Kinetic analysis: Optimization

[0476] A series of experiments were initially performed to optimize the analysis parameters (data not shown) to obtain the following parameters:

[0477] ● Summary of optimized kinetic screening parameters for characterizing IgG:antigen interactions

[0478] Antibody loading concentration 0.15μg / ml Antigen stability at 25°C, shaken at 1000 rpm <4 hours Association / dissociation time 900 / 1200 seconds Required dynamic screening protocol Modification Antigen screening range (seven concentrations, diluted 3 - fold) 30 - 0.0412 nM Interaction / fitting model 1:1

[0479] Based on the optimization experiments using a chimeric control antibody, when using a standard IgG capture level (0.5 - 1.0 nm), the dissociation rate (kd) of the antigen was observed to be very slow (<1.0x 10 -4 )

[0480] Kinetic analysis: Experimental parameters

[0481] Kinetic analysis was performed by first capturing IgG using an anti-human Fc biosensor. Then the mAb capture biosensor was immersed in wells containing different concentrations of antigen (association phase), followed by a dissociation step in an electrophoresis buffer. To perform reference correction, the IgG-captured sensor was immersed in a well containing only buffer. This reference provided a method to compensate for the natural dissociation of the captured IgG. These steps were performed at a constant flow rate of 1000 rpm at 25°C. A new sensor was used for each sample. The dissociation rate constant (K D)。All consumables used are those recommended by ForteBio.

[0482] Kinetic analysis: Results for Ab 1126 variants

[0483] All samples were diluted in freshly prepared electrophoresis buffer. Antibody variants were immobilized on the surface of a series of biosensors using the capture method described (see Materials and Methods). Antigen was passed over the surface to generate a binding reaction. Binding data for the IgG:antigen interaction were collected on the biosensor at 25 °C. A dilution series of antigen was used in the association step to fit the results globally and obtain the best values for ka, kd, and K D . The reaction data for antigen binding to surface-immobilized IgG were fit to a 1:1 binding model, generating a data trace (red - see Figure 11). Kinetic parameters are summarized in Table 2 below.

[0484] Table 2: Kinetic parameters for the interaction of Ab 1126 variants with antigen (fit to a 1:1 interaction model).

[0485]

[0486] R 2 values indicate the degree of correlation between the fit and the experimental data, and values above 0.95 are considered a good fit; X 2 is the sum of the squared deviations and should generally be below 3 (ideally <0.5). X 2 is a measure of the error between the experimental data and the fit line. X 2 The smaller the X max , the better the fit. The average R D should be similar (±20%) to the value for the chimeric control. K

[0487] Conclusion

[0488] All Ab 1126 variants have been successfully expressed and purified.

[0489] According to SDS-PAGE analysis, all antibodies were expressed with sufficient purity. Under denaturing and reducing conditions, the heavy and light chains of the antibody were visible and were observed at the expected molecular weights of ~50 and 25 kDa, respectively. Under denaturing and non-reducing conditions, one major band and several minor bands were observed. The additional bands (impurities) may be the result of non-glycosylated IgG and IgG degradation products (e.g., combinations of single [partial] light chains, two heavy chains and one light chain, two heavy chains, two heavy chains and one light chain).

[0490] Kinetics (Octet) analysis: Both HCX LC1 and HCX LC2 showed binding to the antigen, and in most cases, the experimental data fit very well to a 1:1 binding model. Under the experimental conditions used here, HC5 LC2 and HC4 LC2 showed binding characteristics similar to those of the chimeric control antibody HC0 LC0, with dissociation constants within 2-fold of the chimeric HC0 LC0 control antibody.

[0491] Example 3: Transient expression of Ab 1126 variants HC0 LC0, HC4 LC2, and HC5 LC5 (20 mg increase)

[0492] Summary

[0493] The Ab 1126 variants HC0 LC0, HC4 LC2, and HC5 LC2 are 150 kilodalton (kDa) antibodies that contain two heavy chains and two light chains complexed together by disulfide bonds. Mammalian expression vectors encoding each antibody were transfected into CHO cells. Subsequently, the expressed antibodies were purified from the cell culture supernatant using standard chromatography techniques. The concentration, purity, and endotoxin of the purified antibody products were determined.

[0494] Expression and purification of Ab 1126 variants

[0495] DNA encoding the amino acid sequences of each antibody was synthesized and cloned into the mammalian transient expression plasmid pETEV2 (characteristics of the fusion antibody). A CHO-based transient expression system was used to express the antibodies, and the resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. The antibodies were purified from the cell culture supernatant via affinity chromatography and preparative size exclusion chromatography (SEC column pre-equilibrated in phosphate-buffered saline) using state-of-the-art AKTA chromatography equipment. The purified antibody was buffer-exchanged into phosphate-buffered saline solution. The purity of the antibody was determined to be >95% as judged by reducing and denaturing sodium dodecyl sulfate polyacrylamide gel Figures 12 to 14 ).

[0496] Antibody samples were analyzed by size exclusion chromatography (SEC). The chromatograms each showed a single major peak (>95% of the total area Figures 15 to 17 ). Using -PTS system and PTS cassette (Charles River Laboratories), the bacterial endotoxin levels were determined.

[0497] The antibody concentration was determined by measuring the absorbance at 280 nm, and the antibody concentration was calculated using the standard extinction coefficient of the antibody, 205,500 M-1cm-1 (or A280 of 1.0 mg / ml = 1.37 [assuming MW = 150,000 Da]). Details of the purified antibody products provided are summarized in Table 3.

[0498] Table 3: Purification summary: Ab 1126 variant.

[0499]

[0500] *Purity was determined by analysis of (relative band intensity) of reducing and denaturing SDS polyacrylamide gels stained with Coomassie blue.

[0501] Conclusion

[0502] The antibody was successfully expressed and purified.

[0503] SEC analysis was successfully performed on all antibodies. The chromatogram showed a single major peak (>98% of total area).

[0504] According to SDS-PAGE analysis, all antibodies were expressed with sufficient purity under reducing and denaturing conditions. Under reducing conditions, both the heavy and light chains of the antibody were visible and were observed at the expected molecular weights of ~50 and 25 kDa, respectively. Under non-reducing conditions, one major band and several minor bands were observed. The additional bands (impurities) may be the result of non-glycosylated IgG and IgG degradation products (e.g., combinations of single [partial] light chains, two heavy chains and one light chain, two heavy chains, two heavy chains and one light chain).

[0505] Example 4: Evaluation of the applicability of a novel humanized monoclonal antibody against VEGF-A (VEGF-A 165 b) specific isoform as an antibody therapy for peripheral arterial disease

[0506] Abbreviations and Acronyms

[0507]

[0508]

[0509]

[0510] Background

[0511] Peripheral artery disease (PAD) is a major unmet clinical need that afflicts approximately 10 million people in the United States. Vascular insufficiency in PAD results in tissue ischemia. In response, circulating monocytes produce vascular endothelial growth factor (VEGF-A). Patients with PAD and murine models of metabolic ischemia have reduced soluble frizzled-related protein 5 (sfrp5), leading to increased Wnt5a activity, resulting in overexpression of the anti-angiogenic isoform VEGF-A 165 b. It has previously been shown that an antibody against VEGF-A 165 b can reverse impaired angiogenesis in a murine model of hindlimb ischemia. Lower limb ischemia causes pain and disability, creates non-healing ulcers, and results in 200,000 amputations annually in the United States alone. Insufficient angiogenesis and collateral vessel formation exacerbate limb ischemia caused by arterial obstructive lesions.

[0512] Recent studies have shown that the anti-angiogenic splice variant of VEGF-A, VEGF-A 165 b, is expressed in clinical and experimental settings associated with impaired vascular formation 1 . VEGF-A 165 b is a competitive inhibitor of VEGF-A 165 a-mediated angiogenesis. Circulating VEGF-A in PAD patients predominantly consists of the VEGF-A 165 b isoform. In murine experimental models, mVEGF-A 165 b expression is upregulated in the context of systemic metabolic dysfunction. In experimental models of PAD 2 , the VEGF-A 165 b isoform impairs angiogenesis, and acute antibody-mediated neutralization of the mVEGF-A 165 b isoform promotes angiogenesis in ischemic tissues in the setting of impaired regenerative angiogenesis. These findings support that VEGF-A 165 b can serve as a new pharmacological target for treating limb ischemia in PAD patients, and its neutralization can enhance the activity of pro-angiogenic growth factors 3 .

[0513] Introduction

[0514] The aim was to develop the first humanized monoclonal antibody against the VEGF-A isoform VEGF-A 165 b for the treatment of PAD. By immunizing BALB / c mice with a synthetic peptide fragment of the nine-amino acid carboxyl-terminal sequence of VEGF-A 165 b, murine monoclonal antibodies specific for VEGF-A 165 b were generated, where the last six (CDKPRR) are specific for VEGF-A 165b is specific and conjugated to keyhole limpet hemocyanin (KLH). 4 One of the resulting hybridomas specifically detected VEGF-A 165 b, rather than VEGF-A 165 a. Twenty-five humanized variants of a murine anti-VEGF-A 165 b neutralizing mAb were generated. The variants were transiently expressed in CHO cells and purified from the culture supernatant by single-column protein A affinity chromatography.

[0515] As part of this study, wild-type C57BL6 mice were fed a high-fat diet for 12 weeks and underwent left femoral artery ligation. Before and after resection of the left femoral artery, and on days 3, 7, 14, 21, and 28 postoperatively, blood flow to the paw was measured by Moor FLPI-2 laser speckle imaging. Animals received humanized anti-VEGF-A 165 b (1 mg / kg, i.p.) or a control antibody (human IgG) on days 0, 3, and 7 (see schematic in Figure 25 ). The ratio of ischemic to non-ischemic LDBF was calculated and plotted against time to determine blood flow recovery. Muscle was stained to measure capillary and arteriole density. Animals with less than 60% blood flow inhibition were excluded. ZDF diabetic rats were treated similarly and blood flow was measured.

[0516] Results

[0517] The affinities of the variants for a commercially available VEGF-A 165 b protein were ranked by biolayer interferometry (BLI). The affinities (KD) of two variants, HC4 LC2 and HC5 LC2, were within two-fold of that of the HC0 LC0 chimeric control antibody (KD of 600, 600, and 300 pM, respectively), and the new antibodies showed >100x higher affinity for VEGF 165 b than the original murine antibody, with a slower off-rate and comparable on-rate, see Figure 11.

[0518] In addition, the exemplary antibody clone HC4 LC2 was able to reduce the affinity of VEGF 165 b for the VEGF receptor 2 (VEGFR2) to a degree slightly less than that of the positive control of G6-31 (an antibody that binds the receptor binding site) (see Figure 21 B).

[0519] These two humanized variants were also able to more effectively block VEGF-A 165 b-mediated inhibition of cell migration in vitro compared to the parental murine monoclonal and chimeric antibodies (see Figure 22 and Figure 37 , and Figure 23)。In addition, the humanized variant was also able to reverse the inhibition of endothelial cell migration mediated by human monocytes in diabetic peripheral arterial disease (PAD) (see Figure 24 B).

[0520] Further studies showed that at 1 μg / ml HC4 LC2, the inhibition of endothelial cell (using human umbilical vein cells as an example) transwell migration driven by human monocytes was significantly reversed (see Figure 33A). Monocytes from 7 patients with peripheral vascular disease inhibited migration towards VEGF-A 165 a (see Figure 33B). Migration increased at 1 μg / ml (see Figure 33C) and 5 μg / ml (see Figure 33D) HC4 LC2.

[0521] Blood flow recovery in IgG-treated mice did not return to normal after 28 days (66 ± 6% on day 14 of the contralateral side, 29 ± 8.7% on day 21, 75 ± 8% on day 28, N = 6). The humanized anti-VEGF-A 165 b antibody rescued this impaired angiogenesis (78 ± 5% on day 14, 90 ± 4.6% on day 21, 102 ± 3.5% on day 28, N = 7), see Figure 26 and Figure 27 . In addition, improvement in angiogenesis and arteriogenesis was observed in HFHS obese mice (see Figures 28A to 28C).

[0522] This effect was also observed in diabetic rats, the schematic diagram of which is shown in Figure 29 ; the resulting blood flow recovery is shown in Figure 30 and Figure 31 ; and improvement in angiogenesis and arteriogenesis in Figures 32A to 32C.

[0523] Conclusion

[0524] Obesity leads to impaired collateralization and insufficient angiogenesis in the PAD model. The humanized anti-VEGF-A 165 b reversed this phenomenon, indicating that impaired angiogenesis in PAD patients can be treated with this antibody.

[0525] Example 5: Humanization of anti-VEGF-A165b mα165b

[0526] The sequences of the heavy chain (HC) variable domain (mVH) and the light chain (LC) variable domain (mVL) of the parental murine monoclonal antibody (clone 58 / 8 / 31 / 1 / 13) are as follows:

[0527] Amino acid sequence of clone 58 / 8 / 31 / 1 / 13 mVH (SEQ ID NO:15)

[0528] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA

[0529] Amino acid sequence of clone 58 / 8 / 31 / 1 / 13 mVL (SEQ ID NO:23)

[0530] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR

[0531] Blast analysis of the IMGT database against the human antibody sequence 6 It has been disclosed that the heavy chain CDRs (HCDR - SEQ ID NO:1 - 3) from a mouse antibody (IGHV4 - 1*02 - see Blast 1 below) have been transplanted into the human IGHV3 - 11 framework (see Blast 2 below).

[0532] Blast 1 (Blast of the HC1 VH region against the IMGT database shows 82% identity with the closest mouse germline):

[0533] >9323AJ851868 Mus musculus IGHV4 - 1*02 VH

[0534] Length = 98

[0535] Score = 179 bits (453), Expect = 5e - 47

[0536] Identity = 81 / 98 (82%), Positive = 94 / 98 (95%)

[0537] Query: 1EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPG

[0538] KGLEWVSEIHPYSSTINY 60

[0539] EV+LLESGGGLV+PGGSL+LSCAASGFDFSRYWMSW+RQAPGKGLEW+EI+PSSTINY

[0540] Subject: 1EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPG

[0541] KGLEWIGEINPDSSTINY 60

[0542] Query: 61TPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 98(SEQ ID NO:83)

[0543] TPS+KD+F ISRDNAKN+LYLQM++R+EDTA+YYCAR(SEQ ID NO:122)

[0544] Subject: 61TPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR 98(SEQ ID NO:84)

[0545] Blast 2 (Blast of the HC1 VH region against the IMGT database shows 86% identity with the closest human germline):

[0546] >2883X92287 Homo sapiens IGHV3-11*03 VH

[0547] Length = 98

[0548] Score = 169 bits (429), Expect = 3e-44

[0549] Identity = 85 / 98 (86%), Positives = 87 / 98 (88%)

[0550] Query: 1EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPG

[0551] KGLEWVSEIHPYSSTINY 60

[0552] +VQLLESGGGLVKPGGSLRLSCAASGF FS Y+MSWIRQAPGKGLEWVS ISS NY

[0553] Subject: 1QVQLLESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG

[0554] KGLEWVSYISSSSSYTNY 60

[0555] Query: 61TPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 98 (SEQ ID NO:83)

[0556] SVK RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:123)

[0557] Sbjct: 61ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 98 (SEQ ID NO:85)

[0558] The Vκ light chain of the parental antibody belongs to the IGKV1-135*01 family. Blast analysis of the IMGT database of human antibody sequences 7 revealed that the light chain CDR from the mouse antibody has been transplanted into the human IGKV2-30 framework (see Blast 3 below).

[0559] Blast 3 (LC1 VH region blast against the IMGT database showed 91% identity with the closest human antibody VH sequence (IGKV2-30*02):

[0560] >11075FM164408 Homo sapiens IGKV2-30*02 V-κ

[0561] Length = 100

[0562] Score = 186 bits (472), Expect = 3e-49

[0563] Identity = 91 / 100 (91%), Positives = 93 / 100 (93%)

[0564] Query: 1DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQ

[0565] RPGQSPRRLIYLVSKLD 60

[0566] DVVMTQSPLSLPVTLGQPASISC+SSQSL+SDG TYLNWFQQRPGQSPRRLIY VSD

[0567] Sbjct: 1DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQ

[0568] RPGQSPRRLIYKVSNRD 60

[0569] Query: 61SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHEP 100 (SEQ ID NO:86)

[0570] SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QGTH P (SEQ ID NO:124)

[0571] Sbjct: 61SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWP 100 (SEQ ID NO:87)

[0572] Humanized check

[0573] Check the humanized variants to determine if they have been humanized according to the WHO definition of humanized antibodies 11 : The variable domain of the humanized chain has a V-region amino acid sequence which, when analyzed as a whole, is more similar to humans than to other species (evaluated using the ImmunoGenetics information DomainGapAlign tool). 12 The results are shown in Table 5. All variants can be considered humanized. The approved INN name of the candidate therapeutic is visobizumab.

[0574] Table 5: WHO-designated antibody INNs for murine and humanized variants

[0575]

[0576] # Equally close to humans as mice

[0577] ** Equally close to humans as macaques

[0578] Characterization of antibody

[0579] Twenty-five humanized full-length antibody variants, named HCX LCX (X = 1 - 5), were generated by combining the light chains (LC1 - 5) of each variant with the heavy chains (HC1 - 5) of each variant. In addition, a chimeric version of the antibody (named HC0 LC0) was prepared by transplanting the full-length murine VH and VL domains onto the human IgG1 heavy chain constant domain (CH) and the human κLC constant domain (CL), respectively. The antibodies were transiently expressed in CHO cells, purified from the culture supernatant by protein A affinity chromatography, and dialyzed / buffer-exchanged into PBS for in vitro testing.

[0580] Except for HC1 LC1 and HC3 LC4, the antibody yields (in mg / L) were equivalent to or higher than those of the chimeric antibody HC0LC0. Antibodies expressed with LC3, LC4, or LC5 light chains generally had yields that were approximately 16% lower than those of LC1 and LC2 antibodies (n = 1). By SDS-PAGE, all antibodies appeared intact, with some degradation products or contaminants observed for HC1 LC2, HC1 LC4, HC3LC4, HC4 LC2, and HC4 LC5 on non-reducing gels.

[0581] Using the Fortebio Octet system, kinetic analysis of each of the antibodies was performed by biolayer interferometry (BLI). Antibodies were captured on anti-human Fc biosensors and then incubated with commercially available human VEGF-A 165 b. The capture levels of each antibody on the Fc biosensor were comparable. Kinetic analysis showed that all HCX LC1 and HCX LC2 antibodies expressed binding characteristics similar to those of the chimeric control antibody HC0 LC0. Using a 1:1 binding model, it was not possible to determine the ka and kd values of the other antibodies. Under the assay conditions, the affinity of HC0 LCO (0.3 nM) for binding to VEGF-A 165 b was 2x higher than that of HC4 LC2 and HC5 LC2 (0.6 nM) and 3x higher than that of HC4 LC1 (0.9 nM). Antibodies with affinities > 1 nM were HC1 LC1 (5.0 nM), HC1 LC2 (2.1 nM), HC2 LC1 (4.4 nM), HC2 LC2 (3.6 nM), HC3 LC1 (1.5 nM), HC3 LC2 (1.17 nM), and HC5 LC1 (3.2 nM).

[0582] Subsequent increases were made to HC0 LC0, HC4 LC2, and HC5 LC2. Antibodies were transiently expressed in CHO cells and purified from the culture supernatant by protein A affinity chromatography. The purified mAb buffer was exchanged into PBS and analyzed by SDS-PAGE and size exclusion chromatography. By non-reducing SDS-PAGE, the purity was estimated to be > 95%. By SEC (n = 1), a single peak corresponding to the monomer fraction (> 98%) was detected for all antibodies with similar retention times. The endotoxin content was measured to be less than 1.0 EU / mg, meeting the standards for in vivo administration.

[0583] In vitro and in vivo tests

[0584] In the endothelial cell migration assay, when tested at 1 μg / ml, VEGF-A 165The rank order of the neutralizing potency of b's anti-angiogenic effect was HC4 LC2 > HC5 LC2 > HC0 LC0 > mAb 58 / 6. For HC0 LC0, HC4 LC2, and HC5 LC2, the inhibition of VEGF-A 165 The IC50 values (% activity) of b were 442 ng / ml, 137 ng / ml, and 114 ng / ml, respectively.

[0585] When administered at 1 mg / kg i.p. on days 1, 3, and 7 after ischemia, the chimeric antibody HC0 LC0 was able to reverse obesity-dependent impaired angiogenesis in a murine femoral artery ligation ischemia model for up to 30 days. The humanized antibody has not been tested in this model. Navarro et al. recently also demonstrated proof of concept 14 . In a reperfused myocardial infarction model, in vivo blockade of VEGF-A 165 b increased angiogenesis, reduced infarct size, and enhanced systolic function, but not in a non-reperfused myocardial infarction model.

[0586] References

[0587] 1. Cébe-Suarez, S., Zehnder- A. and Ballmer-Hofer, K. The role of VEGFreceptors in angiogenesis; complex partnerships. Cell. Mol. Life Sci. 63, 601-615 (2006).

[0588] 2. Kikuchi, R. et al. An antiangiogenic isoform of VEGF-Acontributes toimpaired vascularization in peripheral artery disease. Nat. Med. 20, 1464-1471 (2014).

[0589] 3. Carter, J.G. et al. The carboxyl terminus of VEGF-A is a potentialtarget for anti-angiogenic therapy. Angiogenesis 18, 23-30 (2015).

[0590] 4. Woolard, J. et al. VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression. Cancer Res. 64, 7822 - 7835 (2004).

[0591] 5. Beck, A. and Liu, H. Macro - and Micro - Heterogeneity of Natural and Recombinant IgG Antibodies. (2019). doi:10.3390 / antib8010018

[0592] 6. Lefranc, M.-P. IMGT, the international ImMunoGeneTics information system, http: / / imgt.cines.fr. Novartis Found. Symp. 254, 126 - 142, 216 - 222, 250 - 252 (2003).

[0593] 7. Lefranc, M.-P. et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V - like domains. Dev. Comp. Immunol. 27, 55 - 77 (2003).

[0594] 8. Jefferis, R. Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action. Trends Pharmacol. Sci. 30, 356 - 362 (2009).

[0595] 9. van de Bovenkamp, F. S. et al. Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability. Front. Immunol. 9, 740 (2018).

[0596] 10. Jarasch, A. et al. Developability assessment during the selection of novel therapeutic antibodies. J. Pharm. Sci. 104, 1885 - 1898 (2015).

[0597] 11. Jones, T. D. et al. The INNs and outs of antibody nonproprietary names. MAbs 8, 1 - 9 (2016).

[0598] 12. Ehrenmann, F., Kaas, Q. and Lefranc, M.-P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF. Nucleic Acids Res. 38, D301 - 7 (2010).

[0599] 13. Paul, S. et al. Development and validation of a broad scheme for prediction of HLA class II restricted T cell epitopes. J. Immunol. Methods 422, 28 - 34 (2015).

[0600] 14. Ríos-Navarro, C. et al. Role of antiangiogenic VEGF-A(165)b in angiogenesis and systolic function after reperfused myocardial infarction. Rev. Esp. Cardiol. (Engl. Ed). 74, 131 - 139 (2021).

[0601] 15. Haberger, M. et al. Functional assessment of antibody oxidation by native mass spectrometry. MAbs 7, 891 - 900 (2015).

[0602] 16. Brader, M.L. et al. Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies. Mol. Pharm. 12, 1005 - 1017 (2015).

[0603] 17. Igawa, T. et al. Engineering the variable region of therapeutic IgG antibodies. MAbs 3, 243 - 252 (2011).

[0604] 18. Liu, H., Saxena, A., Sidhu, S.S. and Wu, D. Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds. Front Immunol 8, 38 (2017).

[0605] 19. Temel, D. B., Landsman, P., and Brader, M. L. Orthogonal Methods for Characterizing the Unfolding of Therapeutic Monoclonal Antibodies: Differential Scanning Calorimetry, Isothermal Chemical Denaturation, and Intrinsic Fluorescence with Concomitant Static Light Scattering. Methods Enzymol. 567, 359 - 389 (2016).

[0606] 20. Chai, Q., Shih, J., Weldon, C., Phan, S., and Jones, B. E. Development of a high - throughput solubility screening assay for use in antibody discovery. MAbs 11, 747 - 756 (2019).

[0607] 21. Bhirde, A. et al. High Performance Size Exclusion Chromatography and High - Throughput Dynamic Light Scattering as Orthogonal Methods to Screen for Aggregation and Stability of Monoclonal Antibody Drug Products. J. Pharm. Sci. 109, 3330 - 3339 (2020).

[0608] 22. Avery, L. B. et al. Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics. MAbs 10, 244 - 255 (2018).

[0609] 23. Schlothauer, T. et al. Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies. MAbs 5, 576 - 586 (2013).

[0610] 24. Kraft, T. E. et al. Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis. MAbs 12, 1683 - 432 (2020).

[0611] 25. Nugent, P., Duncan, J. N. and Colagiovanni, D. B. The Preparation of a Preclinical Dossier to Support an Investigational New Drug (IND) Application and First - in - Human Clinical Trial. A Comprehensive Guide to Toxicology in Preclinical Drug Development 309 - 334 (Elsevier Inc., 2013). doi:10.1016 / B978 - 0 - 12 - 387815 - 1.00012 - 5

[0612] 26. Avery, L. B. et al. Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies. MAbs 8, 1064 - 1078 (2016).

[0613] 27. Kuppuswamy et al., Targeting Anti-Angiogenic VEGF165b-VEGFR1 Signaling Promotes Nitric Oxide Independent Therapeutic Angiogenesis in Preclinical Peripheral Artery Disease Models, Cells 2022, 11, 2676.

[0614] Example 6: Sequences of Site-Directed Mutagenesis

[0615] Overview

[0616] The chimeric version HC0 LC0, an anti-VEGF-A 165 b mAb, containing the VH and VL of the original murine antibody (Ab 1126), was generated. Twenty-five humanized variants were generated, and the binding affinities of HC4 LC2 and HC5 LC2 were within 2X of the chimeric antibody. Many sequence defects were detected in both the VH and VL of the humanized variants. The next step in humanized antibody development was to attempt to remove potential sequence defects by site-directed mutagenesis of the VH and VL regions without significant loss of affinity or potency compared to the chimeric antibody.

[0617] In addition, mutations in the Fc region of IgG1 will also be tested: LALA = L234A L235A*, to remove Fc receptor binding, and YTE = M252Y S254T Y256* for extended half-life. The chimeric antibody HC0 LC0 was tested together with only the YTE and LALA+YTE variants to ensure that the changes in Fc did not affect the binding of the antibody to the target. The sequence defect mutants were first tested in the wild-type IgG1 form to identify tolerated mutations (without loss of binding or functional activity), and then another set of antibody variants combining HC and LC mutations was prepared. (*Specification number.)

[0618] List of expressed antibodies:

[0619] ● Heavy chain Light chain

[0620] ● HC0 YTE LC0

[0621] ● HC0 LALA+YTE LC0

[0622] ● HC4 YTE LC2

[0623] ● HC5 YTE LC2

[0624] ● HC4 N59Q LC1

[0625] ●HC5 N59Q LC2

[0626] ●HC4 N59Q LC2

[0627] ●HC4 T61A LC2

[0628] ●HC5 T61A LC2

[0629] ●HC4 LC1 S32A

[0630] ●HC4 LC2 S32A

[0631] ●HC5 LC2 S32A

[0632] ●HC4 LC1 D33E

[0633] ●HC4 LC2 D33E

[0634] ●HC5 LC2 D33E

[0635] ●HC4 LC1 G34A

[0636] ●HC4 LC2 G34A

[0637] ●HC5 LC2 G34A

[0638] ●HC0 LC0

[0639] ●HC4 LC1

[0640] ●HC4 LC2

[0641] ●HC5 LC2

[0642] Antibodies were cloned, expressed, and purified at the milligram scale. The parameters measured included: yield, SDS-PAGE (reducing and non-reducing), analytical SEC, and binding affinity for VEGF-A 165 b compared to parental antibodies (HC0 LC0, HC4 LC2, HC5 LC2, and HC4 LC1).

[0643] Antibody sequences without signal peptides

[0644] Residues to be mutated are shown underlined (Fc domain) or double underlined (variable domain).

[0645] >HC0(SEQ ID NO:99)

[0646] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPS

[0647] VKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAP

[0648] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0649] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LL GGPSVFLFPPKPKDTL M I S R T

[0650] PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0651] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0652] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0653] GK

[0654] >HC0 YTE(SEQ ID NO:100)

[0655] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPS

[0656] VKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAP

[0657] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0658] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTL Y I T R E

[0659] PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0660] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0661] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0662] GK

[0663] >HC0 LALA YTE(SEQ ID NO:101)

[0664] EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPS

[0665] VKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAP

[0666] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0667] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTL Y I T R E

[0668] PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0669] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0670] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0671] GK

[0672] Heavy chain mutant:

[0673] >HC4 YTE(SEQ ID NO:102)

[0674] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTI N Y T PS

[0675] VKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAP

[0676] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0677] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LL GGPSVFLFPPKPKDTL Y I T R E

[0678] PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0679] KCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0680] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0681] GK

[0682] >HC5 YTE(SEQ ID NO:103)

[0683] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTI N Y T PS

[0684] VKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAP

[0685] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0686] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LL GGPSVFLFPPKPKDTL Y I T R E

[0687] PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0688] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0689] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0690] GK

[0691] >HC4 N59Q(SEQ ID NO:104)

[0692] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTI Q YTPS

[0693] VKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAP

[0694] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0695] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LL GGPSVFLFPPKPKDTL M I S R T

[0696] PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0697] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0698] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0699] GK

[0700] >HC5 N59Q(SEQ ID NO:105)

[0701] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTI Q YTPS

[0702] VKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAP

[0703] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0704] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LL GGPSVFLFPPKPKDTL M I S R T

[0705] PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0706] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0707] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0708] GK

[0709] >HC4 T61A (SEQ ID NO:106)

[0710] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINY A PS

[0711] VKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSSASTKGPSVFPLAP

[0712] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0713] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LL GGPSVFLFPPKPKDTL M I S R T

[0714] PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0715] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0716] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0717] GK

[0718] >HC5 T61A (SEQ ID NO:107)

[0719] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINY A PS

[0720] VKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSAASTKGPSVFPLAP

[0721] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0722] GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LL GGPSVFLFPPKPKDTL M I S R T

[0723] PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0724] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0725] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0726] GK

[0727] Light chain mutant:

[0728] >LC0(SEQ ID NO:108)

[0729] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGV

[0730] PDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPS

[0731] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0732] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0733] >LC1(SEQ ID NO:109)

[0734] DVVMTQSPLSLPVTLGQPASISCKSSQSLL DSDG KTYLNWFQQRPGQSPRRLIYLVSKL D SGV

[0735] PDRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPS

[0736] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0737] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0738] >LC2(SEQ ID NO:110)

[0739] DVVMTQSPLSLPVTLGQPASISCRSSQSLL DSDG KTYLNWFLQRPGQSPRRLIYLVSKL D SGV

[0740] PDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKRTVAAPSVFIFPPS

[0741] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0742] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0743] >LC1 S32A(SEQ ID NO:111)

[0744] DVVMTQSPLSLPVTLGQPASISCKSSQSLLD A DGKTYLNWFQQRPGQSPRRLIYLVSKLDSGV

[0745] PDRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPS

[0746] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0747] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0748] >LC2 S32A(SEQ ID NO:112)

[0749] DVVMTQSPLSLPVTLGQPASISCRSSQSLLD A DGKTYLNWFLQRPGQSPRRLIYLVSKLDSGV

[0750] PDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKRTVAAPSVFIFPPS

[0751] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0752] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0753] >LC1 D33E(SEQ ID NO:113)

[0754] DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSE GKTYLNWFQQRPGQSPRRLIYLVSKLDSGV

[0755] PDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPS

[0756] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0757] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0758] >LC2 D33E(SEQ ID NO:114)

[0759] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDS E GKTYLNWFLQRPGQSPRRLIYLVSKL D SGV

[0760] PDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKRTVAAPSVFIFPPS

[0761] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0762] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0763] >LC1 G34A(SEQ ID NO:115)

[0764] DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSD A KTYLNWFQQRPGQSPRRLIYLVSKLDSGV

[0765] PDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPS

[0766] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0767] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0768] >LC2 G34A(SEQ ID NO:116)

[0769] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSD A KTYLNWFLQRPGQSPRRLIYLVSKLDSGV

[0770] PDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKRTVAAPSVFIFPPS

[0771] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0772] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0773] >LC1 D60E(SEQ ID NO:117)

[0774] DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRL IYLVSKL E SGV

[0775] PDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKRTVAAPSVFIFPPS

[0776] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0777] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0778] >LC2 D60E(SEQ ID NO:118)

[0779] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFLQRPGQSPRRLIYLVSKL E SGV

[0780] PDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKRTVAAPSVFIFPPS

[0781] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[0782] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0783] Example 7: Anti-VEGF 165 b antibody inhibits VEGF 165 Binding of b to VEGFR2

[0784] Method

[0785] Coat a 96-well clear microplate (high-sensitivity thermo immulon or costar 9018) overnight with 1 μg / μl rh-Fc-VEGFR2 (357-KD-050 / CF, R&D Systems) at 100 μl per well, shaking at room temperature. Anti-VEGF 165 b antibody / VEGF 165 b mixture consists of 4 ng / ml VEGF-A 165 b and increasing concentrations of each body, and incubate at 4 °C for 2 hours. Wash the VEGFR2-coated plate three times with 0.05% tween20 in PBS (PBST). Block the plate with Suberblock buffer and wash 3 times. Add 100 μl of anti-VEGF 165 b antibody / VEGF 165 b mixture, and incubate for 1 hour, then wash 3 times. Add 100 μl of biotinylated anti-VEGF-A (BAF293, R&D Systems) in 1% BSA / PBS and incubate at room temperature for 2 hours. Wash it 3 times, then incubate with 100 μl of streptavidin-HRP in filtered 1% BSA / PBS. Cover the plate with foil and shake at room temperature for 30 minutes. After washing 3 times, add the tetramethylbenzidine (TMB) substrate until color develops. Terminate the reaction with 50 μl of 1M HCL. Measure the optical density using a microplate analyzer set at 450 nm, with a reference wavelength of 620 nm.

[0786] Results

[0787] To determine the neutralizing effect of the antibody, bind VEGFR2 to the ELISA plate, and the anti-VEGF-A 165 b antibody and rhVEGF-A165 b was mixed at increasing concentrations of the VEGF-A 165 b antibody. Both the humanized (HC4LC2) and chimeric (HC0LC0) anti-VEGF 165 b antibodies were able to significantly reduce the VEGF 165 b affinity for the VEGF receptor 2 (VEGFR2), also to a slightly lesser extent than the positive control of G6-31 (a pan-VEGF antibody that binds to the VEGF-A receptor binding site)( Figure 36 ). These results support the findings discussed in Example 4.

[0788] Example 8: Additional Sequences of Site-Directed Mutagenesis

[0789] Background

[0790] Induction of the complement cascade is associated with adverse reactions at the site of antibody injection and off-target cell lysis, also known as complement-dependent cytotoxicity (CDC). Interaction of the Fc domain of an antibody that recognizes a target on the cell surface can lead to the binding of the complement factor C1q, which in turn can activate the complement cascade and mediate complement-dependent cytotoxicity (CDC), resulting in the destruction of the antibody-bound cells. C1q binding is the first step in the initiation of the complement cascade. C1q forms a complex with the serine proteases C1r and C1s to form the C1 complex. C1q is capable of binding six antibodies, however, binding of two IgGs is sufficient to activate the complement cascade. Thus, elimination of C1q binding to Fc - the initial event in antibody-dependent complement cytotoxicity activation - can be achieved by site-directed mutagenesis of specific residues in the antibody Fc domain.

[0791] Context

[0792] Vesobizumab is a humanized IgG1 antibody. It is known that IgG1 activates complement. However, activation requires the antibody to cluster on the cell surface via interaction with its specific target (receptor) expressed on the cell surface. Theoretically, vesobizumab should not fix complement because its target VEGF-A 165 b is a soluble molecule. However, non-specific binding of VEGF-A may occur by binding to heparan sulfate proteoglycans (HSPG) expressed on the surface of non-immune cells such as endothelial cells, epithelial cells, and fibroblasts. Different from the pro-angiogenic form of VEGF-A (VEGF-A 165 a), it has been reported (data not shown) that the VEGF-A 165 b splice variant binds little or no heparin (although in fact it is predicted to contain a heparin-binding domain), so it should not bind to heparan sulfate proteoglycans on the cell surface, making it vulnerable to complement-mediated lysis. On the other hand, it is not clear whether VEGF-A 165Whether the binding of b to its receptor VEGFR2 mediates receptor internalization, and thus the receptor-ligand antibody complex present on the cell surface (transiently) may trigger Fc-mediated complement activation.

[0793] Mutations can be introduced to reduce the risk of possible cytopathic off-target effects on specialized epithelial cells in the eye and kidney due to the presence of surface-bound VEGF-A 165 b. These cells are located distal to a semipermeable barrier that normally excludes antibody-sized molecules and thus may not be a problem. These mutations may include single amino acid mutations in the Fc domain that have been reported to abrogate C1q binding and thus limit complement activation without affecting binding to the neonatal Fc receptor (FcRn).

[0794] Sequence

[0795] The following single amino acid mutations in the human IgG1 heavy chain of HC4 LC2 are proposed:

[0796] ● D270A (Lazar GA, Dang W, Karki S, Vafa O, Peng JS, Hyun L, et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. (2006) 103:4005-10. doi:10.1073 / pnas.0508123103).

[0797] ● P329A (Idusogie EE, Presta LG, Gazzano-Santoro H, Totpal K, Wong PY, Ultsch M, et al. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J Immunol. (2000) 164:4178-84. doi:10.4049 / jimmunol.164.8.4178).

[0798] ●P331S (Xu Y, Oomen R, Klein MH. Residue at position 331 in the IgG1 and IgG4 CH2 domains contributes to their differential ability to bind and activate complement. J Biol Chem. (1994) 269:3469-74).

[0799] Key:

[0800] - Bold highlight = VH

[0801] - Underline = mutated amino acid

[0802] >HC4 (SEQ ID NO:129)

[0803]

[0804] >HC4 D270A (Kabat number) (SEQ ID NO:161)

[0805]

[0806] >HC4 P329A (Kabat number) (SEQ ID NO:162)

[0807]

[0808] >HC4 P331S (Kabat number) (SEQ ID NO:163)

[0809]

[0810] > Mutated Fc with D270A (Kabat number) (SEQ ID NO:164)

[0811] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

[0812] SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

[0813] PPKPKDTLMISRTPEVTCVVVDVSHE APEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[0814] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV

[0815] KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

[0816] HNHYTQKSLSLSPGK

[0817] > Mutated Fc with P329A (Kabat number) (SEQ ID NO:165)

[0818] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

[0819] SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

[0820] PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[0821] TVLHQDWLNGKEYKCKVSNKAL A APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV

[0822] KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

[0823] HNHYTQKSLSLSPGK

[0824] > Mutated Fc with P331S (Kabat number) (SEQ ID NO:166)

[0825] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

[0826] SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

[0827] PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[0828] TVLHQDWLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV

[0829] KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

[0830] HNHYTQKSLSLSPGK

Claims

1. An antibody or antigen-binding fragment thereof that is specific for a splice variant of vascular endothelial growth factor (VEGF), wherein the splice variant is VEGF-A 165 b or any VEGF sequence containing the sequence encoded by exon 8b of the VEGF gene (such as VEGF-Ax and VEGF 189 b).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is: a. a full antibody or antigen-binding fragment selected from the group consisting of Fv fragments (e.g., single-chain Fv, disulfide-bonded Fv, and domain antibodies) and Fab-like fragments (e.g., Fab fragments, Fab' fragments, and F(ab)2 fragments); b. recombinant; c. monoclonal or polyclonal; and / or d. murine, chimeric, human, or humanized.

3. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises or consists of the following CDRs (IMGT format): a.V H CDR1: GFDFSRYW (SEQ ID NO:1); b.V H CDR2: IHPYSSTI (SEQ ID NO:2); c.V H CDR3: ARAFAY (SEQ ID NO:3); d.V L CDR1: QSLLDSDGKTY (SEQ ID NO:4); e.V L CDR2: LVS (SEQ ID NO:5); and / or f.V L CDR3: WQGTHFPYT (SEQ ID NO:6).

4. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises or consists of the following CDRs (Kabat format): a. VH CDR1: RYWMSW (SEQ ID NO:7); b. VH CDR2: EIHPYSSTINYTPSVKD (SEQ ID NO:8); c. VH CDR3: AFAY (SEQ ID NO:9); d. VL CDR1: RSSQSLLDSDGKTYLN (SEQ ID NO:10) or KSSQSLLDSDGKTYLN (SEQ ID NO:11); e. VL CDR2: LVSKLDS (SEQ ID NO:12); and / or f. VL CDR3: WQGTHFPYT (SEQ ID NO:13).

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises or consists of the following variable heavy chains: a. EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA (SEQ ID NO:15); b. EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO:16); c. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFIISRDNAKNSVYLQLNSLRAEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO:17); d. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWISEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRDEDTALYYCARAFAYWGQGTLVTVSS (SEQ ID NO:18); e. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO:19); or f. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSA (SEQ ID NO:20).

6. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises or consists of the following variable light chains: a. DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO:23); b. DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO:24); c. DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWELQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKR (SEQ ID NO:25); d. DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYCWQGTHFPYTFGGGTKVEVKR (SEQ ID NO:26); e. DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGKTYLNWLQQRPGQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR (SEQ ID NO:27); or f. DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO:28).

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises and / or consists of a VH4-VL2 sequence and / or a VH5-VL2 sequence.

8. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is not specific for other VEGF splicing variants lacking exon 8b, such as VEGF-A 165 a.

9. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which further comprises a moiety for increasing the in vivo half-life of the antibody or antigen-binding fragment thereof, such as an amino acid sequence motif, Optionally, the moiety selected from the group of post-translational modifications consists of polyethylene glycol (PEG), glycosylation, fatty acids, and dextran or a gene fusion protein such as, but not limited to, human serum albumin and cytokines (i.e., immunocytokines).

10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is pegylated.

11. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which further comprises a cytotoxic moiety, optionally wherein the cytotoxic moiety comprises and / or consists of the following: a. A radioisotope, such as a radioisotope selected from the group consisting of astatine-211, bismuth-212, bismuth-213, iodine-131, yttrium-90, lutetium-177, samarium-153, and palladium-109; b. A toxin, such as, but not limited to, saporin or calicheamicin; and / or c. A chemotherapeutic agent, such as an antimetabolite.

12. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which further comprises a detectable moiety, optionally wherein the detectable moiety comprises and / or consists of the following: a. A radioisotope, such as a radioisotope selected from the group consisting of technetium-99m; indium-111; gallium-67; gallium-68; arsenic-72; zirconium-89; iodine-124; thallium-201; and / or b. A paramagnetic isotope, such as a paramagnetic isotope selected from the group consisting of gadolinium-157; manganese-55, dysprosium-162, chromium-52; iron-56.

13. A pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof according to any one of the preceding claims, and a pharmaceutically acceptable diluent, carrier, or excipient.

14. The pharmaceutical composition according to claim 13, which is suitable for subcutaneous, intravenous, intramuscular, intracranial, or intraocular delivery.

15. A kit, which comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13 or 14, which is used as a medicine.

17. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or a pharmaceutical composition as claimed in claim 13 or 14 for the manufacture of a medicament for the treatment of a disease, syndrome or condition selected from the group consisting of: VEGF-related diseases (preferably VEGF-A, even more preferably VEGF-A 165 b); ischemia (peripheral, intestinal / mesenteric, coronary / heart, cerebral / brain, retinal, limb, or renal); peripheral artery disease (PAD); atherosclerosis; diabetes-related conditions (such as diabetic retinopathy, diabetic nephropathy, painful diabetic neuropathy, diabetic neuropathy); sclerosis (e.g., systemic sclerosis / scleroderma); Raynaud's syndrome; arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, osteoarthritis); ischemia-related skin conditions (such as cyanosis and gangrene); retinal ischemic diseases (such as rhegmatogenous retinal detachment and proliferative vitreoretinopathy, retinal ischemia, central vein occlusion, branch vein occlusion, non-proliferative diabetic retinopathy); lung diseases associated with altered angiogenesis (e.g., asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease); inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease); neuronal ischemic conditions (e.g., chronic pain, peripheral neuropathy, traumatic neuropathy, chemotherapy-induced peripheral neuropathy); stroke; preeclampsia; hypertension; obesity; hair loss; renal failure (e.g., IgA nephropathy, hereditary kidney diseases such as Denys-Drash syndrome or Fraser syndrome, chronic kidney disease, acute kidney disease, glomerular nephropathy); angiogenesis / vasculogenesis (including tumor-related angiogenesis and VEGF-A 165 a-mediated angiogenesis); cancer (e.g., solid tumors); deep vein thrombosis (DVT); refractory angina; and myocardial infarction (MI) or post-MI conditions.

18. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or a pharmaceutical composition according to claim 13 or 14, for use in the treatment or prevention of a disease, syndrome, or condition selected from the group consisting of: VEGF-related diseases (preferably VEGF-A, even more preferably VEGF-A 165 b); ischemia (peripheral, intestinal / mesenteric, coronary / heart, cerebral / brain, retinal, limb, or renal); peripheral arterial disease (PAD); atherosclerosis; diabetes-related conditions (such as diabetic retinopathy, diabetic nephropathy, painful diabetic neuropathy, diabetic neuropathy); sclerosis (e.g., systemic sclerosis / scleroderma); Raynaud's syndrome; arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, osteoarthritis); ischemia-related skin conditions (such as cyanosis and gangrene); retinal ischemic diseases (such as rhegmatogenous retinal detachment and proliferative vitreoretinopathy, retinal ischemia, central vein occlusion, branch vein occlusion, non-proliferative diabetic retinopathy); lung diseases associated with altered angiogenesis (e.g., asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease); inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease); neuronal ischemic conditions (e.g., chronic pain, peripheral neuropathy, traumatic neuropathy, chemotherapy-induced peripheral neuropathy); stroke; preeclampsia; hypertension; obesity; alopecia; renal failure (e.g., IgA nephropathy, hereditary kidney diseases such as Denys-Drash Syndrome or Frasier Syndrome, chronic kidney disease, acute kidney disease, glomerular nephropathy); angiogenesis / vasculogenesis (including tumor-related angiogenesis and VEGF-A 165 a-mediated angiogenesis); cancer (e.g., solid tumors); deep vein thrombosis (DVT); refractory angina; and myocardial infarction (MI) or post-MI conditions.

19. A method of treating or diagnosing a disease, syndrome, or condition selected from the group consisting of: VEGF-related diseases (preferably VEGF-A, even more preferably VEGF-A 165 b); ischemia (peripheral, intestinal / mesenteric, coronary / heart, cerebral / brain, retinal, limb, or renal); peripheral artery disease (PAD); atherosclerosis; diabetes-related conditions (such as diabetic retinopathy, diabetic nephropathy, painful diabetic neuropathy, diabetic neuropathy); sclerosis (e.g., systemic sclerosis / scleroderma); Raynaud's syndrome; arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, osteoarthritis); ischemia-related skin conditions (such as cyanosis and gangrene); retinal ischemic diseases (such as rhegmatogenous retinal detachment and proliferative vitreoretinopathy, retinal ischemia, central vein occlusion, branch vein occlusion, non-proliferative diabetic retinopathy); lung diseases related to altered angiogenesis (e.g., asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease); inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease); neuronal ischemic conditions (e.g., chronic pain, peripheral neuropathy, traumatic neuropathy, chemotherapy-induced peripheral neuropathy); stroke; preeclampsia; hypertension; obesity; hair loss; renal failure (e.g., IgA nephropathy, hereditary kidney diseases such as Denys-Drash syndrome or Fraser syndrome, chronic kidney disease, acute kidney disease, glomerular nephropathy); angiogenesis / vasculogenesis (including tumor-related angiogenesis and VEGF-A 165 a-mediated angiogenesis); cancer (e.g., solid tumors); deep vein thrombosis (DVT); refractory angina; and myocardial infarction (MI) or post-MI conditions, wherein the method comprises administering an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13 or 14.

20. An in vitro / in ex vivo diagnostic method, wherein the method comprises subjecting an isolated sample to the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13 or 14.

21. An antibody or antigen-binding fragment thereof, which is substantially as described herein for use in a medicine.

22. A pharmaceutical composition, which is substantially as described herein.

23. Use of an antibody or antigen-binding fragment thereof, which is substantially as described herein.

24. A method of treatment or diagnosis, which is as described herein.

25. A kit, which is substantially as defined herein.

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