VEGF antibodies

Abstract

The present invention relates to anti-VEGF-A 165 The present invention also relates to pharmaceutical compositions, kits, and methods of use in therapy.

Description

[Technical Field]

[0001] The present invention relates to VEGF-A 165 Targeting of b, and VEGF-A 165 b. [Background technology]

[0002] Peripheral vascular disease is a leading cause of amputation and a significant contributor to cardiovascular death. Defective angiogenesis has been implicated in obese and diabetic patients due to increased monocyte-derived Wnt5a signaling, which increases the anti-angiogenic form of VEGF (Kikuchi et al. 2014, Nat Med).

[0003] The growth of new blood vessels (angiogenesis), necessary for all tumor growth, is stimulated by the expression of vascular endothelial growth factor (VEGF). VEGF expression is increased in all known solid tumors, as well as in atherosclerosis, diabetic retinopathy, arthritis, and many other conditions. Classical VEGF isoforms have been widely described as pro-angiogenic cytokines. VEGF-A 165 A splice variant of VEGF-A, designated b, is expressed as a protein in normal cells and tissues and circulates in human plasma (see Figure 34). 165 b is VEGF-A 165 Although it binds to VEGF receptor 2 with the same affinity as VEGF-A, it does not activate it or stimulate the same downstream signaling pathways. 165 Inhibits VEGF-A-mediated VEGF receptor 2 phosphorylation and signal transduction. 165 b has no angiogenic effect, but inhibits VEGF-A in rabbit cornea, rat mesentery, mouse mammary gland, dorsal ventricle, and ovary, as well as in proliferative oxygen-induced retinopathy, choroidal neovascularization, and cancer growth. 165 Inhibits VEGF-A mediated angiogenesis. 165 aExpressing tumors are VEGF-A 165 a) The tumors grew significantly slower than VEGF-A expressing tumors. 165a to VEGF-A 165 It has been shown that switching splice variant expression to b can inhibit tumor growth.

[0004] In addition, anti-VEGF-A 165 b Mouse monoclonal antibodies stimulate revascularization in a hindlimb ischemia model in genetically obese (ob / ob) or high-fat, high-sucrose diet-fed mice for 12 weeks (Kikuchi et al 2014. Nat Med.;20(12):1464-1471, which is incorporated herein by reference), and anti-VEGF-A 165 b Mouse monoclonal antibodies have also previously been found to promote revascularization in a model of hindlimb ischemia in mice genetically deficient in eNOS or overexpressing myoglobin (Kuppuswamy et al., Cells 2022, 11, 2676). However, therapeutically effective antibodies, preferably those effective for human use, are needed. Summary of the Invention

[0005] The object of the present invention is to 165 The objective of the present invention is to provide a therapeutic monoclonal antibody against b.

[0006] A first aspect of the present invention relates to an antibody or antigen-binding fragment thereof having specificity 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 (VEGF-Ax and VEGF 189 b, etc.).

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

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

[0009] In some embodiments, the antibody or antigen-binding fragment thereof has the following complementarity determining regions (CDRs) as determined by the IMGT determination method: aV H CDR1: GFDFSRYW (SEQ ID NO: 1), bV H CDR2: IHPYSSTI (SEQ ID NO: 2), cV H CDR3: ARAFAY (SEQ ID NO: 3), dV L CDR1: QSLLDSDGKTY (SEQ ID NO: 4), eV L CDR2: LVS (SEQ ID NO: 5), and / or fV L CDR3: WQGTHFPYT (SEQ ID NO: 6), comprising, or alternatively consisting of,

[0010] In some embodiments, the antibody or antigen-binding fragment thereof has the following CDRs as determined by Kabat determination: aV H CDR1: RYWMSW (SEQ ID NO: 7), bV H CDR2: EIHPYSSTINYTPSVKD (SEQ ID NO: 8), cV H CDR3: AFAY (SEQ ID NO: 9), dV L CDR1: R SSQSLLDSDGKTYLN (SEQ ID NO: 10) or K SSQSLLDSDGKTYLN (SEQ ID NO: 11), eV L CDR2: LVSKLDS (SEQ ID NO: 12), and / or fV L CDR3: WQGTHFPYT (SEQ ID NO: 13), or alternatively consisting of:

[0011] In some embodiments, the V determined by the Kabat method L CDR1 is * SSQSLLDSDGKTYLN (SEQ ID NO: 14), wherein * is R or K.

[0012] In some embodiments, the CDR sequences are determined by the Kabat determination method. In some embodiments, the CDR sequences are determined by the IMGT determination method.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following variable heavy chain: a.EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA (SEQ ID NO: 15, herein the V H also called a chain), b. EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA (SEQ ID NO: 15), also referred to herein as "VH0"); c. EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO: 16, also referred to herein as "VH1"); d. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFIISRDNAKNSVYLQLNSLRAEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO: 17, also referred to herein as "VH2"); e.EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWISEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRDEDTALYYCARAFAYWGQGTLVTVSS (SEQ ID NO: 18, also referred to herein as "VH3"); f. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO: 19, also referred to herein as "VH4"), or g.EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSA (SEQ ID NO: 20, also referred to herein as "VH5").

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable heavy chain sequence, with substitutions or deletions at specific positions indicated in brackets, with deletions indicated as an "X": EVKLLESGGGLVQPGGS(L / P)KLSCAASGFDFSRYW(M / R)SWVRQAPGK(G / E)LEWIGEIHPYSSTINYTPSVKDK(F / L)IISRD(N / S)AKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA (SEQ ID NO: 21)

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus heavy chain sequence (SEQ ID NO:22), with the most common amino acid at each position indicated (variable domains are highlighted in bold and CDRs are double underlined): [Table 1]

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable heavy chain sequence (SEQ ID NO: 15), with the most common amino acid at each position indicated: [Table 2]

[0017] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of a variable light chain as follows: a. DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 23, herein the V L also called a chain), b. DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 23, also referred to herein as "VL0"); c. DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 24, also referred to herein as "VL1"); d. DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWELQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKR (SEQ ID NO: 25, also referred to herein as "VL2"); e. DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYCWQGTHFPYTFGGGTKVEVKR (SEQ ID NO: 26, also referred to herein as "VL3"); f. DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGKTYLNWLQQRPGQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR (SEQ ID NO: 27, also referred to herein as "VL4"), or g. DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 28, also referred to herein as "VL5").

[0018] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable light chain sequence, with substitutions or deletions at specific positions indicated in brackets, with deletions indicated as an "X": ·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(SEQ ID NO:29)

[0019] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable chain sequence (SEQ ID NO:30), with the most common amino acid at each position indicated (variable domains are highlighted in bold, CDRs are determined by IMGT and double underlined): [Table 3]

[0020] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable light chain sequence (SEQ ID NO:31), with the most common amino acid at each position indicated: [Table 4]

[0021] In some embodiments, the variable heavy and / or variable light chain sequences further comprise a signal peptide sequence, eg, MGWTLVFLFLLSVTAGVHS (SEQ ID NO: 32).

[0022] Thus, an antibody or antigen-binding fragment thereof can be formed by combining any of the above VHs with any of the above VLs, for example, VH4 or VH5 can be combined with VL2 as follows (which may optionally include a linker between the two domains, such as (G4S)3): VH4-VL2 (SEQ ID NO: 33) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSS[Linker]DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWELQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKR VH5-VL2 (SEQ ID NO: 34) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSA[Linker]DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWELQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKR

[0023] It will be understood that any VH domain may be combined with any VL domain. 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.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a constant domain. For example, the constant domain may correspond to the following sequence: Heavy chain constant domain: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35) Light chain constant domain: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 36)

[0025] When VH# or VL# further comprises the corresponding heavy or light chain constant domain, it is referred to as HC# or LC#, respectively. For example, taking VH4 and including a heavy chain constant domain results in HC4, and taking VL2 and including a light chain constant domain results in LC2. Thus, combining the heavy and light chain domains for this example results in HC4-LC2 (also referred to herein as "HC4LC2" or "HC4 LC2," all of which are used interchangeably).

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of a VH4-VL2 CDR sequence and / or comprises or consists of a VH4-VL2 VH and / or VL sequence, optionally further comprising a heavy chain constant domain and / or a light chain constant domain as defined herein (i.e., the antibody or antigen-binding fragment thereof may comprise or consist of an HC4-LC2 sequence). In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of a VH5-VL2 CDR sequence and / or comprises or consists of a VH5-VL2 VH and / or VL sequence, optionally further comprising a heavy chain constant domain and / or a light chain constant domain as defined herein (i.e., the antibody or antigen-binding fragment thereof may comprise or consist of an HC5-LC2 sequence).

[0027] In some embodiments, the antibody or antigen-binding fragment thereof does not have specificity for any other VEGF splice variants that do not contain exon 8b. Examples of other VEGF splice variants that do not contain exon 8b include VEGF-A 165 a. Thus, in some embodiments, the antibody or antigen-binding fragment thereof is 165 It has no specificity for a.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a moiety. The moiety may be an amino acid sequence motif optionally contained in the heavy chain constant region. The moiety may be for increasing the in vivo half-life of the antibody or antigen-binding fragment thereof. The moiety may be selected from the group of post-translational modifications consisting of polyethylene glycol (PEG), glycosylation, fatty acids, and dextran. The moiety may also be a gene fusion protein. For example, the gene fusion protein may be to human serum albumin. Additionally or alternatively, the gene fusion protein may be to a cytokine that may form an immune cell kine. In some embodiments, the moiety is a half-life extending motif, and optionally, the half-life extending motif is contained in the Fc domain rather than the variable domain.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof is PEGylated.

[0030] In some embodiments, the moiety is a cytotoxic moiety. For example, the cytotoxic moiety may comprise or consist of a radioisotope, such as, for example, 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 in addition, the cytotoxic moiety may comprise or consist of a toxin, such as saporin or calicheamicin. Alternatively, or in addition, the cytotoxic moiety may comprise or consist of a chemotherapeutic agent, such as an antimetabolite.

[0031] In some embodiments, the moiety is a detectable moiety. For example, the detectable moiety may 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-12, and thallium-201. Alternatively, or in addition, the detectable moiety may 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, and iron-56.

[0032] 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.

[0033] In some embodiments, the pharmaceutical composition is adapted for a particular route of delivery, for example, delivery can be subcutaneous, intravenous, intramuscular, intracranial, or intraocular.

[0034] A third aspect of the present invention relates to a kit comprising an antibody or 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 a vial requiring reconstitution and / or mixing with other components before use. Any one or more parts of the kit may be in lyophilized form.

[0035] A fourth aspect of the present invention relates to an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention, for use as a medicament.

[0036] A fifth aspect of the invention relates to the use of an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention, for the manufacture of a medicament for the treatment of a disease, syndrome or condition as defined herein.

[0037] A sixth aspect of the present invention relates to an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention, for use in the treatment or prevention of a disease, syndrome or condition as defined herein.

[0038] A seventh aspect of the invention relates to a method of treatment or diagnosis, comprising administering an effective amount of an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention.

[0039] An eighth aspect of the present invention relates to a method of diagnosis, comprising subjecting a sample to an antibody or 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 some embodiments, the method is an in vitro method or an 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 a body fluid, a cell, a cell population, a tissue, an organ, plasma, and serum.

[0040] In some embodiments, the disease, syndrome, or condition is: - VEGF-related diseases (preferably VEGF-A, more preferably VEGF-A 165 b). - ischemia (peripheral, intestinal / mesenteric, coronary / cardiac, cerebral / brain, retinal, limb, or renal), -Peripheral artery disease (PAD), - atherosclerosis, -Conditions related to diabetes (e.g., diabetic retinopathy, diabetic nephropathy, diabetic neuropathic pain, diabetic neuropathy), - sclerosis (e.g. systemic sclerosis / scleroderma), -Raynaud's syndrome, - arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, osteoarthritis), -Ischemia-related skin symptoms (e.g., cyanosis and gangrene), -Retinal ischemic disease (rhegmatogenous retinal detachment and proliferative vitreoretinopathy, retinal ischemia, central vein occlusion, branch vein occlusion, non-proliferative diabetic retinopathy, etc.), - pulmonary conditions associated with altered angiogenesis (e.g., asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease), -inflammatory bowel disease (such as ulcerative colitis and Crohn's disease), - Neuroischemic 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 renal conditions such as Dennis-Drash or Fraser syndrome, chronic kidney disease, acute kidney disease, glomerular nephropathy), - Angiogenesis / revascularization (tumor-associated 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 symptoms. DETAILED DESCRIPTION OF THE INVENTION

[0041] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0042] It should be understood that different applications of the disclosed antibodies, uses, methods, pharmaceutical compositions, and kits can be tailored to the particular needs of the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0043] Additionally, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. 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, etc.

[0044] The present invention relates to antibodies or antigen-binding fragments thereof with specificity 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 any VEGF sequence containing the sequence encoded by exon 8b of the VEGF gene (VEGF-Ax, VEGF-A isoform 15 and VEGF 189 b, etc.) By "splice variant" we mean that the gene encoding VEGF may be alternatively spliced, thereby generating different isoforms or versions that translate into different VEGF molecules.

[0045] Advantageously, the described anti-VEGF-A 165 The human VEGF-A antibody is effective in reversing the anti-angiogenic effects of monocytes from patients with peripheral arterial disease in vitro and in vivo. It has an affinity of less than 10 nM and inhibits human VEGF-A mediated angiogenesis in endothelial cells in culture. 165 VEGF-A in a 165 It was desirable to generate a panel of humanized antibodies capable of inhibiting VEGF-A b-mediated suppression. 165These include chimeric antibodies (murine variable domains VH and VL and human heavy and light chain constant domains, HCLC0) and humanized versions against b. The affinity of the chimeric antibody is 300 pM, while the affinities of the humanized versions are 600 pM (two clones - HC4LC2 and HC5LC2), 900 pM (HC4 LC1), 5.0 nM (HC1 LC1), 2.1 nM (HC1 LC2), 4.4 nM (HC2 LC1), 3.6 nM (HC2 LC2), 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-based assays and showed IC values ​​higher than those of the parent murine antibody (IC 50 >1 μg / ml) 50 HC0 LC0, 0.44 μg / ml, HC4 LC2, 0.137 μg / ml, HC5 LC2, 0.114 μg / ml). 165 This represents a significant advance in antibody targeting of b and offers the potential for novel treatments for diseases associated with poor vasculature, such as ischemia.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof has an affinity of 100 to 1000 pM, e.g., 200 to 800 pM, preferably 300 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. "Higher" with respect to affinity refers to increased affinity. For example, 1 pM is a higher (i.e., increased, improved, better, stronger) affinity than 1 nM.

[0047] The antibody or antigen-binding fragment thereof is 165 The antibody or antigen-binding fragment thereof may be specific for VEGF-A. 165 Preferably, the antibody or antigen-binding fragment thereof does not bind or does not significantly bind to VEGF-A.165 b binds to VEGF-A 165 The antibody does not bind or significantly bind to VEGF-A or other splice variants of VEGF that use the proximal splice site of exon 8 that encodes CDKPRR. 165 a or other exon 8 proximal splice variants of VEGF with at least 50-fold or 100-fold greater affinity than VEGF-A 165 In some embodiments, the antibody or antigen-binding fragment thereof does not bind if the splice variant excludes exon 8b.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof is 165 b Affinity / dissociation constant (k) of less than 1 nanomolar for binding D The antibody or antigen-binding fragment thereof may have a VEGF-A 165 b affinity / dissociation constant (k) of less than about 0.8 nM for binding D In another embodiment, the antibody or antigen-binding fragment thereof may have a VEGF-A 165 b) Affinity / dissociation constant (k) of less than 0.6 nM (600 pM) for binding D The antibody or antigen-binding fragment thereof may have a VEGF-A 165 b Affinity / dissociation constant (k) between about 0.3 nM and about 0.8 nM for binding D The antibody or antigen-binding fragment thereof may have a VEGF-A 165 b Affinity / dissociation constant (k) between about 0.5 nM and about 0.7 nM for binding D The antibody or antigen-binding fragment thereof may have a VEGF-A 165 b Affinity / dissociation constant (k) between about 0.55 nM and about 0.65 nM for binding D ) may be present.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof has one or more of the following properties: a. VEGF-A to VEGF receptor 2 (VEGFR2) 165 b reduces the binding affinity, b.VEGF-A 165 b-mediated inhibition of cell migration, e.g., when tested in vitro, the antibody or antigen-binding fragment thereof inhibits cell migration as effectively as or more effectively than the parental murine monoclonal antibody and chimeric antibody as controls; c. Reverse diabetic peripheral arterial disease (PAD) human monocyte-mediated endothelial migration inhibition (eg, by the assay described in Example 4 compared to a control).

[0050] The present invention advantageously provides VEGF-A in the picomolar range. 165 The present invention provides an antibody or antigen-binding fragment thereof with significantly higher specificity for b, which is humanized for efficacy and safety in human therapy.

[0051] In a preferred embodiment, the antibody or antigen-binding fragment thereof 165 bTarget is human VEGF-A 165 b. VEGF-A 165 The sequence of b may comprise the C-terminal sequence of TCRSLTRKD (SEQ ID NO: 37). The antibody or antigen-binding fragment thereof may have specific affinity for a peptide comprising or consisting of the sequence TCRSLTRKD (SEQ ID NO: 37), optionally linked to keyhole limpet hemecyanin (KLH). The sequence TCRSLTRKD (SEQ ID NO: 37) corresponds to the last 9 amino acids of human VEGF165b used as an immunogen to generate the antibody.

[0052] Variants of VEGF-A that contain the TCRSLTRKD sequence, either completely or partially, include vascular endothelial growth factor A VEGFA_HUMAN isoform 15 and vascular endothelial growth factor A isoform VEGF-Ax precursor, as follows: >sp|P15692-15|VEGFA_HUMAN isoform 15 of vascular endothelial growth factor A, OS=Homo sapiens, OX=9606, GN=VEGFA (SEQ ID NO: 159) MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSASRAGPGRASETMNFLL SWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCERPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRSLTRKD >NP_001303939.1 Vascular endothelial growth factor A isoform, VEGF-Ax precursor [Homo sapiens] (SEQ ID NO: 160) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQI MRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRRSAGQEEGASLRVSGTRSLTRKD

[0053] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding portion (i.e., "antigen-binding fragment") or single chains thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of 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 antigens. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with highly conserved regions called framework regions (FRs). The constant regions 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).

[0054] Heavy chains can be of any isotype, including IgG (IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM, and IgE. Light chains include kappa and lambda chains.

[0055] Thus, in some embodiments, the variable regions (VH and VL) of the antibodies of the present invention may further comprise (e.g., be linked to) an antibody constant region or portion thereof. For example, the antibody variable region may be linked at its C-terminus to an antibody light chain constant domain comprising a human Cκ chain or Cλ chain. Similarly, the antibody variable region may be linked 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 isotype subclass, particularly IgG1, IgG2, and IgG4.

[0056] Related 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 it may occur in nature, or that have been modified so as to differ in amino acid sequence from naturally occurring antibodies.

[0057] The term "isolated" refers to the state in which specific antibodies or antigen-binding fragments of the invention, or the nucleic acids encoding them, are preferably in accordance with the present invention. Antibodies and antigen-binding fragments thereof, and nucleic acids will generally be free or substantially free from materials with which they are naturally associated, such as other polypeptides (e.g., host cell proteins) or nucleic acids with which they are found in their natural environment or, if their preparation is by recombinant DNA techniques performed 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 a diluent or adjuvant or excipient and still be isolated for practical purposes; for example, members are typically mixed with gelatin or other carriers when used to coat microtiter plates for use in immunoassays, and mixed with pharmaceutically acceptable carriers, diluents, and / or excipients when used diagnostically or therapeutically. A specifically binding antibody or antigen-binding fragment thereof may be glycosylated or aglycosylated by native or heterologous eukaryotic systems (e.g., when produced by expression in prokaryotic or mammalian cells deficient in a particular glycosylation pathway, or when the antibody or antigen-binding fragment has been specifically engineered to remove glycosylation sites).

[0058] The antibody, or antigen-binding portion thereof, may be a polyclonal or monoclonal antibody. The antibody, or antigen-binding portion thereof, may be produced by any suitable method. For example, suitable methods for producing monoclonal antibodies are disclosed in "Monoclonal Antibodies; A manual of techniques" by H. Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Application" by S. G. R. Hurrell (CRC Press, 1982). Recombinant techniques may also be used.

[0059] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to an antibody that binds to VEGF-A 165 The term "antigen-binding portion" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, such as a fragment of a full-length antibody. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a 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 scFvs, and heavy-chain antibodies such as VHHs and camelid antibodies called nanobodies, are also intended to be encompassed within the scope of the term "antigen-binding portion" of an antibody. These antibody fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments may be screened for utility in the same manner as intact (i.e., full-length) antibodies.

[0060] Antibodies for use in the methods of the present invention may be human antibodies. The term "human antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). 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 human framework sequences, i.e., antibodies typically referred to as chimeric or humanized antibodies.

[0061] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues, often referred to as import residues, are usually obtained from an imported variable domain. Humanization can be performed essentially 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 239:1534-15361; U.S. Pat. No. 4,816,567, the disclosure of which is incorporated herein by reference). In practice, humanized antibodies are typically human antibodies in which some complementarity-determining region residues, and possibly some framework residues, are substituted by residues from analogous sites in rodent (e.g., murine) antibodies. For example, a chimeric antibody may be one in which the VH and VL of a rodent antibody are fused to human IgG constant regions (CH and CL or CK). Alternatively, a humanized antibody is one in which the CDRs from a rodent antibody are replaced with human antibody frameworks that share a high level of sequence identity / homology with the parent rodent antibody. This process is known as CDR-grafting. It will be understood that such antibodies are not limited to analogous sites in rodent antibodies. Rather, the analogous sites may be derived from other non-human parent antibody species, including, for example, rabbit, llama, monkey, etc.

[0062] Human antibodies for use in the methods of the present invention are typically human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas containing B cells obtained from transgenic non-human animals, such as transgenic mice, whose genomes contain human heavy and light chain transgenes 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 the antibody with another agent or antibody.

[0063] Alternatively, an antibody or antigen-binding portion thereof according to the invention may be a humanized antibody.

[0064] The term "humanized" refers to antibody molecules, typically prepared using recombinant techniques, that have an antigen-binding site derived from an immunoglobulin from a non-human species and the remainder of their immunoglobulin structure based on the structure and / or sequence of a human immunoglobulin.

[0065] In one embodiment, the antigen-binding site may 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).

[0066] Alternatively, in fully humanized antibodies (referred to herein as "humanized"), only the complementarity-determining regions (CDRs) of such variable domains are grafted onto appropriate human framework regions of a human variable domain. The framework residues of such humanized molecules may be wild-type (e.g., fully human), or they may be modified to contain one or more amino acid substitutions not found in the human antibody whose sequence serves as the basis for humanization. Humanization reduces or eliminates the likelihood that a "humanized antibody" will act as an immunogen in human individuals, although the possibility of an immune response to the foreign variable region remains (LoBuglio, AF et al. (1989) "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (USA) 86:4220-4224).

[0067] Those skilled in the art will understand that for human therapy or diagnosis, human or humanized antibodies are preferably used. Humanized forms of non-human (e.g., murine) antibodies are genetically engineered chimeric antibodies or antibody fragments, preferably with minimal amino acid residues derived from the non-human antibody. Humanized antibodies include antibodies in which the complementarity-determining regions of a human antibody (recipient antibody) are replaced with residues from complementarity-determining regions of a non-human species (donor antibody) such as mouse, rat, or rabbit that have the desired function. In some cases, Fv framework residues of the human antibody are replaced with corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported complementarity-determining regions or framework sequences. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the complementarity-determining regions corresponding to those of a non-human antibody and all or substantially all of the framework regions corresponding to those of the relevant human consensus sequence. A humanized antibody optimally also comprises at least a portion of an antibody constant region, e.g., an Fc region, typically derived from a human antibody (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).

[0068] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues, often referred to as import residues, are usually obtained from imported variable domains. Humanization can be performed essentially as described by substituting 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 239:1534-1536; U.S. Pat. No. 4,816,567). In practice, humanized antibodies will typically be human antibodies in which some complementarity-determining region residues, and possibly some framework residues, are substituted by residues from analogous sites in rodent antibodies.

[0069] Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, e.g., Hoogenboom & Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies and Cancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991, J. Immunol. 147:86-95).

[0070] Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable regions to make them as close to human form as possible. Both heavy and light chain variable regions are known to contain three complementarity-determining regions (CDRs), which change in response to the antigen of interest and determine binding affinity, flanked by four framework regions (FRs), which are relatively conserved in a given species and are predicted to provide a scaffold for the CDRs. When a non-human antibody is prepared for a specific antigen, the variable region can be "reshaped" or "humanized" by grafting CDRs from the non-human antibody onto the FRs present in the modified human antibody. The application of this approach to various antibodies has been reviewed in 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, CA et al. (1991) “Humanization of a Mouse Monoclonal Antibody by CDR-Grafting: The Importance of Framework Residues on Loop Conformation,” Protein Engineering 4:773-783; Maeda, H. et al. (1991) “Construction of Reshaped Human Antibodies with HIV-Neutralizing Activity,” Human Antibodies Hybridoma 2:124-134;Gorman, SDet al. (1991) “Reshaping A Therapeutic CD4 Antibody,” Proc. Natl. Acad. Sci. (USA) 88:4181-4185; Tempest, PRet al.(1991) “Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo,” Bio / Technology 9:266-271; Co, MSet al. (1991) “Humanized Antibodies For Antiviral Therapy,” Proc. Natl. Acad. Sci. (USA) 88:2869-2873; Carter, P. et al. (1992) “Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy,” Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co, MS et al. (1992) “Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen,” J. Immunol. 148:1149-1154. .

[0071] In some embodiments, a humanized antibody preserves all six CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibody). In other embodiments, a humanized antibody has one or more CDRs (1, 2, 3, 4, 5, 6) that are modified relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from 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, and 6,881,557).

[0072] Any antibody referred to herein may be provided in isolated form or, optionally, linked (directly or indirectly) to another moiety, which may be a therapeutic molecule such as a cytotoxic moiety, an antibiotic, or a drug.

[0073] Therapeutic molecules can be directly attached to the antibodies or antigen-binding fragments of the present invention, for example, by chemical conjugation. Methods for conjugating molecules to such antibodies are known in the art. For example, carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 77, 151-159) can be used to conjugate various drugs, including doxorubicin, to antibodies or peptides. The water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), is particularly useful for conjugating functional moieties to binding moieties.

[0074] Other methods for conjugating a moiety to an antibody can also be used. For example, sodium periodate oxidation followed by reductive alkylation with a suitable reagent can be used, as can glutaraldehyde cross-linking. However, regardless of the method selected for producing the conjugate of the present invention, it is recognized that a decision must be made that the antibody maintains its targeting ability and the functional moiety maintains its relevant function.

[0075] A cytotoxic moiety can be directly and / or indirectly cytotoxic. "Directly cytotoxic" means that the moiety is a moiety that is cytotoxic itself. "Indirectly cytotoxic" means that the moiety is not cytotoxic itself, but is able to induce cytotoxicity, for example, by its action on or a further action upon a further molecule. Preferably, the cytotoxic moiety may be cytotoxic only intracellularly, and not extracellularly.

[0076] The antibody or antigen-binding portion thereof may 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 this context of the present invention, the antibody or antigen-binding portion thereof with the cytotoxic chemotherapeutic agent is intended for use in combination with a different chemotherapy. In some embodiments, the antibody or antigen-binding portion thereof is not conjugated to a cytotoxic chemotherapeutic agent.

[0077] Cytotoxic chemotherapeutic agents, whether conjugated to an antibody or its antigen-binding portion or used separately as chemotherapy, such as anti-cancer agents, include alkylating agents, including nitrogen mustards, such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolidine), and chlorambucil; ethylenimines and methylmelamines, such as hexamethylmelamine, thiotepa; alkylsulfonates, such as busulfan; nitrogen ions, such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozotocin (streptozotocin); Anti-metabolites include triazenes such as trosourea and dacarbazine (DTIC, dimethyltriazenoimidazole-carboxamide), 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 (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and matomycin (mitomycin C), enzymes such as L-asparaginase, and biological response modifiers such as interferon alphenomenon. Other agents include platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin, anthracenediones such as mitoxantrone and anthracyclines, substituted ureas such as hydroxyurea, methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH), and adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide, taxol and analogs / derivatives, and hormone agonists / antagonists such as flutamide and tamoxifen.

[0078] The cytotoxic moiety can be a cytotoxic peptide or a polypeptide moiety that leads to cell death. Cytotoxic peptides and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor, etc. Methods for linking 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 useful as cytotoxic agents.

[0079] Certain radioactive atoms can also be cytotoxic when delivered in sufficient doses. Thus, a cytotoxic moiety can include a radioactive atom that delivers a sufficient amount of radioactivity to a target site during use 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 destroy 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, 8000, or 10000 cGy) is delivered to the target site, preferably the cells and their organelles, particularly the nuclei, of the target site.

[0080] The radioactive atom can be attached to the antibody, antigen-binding fragment, variant, fusion, or derivative thereof by known methods. For example, EDTA or another chelating agent can be attached to the binding moiety or used to attach In or Y. Tyrosine residues can be directly labeled with I or I.

[0081] The cytotoxic moiety can be a suitable indirectly cytotoxic polypeptide. The indirectly cytotoxic polypeptide can be a polypeptide that has enzymatic activity and can convert a non-toxic and / or relatively non-toxic prodrug into a cytotoxic drug. With antibodies, this type of system is often referred to as ADEPT (antibody-directed enzyme prodrug therapy). The system involves an antibody placing an enzymatic moiety at a desired site in the patient's body, allowing time for the enzyme to localize at the site, and then administering a prodrug that is a substrate for the enzyme, the end product of which is a cytotoxic compound. The goal of this approach is to maximize the concentration of the drug at the desired site and minimize the concentration of the drug in normal tissues. The cytotoxic moiety can be capable of converting a non-cytotoxic prodrug into a cytotoxic drug.

[0082] The enzymes and prodrugs of the targeted enzyme system described herein can be any of those previously proposed. The cytotoxic agent can be any existing anticancer drug, such as an alkylating agent, a DNA-intercalating agent, a drug that inhibits any important enzyme, such as dihydrofolate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinase, or topoisomerase, or a drug that affects cell death by interacting with any other cellular component. Etoposide is an example of a topoisomerase inhibitor.

[0083] Reported prodrug systems include those listed in Table A. [Table 5]

[0084] Suitable enzymes for forming part of the enzyme moiety include exopeptidases such as carboxypeptidase G, G1, G2 (for glutamylated mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs), and aminopeptidases (for 2-α-aminoacyl MTC prodrugs); undopeptidases such as thrombolicin (for thrombin prodrugs); hydrolases such as phosphatase (such as alkaline phosphatase) or sulfatase (such as arylsulfatase) (for phosphorylated or sulfated prodrugs); amidases such as penicillin amidase and aryl acylamidase; lactamases such as β-lactamase; β-glucuronidase (β glycosidases such as α-glucuronomide anthracyclines, α-galactosidase (for amygdalin), and β-galactosidase (for β-galactose anthracyclines); deaminases such as cytosine deaminase (for 5FC); kinases such as urokinase and thymidine kinase (for ganciclovir); reductases such as nitroreductase (for CB1954 and analogs), 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.

[0085] Preferably, the prodrug is relatively non-toxic compared to the cytotoxic drug, typically having less than 10% toxicity, preferably less than 1%, as measured in an appropriate in vitro cytotoxicity assay.

[0086] The moiety capable of converting a prodrug into a cytotoxic drug is likely to be active in isolation from the remainder of the drug of the invention, but need only be active (a) when combined with the remainder of the drug of the invention, and (b) when the drug of the invention is attached to, adjacent to, or internalized within a target cell.

[0087] When each moiety is a polypeptide, the two moieties can be linked by any conventional method for cross-linking polypeptides. For example, an antibody or antigen-binding portion thereof may be enriched in thiol groups, and the additional moiety is reacted with a bifunctional agent capable of reacting with those thiol groups, such as the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Amide and thioether bonds, achieved with, for example, m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulfide bonds.

[0088] The cytotoxic moiety can be a radiosensitizer. Radiosensitizers include fluoropyrimidines, thymidine analogs, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamides, 3-aminobenzodiamides, etanixadol, pimonidazole, and mizonidazole. Gene delivery into cells can also sensitize them to radiation, such as delivery of the p53 gene or cyclin D. The additional moiety can be a moiety that becomes cytotoxic upon irradiation or releases a cytotoxic moiety. For example, boron-10 isotopes emit cytotoxic alpha particles when appropriately irradiated. Similarly, the cytotoxic moiety can be one useful in photodynamic therapy, such as Photofrin.

[0089] The terms "therapeutically effective amount," "effective amount," or "therapeutically effective" mean that a given substance is administered to a subject suffering from a condition in an amount sufficient to cure, alleviate, or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. The effective amount for a given purpose and a given agent will depend on the severity of the disease or injury, as well as the weight and general condition of the subject. This may be a predetermined amount of active antibody calculated to produce the desired therapeutic effect in association with necessary additives and diluents, i.e., carriers or administration vehicles. It is further intended to mean an amount sufficient to reduce or prevent clinically significant deficits in host activity, function, and response. Alternatively, a therapeutically effective amount is an amount sufficient to cause an improvement in clinically significant symptoms in the host. As will be appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. A suitable dosage may contain a predetermined amount of active composition calculated to produce the desired therapeutic effect in association with necessary diluents. A therapeutically effective amount can be determined by an ordinarily skilled medical or veterinary practitioner based on patient characteristics such as age, weight, sex, condition, comorbidities, other diseases, etc., as is well known in the art.

[0090] The optimal dose can be determined by a physician based on many parameters, such as age, sex, weight, the severity of the condition being treated, the active ingredient being administered, and the route of administration. Generally, a serum concentration of polypeptide and antibody that allows receptor saturation is desirable. A concentration of more than about 0.1 nM is usually sufficient. For example, a dose of 100 mg / kg of antibody provides a serum concentration of about 20 nM for about 8 days. As a rough guideline, antibody doses can be administered weekly in amounts of 10 to 300 mg / kg.

[0091] The dosage of the composition will depend, as is within the skill of a physician, on the characteristics 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, site and rate of administration, the target organ, the clinical tolerance of the patient involved, the symptoms afflicting the patient, etc. For example, a single administration may provide 300 μg of antibody per patient, although the dosage may range from about 10 μg to 6 g per administration. Different dosages are utilized during a series of successive inoculations. A practitioner may administer an initial inoculation followed by boosting with smaller doses of antibody.

[0092] The term "subject" (used interchangeably herein with "patient") includes any animal, including humans, in need of treatment with an antibody, or antigen-binding portion thereof, of the invention. A subject or patient can be a mammal or a non-mammal. Preferably, the subject is a mammal such as a horse, or cow, or sheep, or pig, or camel, or dog, or cat. Most preferably, the mammalian patient is a human.

[0093] In some embodiments, the antibody or antigen-binding portion thereof is formulated and / or adapted for delivery by a route selected from the group including intravenous, intramuscular, subcutaneous, intracranial, and intraocular. For example, the antibody or antigen-binding portion thereof may be formulated and / or adapted for intravenous (i.e., "iv" or "IV") delivery.

[0094] In some embodiments, the antibody or antigen-binding portion thereof is delivered to the subject by a route selected from the group including intravenous, intramuscular, subcutaneous, intracranial, and intraocular. For example, the antibody or antigen-binding portion thereof may be delivered intravenously.

[0095] It is envisioned that injection will be the primary route of therapeutic administration of the composition, although delivery via catheter or other surgical tubes may also be used. Suitable routes of administration include intravenous, subcutaneous, intraperitoneal, intradermal, intramuscular, intracranial, and intraocular administration. Liquid formulations may be utilized after reconstitution from powder or lyophilized formulations.

[0096] For intravenous injection or injection via other parenteral routes, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed, as further described herein.

[0097] The compositions can be administered locally or systemically.

[0098] Depending on the route of administration, the antibody or antigen-binding portion thereof may be coated with a material to protect the agent(s) from the action of acids and other natural conditions, which may inactivate or denature the antibody or antigen-binding portion thereof. Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water, and physiological saline. Other examples of carriers include ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, or sodium chloride in the composition.

[0099] Methods and formulations for various routes of administration are well known in the art.

[0100] Antibodies or antigen-binding fragments thereof can be defined by their 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 the binding of an antibody to its target can be defined in terms of the relative dissociation constant (Kd) of the antibody for its target compared to the dissociation constant for the antibody and another non-target molecule.

[0101] Typically, the Kd of an antibody for a target will be less than 2-fold, preferably 5-fold, and more preferably 10-fold greater than the Kd for other non-target molecules, such as unrelated substances or accompanying substances in the environment. More preferably, the Kd will be less than 50-fold, even more preferably less than 100-fold, for example less than 200-fold, and even more preferably less than 1000-fold.

[0102] The value of this dissociation constant can be determined directly by well-known methods, and can also be calculated for complex mixtures, for example, by methods such as those shown in Caceci et al. (Byte 9:340-362, 1984). For example, it can be established using a double-filter nitrocellulose filter binding assay, such as that disclosed by Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Other standard assays for evaluating 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 (e.g., binding affinity) of an antibody can also be evaluated by standard assays known in the art, such as BIAcore™ (SPR) system analysis or biolayer interferometry (BLI) using the Octet system.

[0103] Competitive binding assays may be performed in which binding of an antibody to a target is compared to binding of the target by another known ligand of that target, such as another antibody. The concentration at which 50% inhibition occurs is known as Ki. Under ideal conditions, Ki is equivalent to Kd. Because the Ki value cannot fall below Kd, the measurement of Ki can be conveniently substituted to provide an upper limit for Kd.

[0104] The antibodies or antigen-binding fragments thereof described herein are preferably capable of binding to their target with an affinity that is at least 2-fold, 10-fold, 50-fold, 100-fold or more than their affinity for binding to another non-target molecule.

[0105] For example, the antibody may be a specific anti-VEGF-A antibody disclosed herein. 165 b antibody, provided that the variant or fragment is a VEGF-A 165 Retains specificity for b.

[0106] The fragment is preferably an antigen-binding portion of an antibody, as described herein. Fragments can be generated by truncation, e.g., removal of one or more amino acids from the N-terminus and / or C-terminus of the polypeptide. In this manner, up to 10, up to 20, up to 30, up to 40, or more amino acids can be removed from the N-terminus and / or C-terminus. Fragments can also be generated by one or more internal deletions.

[0107] The variants include the specific anti-VEGF-A 165b) The antibody sequence may include one or more substitutions, deletions, or additions. Variants may include 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 sequences disclosed herein. "Deletion" variants may include deletion of individual amino acids, deletion of small groups of amino acids, e.g., 2, 3, 4, or 5 amino acids, or deletion of larger amino acid regions, e.g., deletion of specific amino acid domains or other features. "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids, making conservative amino acid substitutions. For example, amino acids may be substituted with alternative amino acids 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.

[0108] In some embodiments, the 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, the 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 and / or light chain sequences. For example, a variant may comprise or consist of a substitution 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 in SEQ ID NOs: 19, 20, and / or 25. Alternatively, or in addition, a variant may comprise or consist of a deletion 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 in SEQ ID NOs: 19, 20, and / or 25.

[0109] In some embodiments, variants comprise or consist of a percentage of sequence identity with the sequences disclosed herein (e.g., variable heavy chain sequences, variable light chain sequences, and / or CDR sequences). In some embodiments, variants comprise or consist of 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% sequence identity to the sequences disclosed herein (e.g., variable heavy chain sequences, variable light chain sequences, and / or CDR sequences). In some embodiments, variants comprise or consist of a percentage of sequence identity with the variable heavy chain sequences and / or variable light chain sequences, while retaining the same CDR sequences as the corresponding variable heavy chain sequences and / or variable light chain sequences, respectively.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof may comprise a variable heavy chain sequence selected from SEQ ID NOs: 15 to 22, or an amino acid sequence having at least 60% sequence identity thereto, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof may comprise a variable light chain sequence selected from SEQ ID NOs: 23 to 31, or an amino acid sequence having at least 60% sequence identity thereto, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof may comprise a CDR sequence selected from SEQ ID NOs: 1 to 6, or an amino acid sequence having at least 60% sequence identity thereto, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof may comprise a CDR sequence selected from SEQ ID NOs: 7 to 13, or an amino acid sequence having at least 60% sequence identity thereto, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof may comprise a CDR sequence selected from SEQ ID NO: 14. LIn some embodiments, the V may comprise a CDR1 sequence or an amino acid sequence having at least 60% sequence identity thereto, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. H The CDR1 sequence comprises or consists of 1, 2, 3, 4, 5, or 6 substitutions. H The CDR2 sequence comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions. H The CDR3 sequence comprises or consists of 1, 2, 3, or 4 substitutions. L The CDR1 sequence comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions. L The CDR2 sequence comprises or consists of 1, 2, 3, 4, 5, 6, or 7 substitutions. L The CDR3 sequence may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 or 9 substitutions.

[0111] 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 thereto, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. 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 thereto, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof may comprise any one or more CDRs of a variable heavy chain sequence selected from SEQ ID NOs: 15 to 22. In some embodiments, the antibody or antigen-binding fragment thereof may comprise any one or more CDRs of a variable light chain sequence selected from SEQ ID NOs: 23 to 31.

[0113] Some properties of the 20 main amino acids that can be used to select suitable substitutes are as follows: [Table 6]

[0114] Preferred "variants" include those in which an amino acid that appears in the sequence instead of a naturally occurring amino acid is its structural analogue. The amino acids used in the sequence may also be derivatized or modified, e.g., labeled, so long as the function of the antibody is not significantly adversely affected.

[0115] Variants may be prepared by modification during antibody synthesis or after production, or if the antibody is in recombinant form, may be prepared using known techniques such as site-directed mutagenesis or enzymatic cleavage and / or ligation of nucleic acids.

[0116] The present invention also encompasses within its scope the polypeptides and polynucleotides described herein, as well as sequences having substantial identity thereto, e.g., at least 70%, 80%, 85%, 90%, 95%, or 99% identity. In one embodiment, the CDR sequences of a sequence may be identical, but the variable regions outside of the CDRs (e.g., any of the VH and / or VL sequences specified herein) may vary, e.g., by at least 70%, 80%, 85%, 90%, 95%, or 99% identity, relative to a given variable sequence. In one embodiment, the CDR sequences of a sequence may be identical, but the framework regions may vary, e.g., by at least 70%, 80%, 85%, 90%, 95%, or 99% identity, relative to a given framework sequence. In another embodiment, the framework regions may be varied by one, two, three, four, or five amino acid modifications, such as substitutions, deletions, or insertions. Variants may retain target binding function.

[0117] The percent identity of two amino acid sequences or two nucleic acid sequences is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the first sequence for optimal alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. "Best alignment" refers to the 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 in the sequences (i.e., % identity = number of identical positions / total number of positions × 100).

[0118] Preferably, the variant antibody has an amino acid sequence with greater than 60%, or greater than 70% (e.g., 75% or 80%), preferably greater than 85%, for example, greater than 90% or 95% amino acid identity to the VL or VH domain of an antibody disclosed herein. This level of amino acid identity can be found over the entire length of the sequence of the relevant SEQ ID NO, or over a portion of the sequence, such as over 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0119] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. One 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) incorporate such an algorithm. To obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention, BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12. To obtain amino acid sequences homologous to the protein molecules of the present invention, BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3. 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 an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilized for sequence comparison is the Myers and Miller algorithm. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. Other algorithms for sequence analysis known in the art include the ADVANCE and ADAM algorithms described in Torellis and Robotti (1994), and the FASTA algorithm described in Pearson and Lipman (1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0120] The antibodies and antigen-binding fragments thereof described herein may be modified by mutation to remove one or more sites of N-linked glycosylation (HCDR2), Met oxidation (HCDR1), Trp oxidation, or Asp isomerization. Advantageously, modifying antibodies and antigen-binding fragments thereof to remove, for example, one or more sites of N-linked glycosylation (HCDR2), Met oxidation (HCDR1), Trp oxidation, or Asp isomerization may avoid problems resulting from such post-translational modifications, which may reduce antigen-binding affinity, lead to product heterogeneity and aggregation, or adversely affect expression levels during production or manufacturing.

[0121] Residue numbers herein may be determined according to the Kabat numbering system. For example, the heavy chain HC4 or HC5 of an antibody 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 chain LC1 or LC2 of an antibody described herein may contain one or more mutations selected from S32A, D33E, G34A, and D60E. Equivalent residues may be mutated in any of the light chains described herein. [Table 7]

[0122] In one embodiment, the antibodies or antigen-binding fragments thereof described herein may be selected from one or more of the following, where the particular mutation in the heavy or light chain is indicated in parentheses following the relevant chain: HC4(N59Q)LC1, HC4(N59Q)LC2, HC5(N59Q)LC2, HC4(T61A)LC2, HC5(T61A)LC2, HC4 LC1(S32A), HC4 LC2(S32A), HC5 LC2(S32A), HC4 LC1(D33E), HC4 LC2(D33E), HC5 LC2(D33E), HC4 LC1(G34A), HC4 LC2(G34A), HC5 LC2(G34A).

[0123] In some embodiments, the antibody or antigen-binding fragment thereof comprises HC4(N59Q). In some embodiments, the antibody or antigen-binding fragment thereof comprises HC5(N59Q). In some embodiments, the antibody or antigen-binding fragment thereof comprises HC4(T61A). In some embodiments, the antibody or antigen-binding fragment thereof comprises HC5(T61A). In some embodiments, the antibody or antigen-binding fragment thereof comprises LC1(S32A). In some embodiments, the antibody or antigen-binding fragment thereof comprises LC2(S32A). In some embodiments, the antibody or antigen-binding fragment thereof comprises LC1(D33E). In some embodiments, the antibody or antigen-binding fragment thereof comprises LC2(D33E). In some embodiments, the antibody or antigen-binding fragment thereof comprises LC1(G34A). In some embodiments, the antibody or antigen-binding fragment thereof comprises LC2(G34A). In some embodiments, the antibody or antigen-binding fragment thereof comprises LC1(D60E). In some embodiments, the antibody or antigen-binding fragment thereof comprises LC2(D60E). In some embodiments, the antibody or antigen-binding fragment thereof comprises HC4(D270A). In some embodiments, the antibody or antigen-binding fragment thereof comprises HC4(P329A). In some embodiments, the antibody or antigen-binding fragment thereof comprises HC4(P331S).

[0124] In some embodiments, the antibody or antigen-binding fragment thereof comprises any combination 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).

[0125] In some embodiments, a linker is present between the HC domain and the LC domain. For example, in some embodiments, the antibody or antigen-binding fragment thereof comprises 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:1 33, 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:1 30 + 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 ID NO: 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 ID NO: 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 ID NO: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 ID NO:130 + linker + SEQ ID NO:117, SEQ ID NO:104 + linker + SEQ ID NO:116 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 ID NO: 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.

[0126] In some embodiments, the heavy chain is selected from any of SEQ ID NOs: 99 to 107. In some embodiments, the light chain is selected from any of SEQ ID NOs: 108 to 118. Any combination of heavy and light chains can be used to generate the antibody or antigen-binding fragment thereof.

[0127] For example, removing the Fc function of an antibody can be beneficial when using receptor agonists to crosslink receptors and induce signal transduction, or when using receptor antagonists to block receptor-ligand interactions and prevent signal transduction. Fc ligation of Fcγ receptors on effector cells or ligation of C1q may be undesirable because it can result in unwanted killing of biologically important cells expressing the receptor by 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 fc and protein a. J Immunol, May 15, 2000, 164(10)5313-5318). Following the discovery of this initial mutation, the combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) was found to abolish 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 abolish detectable binding of both 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, as shown, for example, in Saunders, K. O., Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life 2019 Front. Immunol., 07 June 2019 Sec. Comparative Immunology.

[0128] Thus, in one embodiment in which an antibody or antigen-binding fragment thereof comprises an Fc portion, an antibody or antigen-binding fragment thereof of the invention may comprise a LALA (SEQ ID NO: 121) modification in the CH2 domain of the Fc portion. An antibody of the invention may comprise Leu234Ala and Leu235Ala modifications in the CH2 domain of the Fc portion, or equivalent residues thereof (e.g., equivalent Leu residues that may not be at positions 234 or 235, respectively).

[0129] In one embodiment, the antibody or antigen-binding fragment thereof comprises an Fc portion. 165 The antibody may comprise an Fc portion mutated to reduce cytotoxic off-target effects on specialized epithelial cells of the eye and kidney due to the presence of β. For example, the Fc region may be selected from the group consisting of SEQ ID NOs: 164 to 166. An exemplary HC4 sequence with such an Fc mutation may be selected from the group consisting of SEQ ID NOs: 161 to 163.

[0130] "Sequence identity," in the context of amino acid sequences, refers to sequences having the stated value when assessed using ClustalW (Thompson et al., 1994, supra) using the following parameters: Pairwise alignment parameters - Method: Exact, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10, Multiple alignment parameters - matrix: PAM, gap open penalty: 10.00, % identity of delay: 30, penalty end gap: on, gap separation distance: 0, negative matrix: none, gap extension penalty: 0.20, residue specific gap penalty: on, hydrophobic gap penalty: on, hydrophilic residues: GPSNDQEKR (SEQ ID NO: 120). Sequence identity at specific residues is intended to include identical residues that have only been derivatized.

[0131] The anti-VEGF-A of the present invention 165This is because an antibody may bind to the same epitope as a specific antibody disclosed herein, and such an antibody is likely to mimic the action of the disclosed antibody. Whether an antibody binds to the same epitope as another antibody can be determined by routine methods. For example, the binding of each antibody to a target may be determined using a competitive binding assay. Methods for conducting competitive binding assays are well known in the art. For example, they may involve contacting an antibody and a target molecule together under conditions that allow the antibody to bind to the target molecule. The antibody / target complex may then be contacted with a second (test) antibody, and the extent to which the test antibody can displace the first antibody from the antibody / target complex may be assessed. Such assessment may use any suitable technique, including, for example, surface plasmon resonance, ELISA, or flow cytometry. 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, therefore, binds to the same epitope or region on the target as the first antibody and, therefore, can mimic the action of the first antibody.

[0132] The present invention also provides a kit comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention, for example the kit comprising (a) an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention, comprising: 165 The composition may comprise a therapeutically effective amount of an antibody or antigen-binding portion thereof that specifically binds to b.

[0133] The kits of the present invention may further comprise one or more other reagents or equipment that allow for carrying out any of the above-described embodiments. Such reagents or equipment may include suitable buffer(s) (aqueous solutions), as well as anti-VEGF-A 165 b) a means for administering the antibody (e.g., a container or device containing a needle). The kit may include instructions for carrying out the combination therapy or method described herein.

[0134] Anti-VEGF-A antibodies described herein or provided in the kits of the invention 165The antibody may 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 and absorption delaying agents, and the like that are physiologically compatible and compatible with the desired route of administration.

[0135] Pharmaceutical compositions may contain pharmaceutically acceptable antioxidants. These compositions may also contain excipients and adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures (see above) and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. In addition, prolonged absorption of the injectable dosage form may be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0136] Pharmaceutical compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of active agent (e.g., antibody) in an appropriate solvent, optionally containing one or a combination of the above-listed ingredients, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle containing a basic dispersion medium and the required other ingredients listed above. For sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active agent and any additional desired ingredients from a previously sterile-filtered solution thereof. Pharmaceutical compositions can contain additional active ingredients and those listed above.

[0137] 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 incorporated herein by reference).

[0138] The term "buffer" is intended to include an aqueous solution containing an acid-base mixture for the purpose of stabilizing 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 lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0139] The term "diluent" is intended to include aqueous or non-aqueous solutions for the purpose of diluting a drug in a pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as safflower oil, corn oil, peanut oil, cottonseed oil, olive oil, or sesame oil).

[0140] The term "adjuvant" is intended to include any compound added to a formulation to enhance the biological effect of the agent of the present invention. Adjuvants can be one or more of zinc, copper, or silver salts with different anions, such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates with different acyl compositions. Adjuvants can also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinylimidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

[0141] The excipient may 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 include starch, cellulose ether, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginate, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate with different hydrolysis degrees, and polyvinylpyrrolidone, all of which have different molecular weights, and are added to the formulation, for example, to control viscosity, achieve bioadhesion, or protect lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain lengths and degrees of saturation, egg lecithin, soybean lecithin, hydrogenated egg, and soybean lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide, and titanium oxide, which are added to the composition to obtain benefits such as reduced liquid accumulation or advantageous pigment properties.

[0142] The active antibody-based agents of the present invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for their delivery.

[0143] In one embodiment, the pharmaceutical composition of the present invention may be in the form of a liposome, in which the drug is combined with an amphiphilic agent, such as a lipid, which exists in aggregated form as a micelle, an insoluble monolayer, or a liquid crystal, in addition to other pharmaceutically acceptable carriers. Suitable lipids for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, and the like. Suitable lipids also include the above lipids modified at the polar head group with poly(ethylene glycol) to extend blood circulation time. Preparation of such liposomal formulations can be found, for example, in U.S. Pat. No. 4,235,871.

[0144] 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 are widely used as biodegradable polymers in the production of microparticles. The preparation of such microspheres is described in US 5,851,451 and EP 0 213 303.

[0145] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of nanoparticles, for example based on poly-gamma 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 of the combination therapy of the present invention may be formulated in separate nanoparticles, or both active ingredients may be formulated in the same nanoparticle.

[0146] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of a polymer gel, and polymers such as starch, cellulose ether, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginate, carrageenan, hyaluronic acid and their derivatives, polyacrylic acid, polyvinylimidazole, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinylpyrrolidone are used to thicken the solution containing the drug. The polymer may also include gelatin or collagen.

[0147] Alternatively, the drug may simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil), tragacanth gum, and / or various buffers.

[0148] It will be understood that the pharmaceutical compositions of the present invention may contain ions and a defined pH to enhance the action of the active agent. The compositions may also be subjected to conventional pharmaceutical operations such as sterilization, and / or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.

[0149] According to another aspect of the invention, there is provided an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention, for use in treating or preventing ischemia in a subject.

[0150] According to another aspect of the invention there is provided the use of an antibody or 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 the treatment or prevention of ischemia in a subject.

[0151] 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 antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention.

[0152] Ischemia can be a result of peripheral arterial disease (PAD) (also called peripheral arterial disease). In one embodiment, the condition being treated or prevented is a condition associated with ischemia. In one embodiment, the condition being treated or prevented is peripheral arterial disease (PAD) and / or a condition associated with PAD.

[0153] The ischemia may be selected from cardiac or coronary ischemia, intestinal ischemia, cerebral ischemia, limb ischemia, retinal ischemia, renal ischemia, and associated skin conditions such as cyanosis and gangrene.

[0154] In accordance with another aspect of the present invention, the present invention provides a method for treating a variety of conditions, including but not limited to myocardial infarction (MI), including ST-segment elevation MI, angina pectoris, systemic sclerosis / scleroderma, Raynaud's syndrome, ulcerative colitis, Crohn's disease, inflammatory bowel disease, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy pain, diabetic neuropathy, stroke, preeclampsia, hypertension, obesity, or hair loss, including but not limited to VEGF-A. 165 a vs VEGF-A 165 The monoclonal antibodies of the invention are provided for use in treating or preventing diseases in which b is elevated.

[0155] In accordance with another aspect of the present invention, the present invention provides a method for treating a variety of conditions, including but not limited to myocardial infarction (MI), including ST-segment elevation MI, angina pectoris, systemic sclerosis / scleroderma, Raynaud's syndrome, ulcerative colitis, Crohn's disease, inflammatory bowel disease, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy pain, diabetic neuropathy, stroke, preeclampsia, hypertension, obesity, or hair loss, including but not limited to VEGF-A. 165 a vs VEGF-A 165 There is provided the use of an antibody or 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 the treatment or prevention of a disease in which b is elevated.

[0156] In accordance with another aspect of the present invention, the present invention provides a method for treating a variety of conditions, including but not limited to myocardial infarction (MI), including ST-segment elevation MI, angina pectoris, systemic sclerosis / scleroderma, Raynaud's syndrome, ulcerative colitis, Crohn's disease, inflammatory bowel disease, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy pain, diabetic neuropathy, stroke, preeclampsia, hypertension, obesity, or hair loss, including but not limited to VEGF-A. 165 a vs VEGF-A 165 The present invention provides a method for treating or preventing a disease in which b is elevated, the method comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention.

[0157] A convenient way to produce an antibody or antigen-binding portion thereof according to the invention is to express nucleic acid encoding it using nucleic acid in an expression system.

[0158] Thus, in a further aspect, the present invention provides a nucleic acid comprising a sequence encoding an antibody or antigen-binding fragment thereof, or a heavy and / or light chain thereof, according to the first aspect of the invention.

[0159] The present invention further provides isolated nucleic acids encoding the specific monoclonal antibodies of the present invention. Nucleic acids include DNA and RNA. In a preferred embodiment, the present invention provides nucleic acids encoding the specific antibodies or antigen-binding portions thereof of the present invention as defined above. One skilled in the art will be able to determine substitutions, deletions, and / or additions to such nucleic acids that will still provide antibodies or antigen-binding portions thereof in accordance with the present invention.

[0160] The present invention also provides constructs in the form of plasmids, vectors, transcription, or expression cassettes comprising at least one nucleic acid as described above. The present invention also provides recombinant host cells comprising one or more constructs as described above. As noted above, nucleic acids encoding antibodies or antigen-binding portions of the present invention form an aspect of the present invention, as do methods for producing such, which include expression from encoding nucleic acids therefor. Expression may be conveniently achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, the specific monoclonal antibody may be isolated and / or purified using any suitable technique and then used as appropriate.

[0161] 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 available in the art for expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, and many others. Eukaryotic expression in culture is available to those skilled in the art as an option for producing specific monoclonal antibodies; see, e.g., 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.

[0162] Suitable vectors can be chosen or constructed containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences. Vectors may be plasmids or viral, e.g., phages or phagemids, as appropriate. For further details, see, e.g., Sambrook et al. (1989). Many known techniques and protocols for manipulating nucleic acids, such as preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression, and analyzing proteins, are described in detail in Ausubel et al. (1992).

[0163] Thus, according to 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. The antibody or antigen-binding fragment thereof may be recombinantly expressed in any cell selected from CHO, NS0, Sp2 / 0, HEK293, and PER.C6.

[0164] A further aspect of the present invention provides a host cell comprising a nucleic acid as disclosed herein. In one embodiment, the nucleic acid of the present invention is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by including sequences that facilitate recombination with the genome, according to standard techniques.

[0165] In a further aspect, the invention provides a method for preparing a specific antibody or antigen-binding fragment of the invention, comprising expressing the nucleic acid under conditions that result in expression of the antibody or antigen-binding fragment in a cell, and recovering the antibody or antigen-binding fragment.

[0166] The specific antibodies or antigen-binding fragments according to the invention may be used in methods of treatment of the human or animal body, for example in methods of treating ischemia in a patient (preferably a human), comprising administering to said patient an effective amount of a specific antibody or antigen-binding fragment of the invention. [Brief explanation of the drawings]

[0167] [Figure 1] PCR using several combinations of Ig variable domain primers. [Figure 2] Sequence alignment of heavy chains using Clustal W 2.1 multiple sequence alignment tool (www.expasy.ch). [Figure 3] 2D representation of the V region of the heavy chain CDR loop or collier de perle (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501). Amino acids are indicated by single-letter abbreviations. CDRs assigned using the IMGT numbering system are constrained by amino acids indicated by boxes (anchor positions) belonging to the adjacent FR-IMGT. Hatched circles correspond to missing positions according to the IMGT-specific numbering. Arrows indicate the orientation of the beta strands and their different designations in the 3D structure (from the IMGT repertoire, http: / / imgt.cines.fr). [Figure 4] Light chain sequence alignment using Clustal W 2.1 multiple sequence alignment tool (www.expasy.ch). [Figure 5] 2D representation of the V region of the light chain CDR loop or collier de perle (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501). Amino acids are indicated by single-letter abbreviations. CDRs assigned using the IMGT numbering system are constrained by amino acids indicated by boxes (anchor positions) belonging to the adjacent FR-IMGT. Hatched circles correspond to missing positions according to the IMGT-specific numbering. Arrows indicate the orientation of the beta strands and their different designations in the 3D structure (from the IMGT repertoire, http: / / imgt.cines.fr). [Figure 6]4-20% denaturing, reduced (A) and non-reduced (B) SDS-PAGE analysis of Ab 1126 HC0 LC0 to HC1 LC5 #250719. Molecular weight markers are in kilodaltons. Lanes are as follows: [Table 8] [Figure 7] 4-20% denaturing, reduced (A) and non-reduced (B) SDS-PAGE analysis of Ab 1126 HC2 LC1 to HC2 LC5 #250719 and #080819. Molecular weight markers are in kilodaltons. Lanes are as follows: [Table 9] [Figure 8] 4-20% denaturing, reducing (A) and non-reducing (B) SDS-PAGE analysis of Ab 1126 HC3 LC1 to HC3 LC5 #250719. Molecular weight markers are in kilodaltons. Lanes are as follows: [Table 10] [Figure 9] 4-20% denaturing, reducing (A) and non-reducing (B) SDS-PAGE analysis of Ab 1126 HC4 LC1 to HC4 LC5 #250719. Molecular weight markers are in kilodaltons. Lanes are as follows: [Table 11] [Figure 10] 4-20% denaturing, reduced (A) and non-reduced (B) SDS-PAGE analysis of Ab 1126 HC5 LC1 to HC5 LC5 #250719. Molecular weight markers are in kilodaltons. Lanes are as follows: [Table 12] [Figure 11]Characterization of IgG:antigen interactions. Reference-corrected BLI binding curves (black) monitored on a non-covalently immobilized antibody (variant IDs indicated below the sensorgram) surface for various antigen concentrations in running buffer at 25 °C. The apparent dissociation rate constant (kd) and association rate constant (ka) were determined by globally fitting a 1:1 binding model to the sensorgram using the software provided with the instrument. The results of the global fitting are summarized in Table 2. [Figure 12] 4-20% denaturing, reducing and non-reducing SDS-PAGE analysis of Ab 1126 HC0 LC0 lot #011020. Molecular weight markers are in kilodaltons. Lanes are as follows: [Table 13] [Figure 13] 4-20% denaturing, reducing and non-reducing SDS-PAGE analysis of Ab 1126 HC4 LC2 lot #061020. Molecular weight markers are in kilodaltons. Lanes are as follows: [Table 14] [Figure 14] 4-20% denaturing, reducing and non-reducing SDS-PAGE analysis of Ab 1126 HC5 LC2 lot #061020. Molecular weight markers are in kilodaltons. Lanes are as follows: [Table 15] [Figure 15]Analytical gel filtration elution profile observed for Ab 1126 HC0 LC0. Sample Ab 1126 HC0 LC0 lot 011020 was analyzed and a chromatogram was obtained. The chromatogram is displayed, and integrated results are shown in the corresponding table. The peak corresponding to the monomer fraction (based on the retention of the control protein [see Figures 18 and 19]) is greater than 98%. SEC analysis of Ab 1126 variant HC0 LC0 using a Superdex 200 Increase 10 / 300 GL column (see Figure 18). Acetone control samples were run before and after the samples and were identical within experimental error (see Figure 19). [Figure 16] Analytical gel filtration elution profile observed for Ab 1126 HC4 LC2. Sample Ab 1126 HC4 LC2 lot 061020 was analyzed and a chromatogram was obtained. The chromatogram is displayed, and integrated results are shown in the corresponding table. The peak corresponding to the monomer fraction (based on the retention of the control protein [see Figures 18 and 19]) is greater than 98%. SEC analysis of Ab 1126 variant HC4 LC2 using a Superdex 200 Increase 10 / 300 GL column (see Figure 18). Acetone control samples were run before and after the samples and were identical within experimental error (see Figure 19). [Figure 17] Analytical gel filtration elution profile observed for Ab 1126 HC5 LC2. Sample Ab 1126 HC5 LC2 lot 061020 was analyzed and a chromatogram was obtained. The chromatogram is displayed, and integrated results are shown in the corresponding table. The peak corresponding to the monomer fraction (based on the retention of the control protein [see Figures 18 and 19]) is greater than 98%. SEC analysis of Ab 1126 variant HC5 LC2 using a Superdex 200 Increase 10 / 300 GL column (see Figure 18). Acetone control samples were run before and after the samples and were identical within experimental error (see Figure 19). [Figure 18]HMW calibration kit. Calibration data for Superdex 200 Increase 10 / 300 GL. [Table 16] [Figure 19] SEC analysis of an acetone control using a Superdex 200 Increase 10 / 300 GL column. SEC analysis of an acetone control sample performed before and after sample analysis. [Figure 20] A model of the humanized VH1 / VL1 Fv domain predicted by Abodybuilder. Potential sequence hurdles are circled (N-glycosylation, Met oxidation, Trp oxidation, Asp isomerization). The same potential sequence hurdles are present in all humanized variants. [Figure 21] (A) Illustrative diagram of anti-VEGF-A, anti-VEGF165b, VEGF165b, and rh-Fc-VEGFR2. (B) Inhibitory effect of anti-VEGF165b (HC4 LC2) on the binding of VEGF165b to fc-VEGF receptor 2. [Figure 22] Cell migration assay of chimeric anti-VEGF165b (HC0 LC0) and humanized anti-VEGF165b (HC4 LC2 Vexobicizumab) and mouse anti-VEGF165b antibody (mα165b) in the presence of VEGF165a. [Figure 23] Cell migration assay of mα165b, chimeric anti-VEGF165b (HC0 LC0), humanized anti-VEGF165b (HC4 LC2 Vexobicizumab), and a previously reported murine anti-VEGF165b antibody (MRVL56 / 1, AbCam) in the presence of VEGF165a. B. IC50 of each antibody (Vex = HC4 LC2). [Figure 24] (A) Schematic of cell migration assay for diabetic PAD human monocyte-mediated endothelial cell migration inhibition. (B) Results of cell migration assay. [Figure 25] Schematic diagram of the mouse experiment described in Example 4. [Figure 26]Murine analysis of revascularization during hindlimb ischemia. Example laser speckle blood flow images of a mouse fed a high-fat, high-sucrose (HFHS) diet for 12 weeks, taken before (pre-op) and after (post-op) femoral artery ligation, and on subsequent days. [Figure 27] Graphical quantification of murine analysis of blood flow in hindlimb ischemia. Speckle intensity was calculated as ipsilateral (ischemic) blood flow compared to contralateral flow. Blood flow relative to pre-operative ratio. **=p<0.05 compared to same-day IgG, two-way ANOVA. [Figure 28] (A) Example of mouse gastrocnemius muscle 28 days after ischemia and treatment with either IgG or vexobicizumab. (B) Angiogenesis was calculated as ipsilateral (ischemic) vessels relative to contralateral IB4+ vessels. (C) Arteriogenesis was calculated as ipsilateral (ischemic) vessels relative to contralateral smooth muscle actin (SMA)+ vessels. **=p<0.01, ***=p<0.001 compared to IgG, unpaired t-test. [Figure 29] Schematic of the rat experiment described in Example 4. [Figure 30] Rat analysis of revascularization during hindlimb ischemia. Example laser speckle images of paw blood flow in a rat before (pre-op) and after (post-op) femoral artery ligation. Speckle intensity was calculated as ipsilateral (ischemic) blood flow compared to contralateral flow. [Figure 31] Graphical quantification of rat analysis of blood flow in hindlimb ischemia, compared to same day IgG *=p<0.05, #=p<0.05 vs. pre-op, ###=P<0.001 vs. pre-op, two-way ANOVA. [Figure 32] (A) Example of STZ rat gastrocnemius muscle 28 days after ischemia and treatment with either IgG or vexobicizumab. (B) Angiogenesis was calculated as ipsilateral (ischemic) vessels relative to contralateral IB4+ vessels. (C) Arteriogenesis was calculated as ipsilateral (ischemic) vessels relative to contralateral smooth muscle actin (SMA)+ vessels. **=p<0.01, ***=p<0.001 compared to IgG, unpaired t-test. [Figure 33]A. Human monocytes inhibit endothelial cell migration across a porous membrane. 1 μg / mL of HC4 LC2 significantly reversed this inhibitory effect on human umbilical vein cell migration. N=3, cells from a single subject. One-way ANOVA. B. Monocytes from seven patients with peripheral vascular disease inhibit migration toward VEGF-A165a. C and D. Migration is increased by 1 μg / mL and 5 μg / mL of HC4LC2. Paired t-test. *=p<0.05, **=p<0.01 [Figure 34] A single gene can produce opposing isoforms of VEGF: proangiogenic VEGF-A165a and antiangiogenic VEGF-A165b. [Figure 35] Key showing antibody derivation from parental hybridomas. [Figure 36] To measure the neutralizing effect of antibodies, VEGFR2 was bound to anti-VEGF-A165b antibodies mixed with rhVEGF-A165b on ELISA plates and increasing concentrations of VEGF-A165b antibody. Both humanized (HC4LC2) and chimeric (HC0LC0) anti-VEGF165b antibodies were able to significantly reduce the affinity of VEGF165b for VEGF receptor 2 (VEGFR2), although to a slightly lower extent than the positive control, G6-31 (a pan-VEGF antibody that binds to the receptor-binding site of VEGF-A). [Figure 37] Cell migration assay in the presence of VEGF165a using humanized anti-VEGF165b antibody (HC5 LC2) and mouse anti-VEGF1 65b antibody (mα165b, indicated as "56 / 8" in the figure). [Example]

[0168] Example 1: Monoclonal sequencing mRNA was extracted from the hybridoma cell pellet. Total RNA was extracted from the pellet using Fusion Antibodies' in-house RNA extraction protocol, the Pie method. cDNA was reverse transcribed from the RNA using oligo(dT) primers. PCR reactions were set up using variable domain primers to amplify both the VH and VL regions of the monoclonal antibody DNA. Agarose gel electrophoresis of the amplified PCR products is detailed in Figure 1.

[0169] The VH and VL products were cloned into the Thermo Fisher sequencing vector pCR™2.1. The top 10 cells were transformed with the cloned plasmids and screened by PCR for positive transformants. Selected colonies were picked and analyzed by DNA sequencing on an ABI3130xI genetic analyzer; the results can be seen in Figure 2, and the sequences are reproduced below. heavy chain VH2.2 protein (SEQ ID NO: 38) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPPVYP VH2.2 nucleic acid (SEQ ID NO: 39) GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGC CATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATAGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTTTATCCT ·VH2.4 Protein EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPSVYP ·VH2.4 nucleic acid GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGC CATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATAGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTCTATCC VH2.5 protein EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWRSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLA ·VH2.5 Nucleic acid(SEQ ID NO:43) GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGAGGAGTTGGGTCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGTAGTACGATAAACTATACGCCAT CTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTCTATCCCTTGGC VH2.7 protein EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKLIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF ·VH2.7 nucleic acid GAGGTGAAGCTTCTCGAATCTGGAGGTGGTCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTAAATCATCTCCAGAGAC AACGCCAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGATGCCTTGTCAAAGGTTATTTC ·VH2HB.1 protein(SEQ ID NO:46) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPKGELEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPPVYPLA ·VH2HB.1 nucleic acid(SEQ ID NO:47) GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGAGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTTTATCCATTGGCC · VH2HB.2 protein (SEQ ID NO: 48) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF · VH2HB.2 nucleic acid (SEQ ID NO: 49) GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCCGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAAGGTTATTTC · VH2HB.3 protein (SEQ ID NO: 48) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF · VVH2HB.3 nucleic acid (SEQ ID NO: 50) GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTTGTCAAAGGTTATTTC · VH2HB.4 protein (SEQ ID NO: 51) EVKLLESGGGLVQPGGSPKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF · VH2HB.4 nucleic acid (SEQ ID NO: 52) GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCCGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGAC AACGCCAAAAATAGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGATGCCTGGTCAAAGGTTATTTC VH2HB.5 protein EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPPVYPLV ·VH2HB.5 Nucleic Acid GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCAT CTGTAAAGGATAAATTCATCATTCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTCTATCCCTTTGGTC VH2HB.6 protein EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF ·VH2HB.6 Nucleic acid GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGAC AACGCCAAAAATAGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGGTTATTC VH4HB.1 protein EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDSAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF ·VH4HB.1 Nucleic acid(SEQ ID NO:57) GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGAGAC AGCGCCAAAAATACGCTGTACCTGCAAATGAGCGAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGGGCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTC Consensus (SEQ ID NO: 58) (positions in brackets indicate possible substitutions at that position and X = blank) EVKLLESGGGLVQPGGS(L / P)KLSCAASGFDFSRYW(M / R)SWVRQAPGK(G / E)LEWIGEIHPYSSTINYTPSVKDK(F / L)IISRD(N / S)AKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVS AAKTTPP(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) Consensus sequence (SEQ ID NO: 48) with the most common amino acid indicated at each position (variable domains are highlighted in bold, sequence primers are underlined, and CDRs are double underlined) [Table 17]

[0170] The complementarity-determining regions (CDRs), shown double-underlined, were determined by the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-272 (1999)). A consensus amino acid sequence from VH sequencing was obtained from at least five amino acid sequences, as shown by the alignment in Figure 2. A graphical representation of the variable domains using IMGT labels is shown in Figure 3. Light chain VL2.1 protein (SEQ ID NO: 137) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSEQLTSGGASVVCFLNNF VL2.1 nucleic acid (SEQ ID NO: 149) GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCA GGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCT VL2.2 protein (SEQ ID NO: 138) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYNLWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVC VL2.2 nucleic acid GATATTGTGATCAGCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCA GTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTGCGTGTGC VL2.3 protein DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK VL2.3 nucleic acid GATATTGTGATCAGCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCCGGAGGTGCCTCAGTCGTGTGCTTCTTGAACACTTCTACCCCAAA VL2.6 protein DIVMTQTPLTLSVTIGQPASISCKSSQCLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR VL2.6 nucleic acid GATATTGTGATCGACCGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGTGCCTCTTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACGTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACACTTCTACCCAGA VL2.7 protein DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR VL2.7 nucleic acid GATATTGTGATCAGCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGACCAAGGTGGAAATAAAACGGGCTGTGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACACTTCTACCCCAGA VL2.8 protein DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR VL2.8 nucleic acid GATATTGTGATCAGCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGACCAAGGTGGAAATAAAACGGGCTGTGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACACTTCTACCCCAGA ·VL2HB.2 protein(SEQ ID NO:143) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK VL2HB.2 nucleic acid GATATTGTGATCAGCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTCCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACACTTCTACCCCAAA ·VL2HB.3 protein(sequential number:144) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK VL2HB.3 nucleic acid GATATTGTGATCAGCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTCCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACACTTCTACCCCAAA ·VL2HB.4 protein(sequential number:145) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR VL2HB.4 nucleic acid GATATTGTGATCAGCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGACCAAGGTGGAAATAAAACGGGCTGTGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACACTTCTACCCCAGA ·VL2HB.7 protein(sequential number:146) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPR VL2HB.7 nucleic acid GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGACCAAGGTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGA Consensus (SEQ ID NO: 147) (positions in brackets indicate possible substitutions at that position) 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)LIYLVS KLDSGVPDRF(T / S)GSG(S / A)GTDFTLKISRVE(A / P)ED(L / V)GVYYCWQGTHFPYTFG(G / Q)GTK(V / L)E(I / V)KRADAAPTVSIFPPSEQLTSGGASVVCFLNNFYPR Consensus sequence (SEQ ID NO: 148) with the most common amino acid at each position (variable domains are highlighted in bold, sequence primers are underlined, and CDRs are double underlined) [Table 18]

[0171] The double-underlined CDRs were determined by the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-272 (1999)). A consensus amino acid sequence from VH sequencing was obtained from at least five amino acid sequences, as shown by the alignment in Figure 4. A graphical representation of the variable domains using IMGT labels is shown in Figure 5.

[0172] Example 2: Transient expression, purification, and affinity screening of variants (25+1) of Ab 1126 humanized anti-VEGF-A165b clone mα165b (also referred to herein as 56 / 8 / 31 / 1 / 16) overview The Ab 1126 humanized variants are 150 kilodalton (kDa) antibodies composed of two heavy chains and two light chains conjugated via disulfide bonds. Mammalian expression vectors encoding each variant were transfected into CHO cells, and batch cultures of each variant were performed for up to 7 days. The expressed antibodies were purified from cell culture supernatants via affinity chromatography. Concentration and purity were measured for the purified antibody products. The results of these quality control experiments are detailed below.

[0173] Expression and purification of Ab 1126 DNA encoding the amino acid sequence of Ab 1126 variant (see sequence below) was synthesized and cloned into the mammalian transient expression plasmid pETE V2 (proprietary of Fusion Antibodies). Ab 1126 variant was expressed using a CHO-based transient expression system, and the resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. Ab 1126 variant was purified from the cell culture supernatant via affinity chromatography (using a state-of-the-art AKTA chromatography device). The purified antibody was dialyzed / buffer-exchanged into phosphate-buffered saline solution. The purity of the antibody was determined by reducing and denaturing sodium dodecyl sulfate polyacrylamide gels and was greater than 95% (Figures 6 to 10). The antibody concentration was determined by measuring absorbance at 280 nM, using the antibody's standard extinction coefficient of 205,500 M. -1 cm -1 (or an A280 of 1.0 mg / ml = 1.37 [assuming MW = 150,000 Da]). Details of the purified antibody products supplied are summarized in Table 1, and the derivation of the antibody sequences from the parental hybridomas is summarized in Figure 35.

[0174] Ab 1126 variant sequence (signal peptides used for expression of antibody heavy and light chains are underlined and may be omitted from the heavy and light chain sequences): >Ab 1126 HC0 (SEQ ID NO: 71, with signal peptide) MGWTLVFLFLLSVTAGVHSEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVT VSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC0 (SEQ ID NO: 125, no signal peptide) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVT VSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC1 (SEQ ID NO: 72, with signal peptide) MGWTLVFLFLLSVTAGVHS EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC1 (SEQ ID NO: 126, no signal peptide) EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC2 (SEQ ID NO: 73, with signal peptide) MGWTLVFLFLLSVTAGVHS EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFIISRDNAKNSVYLQLNSLRAEDTAVYYCARAFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC2 (SEQ ID NO: 127, no signal peptide) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFIISRDNAKNSVYLQLNSLRAEDTAVYYCARAFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC3 (SEQ ID NO: 74, with signal peptide) MGWTLVFLFLLSVTAGVHS EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWISEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRDEDTALYYCARAFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC3 (SEQ ID NO: 128, no signal peptide) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWISEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRDEDTALYYCARAFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC4 (SEQ ID NO: 75, with signal peptide) MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC4 (SEQ ID NO: 129, no signal peptide) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC5 (SEQ ID NO: 76, with signal peptide) MGWTLVFLFLLSVTAGVHS EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVT VSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 HC5 (SEQ ID NO: 130, no signal peptide) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVT VSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Ab 1126 LC0 (SEQ ID NO: 77, with signal peptide) MVSSAQFLGLLLLCFQGTRC DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC0 (SEQ ID NO: 131, no signal peptide) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC1 (SEQ ID NO: 78, with signal peptide) MVSSAQFLGLLLLCFQGTRC DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC1 (SEQ ID NO: 132, no signal peptide) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC2 (SEQ ID NO: 79, with signal peptide) MVSSAQFLGLLLLCFQGTRC DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC2 (SEQ ID NO: 133, no signal peptide) DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC3 (SEQ ID NO: 80, with signal peptide) MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYCWQGTHFPYTFGGGTKV EVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC3 (SEQ ID NO: 134, no signal peptide) DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYCWQGTHFPYTFGGGTKV EVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC4 (SEQ ID NO: 81, with signal peptide) MVSSAQFLGLLLLCFQGTRC DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGKTYLNWLQQRPGQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC4 (SEQ ID NO: 135, no signal peptide) DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGKTYLNWLQQRPGQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC5 (SEQ ID NO: 82, with signal peptide) MVSSAQFLGLLLLCFQGTRC DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Ab 1126 LC5 (SEQ ID NO: 136, no signal peptide) DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [Table 19]

[0175] Kinetic analysis: overview IgG antibodies were immobilized on a biosensor using an appropriate capture surface, and binding of soluble antigens to the immobilized antibodies was monitored by BLI (Octet). The resulting sensorgrams were analyzed using the manufacturer's software (ForteBio).

[0176] Kinetic analysis: Materials and methods Antigen: Human VEGF165b, Cyno Biologicals, Catalog No. 29656-HNAB, Lot #LC13JU1402 ** Note ** VEGF165b frequently exists as a disulfide-linked homodimer

[0177] Details of the antibodies tested: ·Antibody identifier=HCX LCX Species = Humanized ·Capture method = anti-human IgG Fc (AHC)

[0178] Kinetic analysis: optimization A series of experiments was first performed to optimize the assay parameters (data not shown), resulting in the following parameters: Overview of optimized kinetic screening parameters for characterization of IgG:antigen interactions [Table 20]

[0179] Standard assay optimization experiments using chimeric control antibodies demonstrated that the antigen dissociation rate (kd) was very slow (<1.0 x 10) when using standard IgG capture levels (0.5-1.0 nM). -4 ) was observed.

[0180] Kinetic analysis: experimental parameters Kinetic assays were performed by first capturing IgG using an anti-human Fc biosensor. The mAb-captured biosensor was then immersed in wells containing different concentrations of antigen (binding step), followed by a dissociation step in running buffer. To allow for reference correction, the IgG-captured sensor was immersed in a well containing buffer only. This reference provided a means to correct for spontaneous dissociation of the captured IgG. The 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 ) was calculated using data analysis software manufactured by ForteBio. All consumables used were those recommended by ForteBio.

[0181] Kinetic analysis: Results for Ab 1126 variants All samples were diluted in freshly prepared running buffer. Antibody variants were immobilized on a series of biosensor surfaces using the described capture method (see Materials and Methods). Antigen was passed over the surface, allowing the binding reaction to occur. Binding data for the IgG:antigen interaction were collected on the biosensor at 25°C. A dilution series of antigen was used for the binding step, allowing results to be globally fitted and the k, kd, and K D The reaction data for antigen binding to surface-immobilized IgG were fitted to a 1:1 binding model to obtain a data trace (red, see Figure 11). The kinetic parameters are summarized in Table 2 below. [Table 21] R 2 The value indicates how well the fit correlates with the experimental data, with values ​​above 0.95 being considered a good fit, and X 2 is the sum of squared deviations, which should generally be less than 3 (ideally <0.5). X 2 is a measure of the error between the experimental data and the fitted line. X 2 A smaller R indicates a better fit. max should be equivalent to that of the chimeric control (±20%).D should be within 2-fold of the chimeric control antibody HC0LC0. Abbreviations are as follows: NF, does not fit to a 1:1 binding model.

[0182] conclusion All Ab 1126 variants have been successfully expressed and purified.

[0183] SDS-PAGE analysis showed that all antibodies exhibited an appropriate level of purity. Under denaturing and reducing conditions, both the antibody heavy and light chains were visible and were observed at the expected molecular weights of approximately 50 kDa and 25 kDa, respectively. Under denaturing and non-reducing conditions, a single major band and several minor bands were observed. Additional bands (impurities) were likely the result of non-glycosylated IgG and IgG degradation products (e.g., a single [partial] light chain, a combination of two heavy chains and one light chain, two heavy chains, two heavy chains and one light chain).

[0184] Kinetic (Octet) Analysis: Both HCX LC1 and HCX LC2 exhibited binding to the antigen, and in most cases, the experimental data fit well to a 1:1 binding model. Under the experimental conditions used here, HC5 LC2 and HC4 LC2 exhibited binding properties similar to those of the chimeric control antibody HC0 LC0, with dissociation constants within 2-fold of those of the chimeric control antibody HC0 LC0.

[0185] Example 3: Transient expression of Ab 1126 variants, HC0 LC0, HC4 LC2, and HC5 LC5 (20 mg scale-up) overview The Ab 1126 variants, HC0 LC0, HC4 LC2, and HC5 LC2, are 150 kilodalton (kDa) antibodies composed of two heavy chains and two light chains linked via disulfide bonds. Mammalian expression vectors encoding each antibody were transfected into CHO cells. The expressed antibodies were then purified from cell culture supernatants using standard chromatography techniques. The purified antibody products were assayed for concentration, purity, and endotoxin.

[0186] Expression and purification of Ab 1126 variants DNA encoding the amino acid sequence of each antibody was synthesized and cloned into the mammalian transient expression plasmid pETE V2 (proprietary of Fusion Antibodies). The antibodies were expressed using a CHO-based transient expression system, and the resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. The antibodies were purified from the cell culture supernatant using affinity chromatography and preparative-grade size-exclusion chromatography (using a modern AKTA chromatography system). The SEC column was pre-equilibrated with phosphate-buffered saline. The purified antibodies were buffer-exchanged into phosphate-buffered saline. The purity of the antibodies was determined to be greater than 95% by reducing and denaturing sodium dodecyl sulfate polyacrylamide gel (Figures 12 to 14).

[0187] Antibody samples were analyzed by size exclusion chromatography (SEC). Each chromatogram showed a single major peak (over 95% of the total area [Figures 15 to 17]). Bacterial endotoxin levels were measured using the Endosafe®-PTS system and Endosafe® PTS cartridges (Charles River Laboratories).

[0188] Antibody concentrations were determined by measuring absorbance at 280 nM and calculated using the antibody's standard extinction coefficient of 205,500 M cm (or an A of 1.0 mg / ml = 1.37 [assuming MW = 150,000 Da]). Details of the purified antibody products supplied are summarized in Table 3. [Table 22] * Purity was determined by analysis (relative band intensity) of Coomassie Blue stained reducing and denaturing SDS polyacrylamide gels.

[0189] conclusion The antibody has been successfully expressed and purified.

[0190] SEC analysis was successfully performed on all antibodies, and the chromatograms show one major peak (over 98% of the total area).

[0191] SDS-PAGE analysis showed that all antibodies exhibited adequate levels of purity under reducing and denaturing conditions. Under reducing conditions, both the antibody heavy and light chains were visible and were observed at the expected molecular weights of approximately 50 kDa and 25 kDa, respectively. Under non-reducing conditions, a single major band and several minor bands were observed. Additional bands (impurities) were likely the result of non-glycosylated IgG and IgG degradation products (e.g., a single [partial] light chain, a combination of two heavy chains and one light chain, two heavy chains, two heavy chains and one light chain).

[0192] Example 4: Specific isoforms of VEGF-A (VEGF-A 165 b) Evaluation of the suitability of a novel humanized monoclonal antibody against IL-1 as an antibody therapy for peripheral arterial disease [Table 23]

[0193] Background technology Peripheral arterial disease (PAD) is a major unmet clinical need affecting approximately 10 million people in the United States. Vascular insufficiency in PAD leads to tissue ischemia. In response, circulating monocytes produce vascular endothelial growth factor A (VEGF-A). In PAD patients and mouse models of metabolic ischemia, a decrease in soluble frizzled-related protein 5 (sfrp5) increases Wnt5a activity, resulting in the production of the antiangiogenic isoform VEGF-A. 165 b is overexpressed. 165Antibodies against b have been shown to reverse impaired revascularization in a mouse model of hindlimb ischemia. Limb ischemia in PAD leads to painful, disabling, and non-healing ulcers, resulting in 200,000 amputations annually in the United States alone. Limb ischemia induced by arterial occlusive lesions is exacerbated by insufficient angiogenesis and collateral formation.

[0194] Recent studies have identified an anti-angiogenic splice variant of VEGF-A, VEGF-A 165 b has been shown to be expressed in clinical and experimental settings associated with impaired angiogenesis 1 VEGF-A 165 b is VEGF-A 165 Circulating VEGF-A in patients with PAD is a competitive inhibitor of VEGF-A 165 In mouse experimental models, mVEGF-A 165 b Expression is elevated under conditions of systemic metabolic dysfunction. VEGF-A 165 The b isoform is PAD 2 In experimental models, mVEGF-A impaired revascularization. 165 Acute antibody neutralization of the b isoform promotes revascularization of ischemic tissues under conditions in which regenerative angiogenesis is impaired. These findings support the role of VEGF-A in the regulation of VEGF-A. 165 These findings support the notion that b may represent a novel pharmacological target for treating limb ischemia in patients with PAD, and its neutralization may enhance the activity of pro-angiogenic growth factors. 3 .

[0195] Introduction For the treatment of PAD, VEGF-A, an isoform of VEGF, 165 The goal is to develop a first-in-class humanized monoclonal antibody against VEGF-A. 165 A mouse monoclonal antibody specific for VEGF-A 165 b, The last six (CDKPRR) are VEGF-A 165A synthetic peptide fragment of the carboxy-terminal sequence of 9 amino acids specific to b was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. 4 One of the resulting hybridomas expressed VEGF-A 165 b specifically detects VEGF-A 165 a was not detected. One mouse anti-VEGF-A 165 Twenty-five humanized variants of the b-neutralizing mAb were generated, transiently expressed in CHO cells, and purified from culture supernatants by single-column protein A affinity chromatography.

[0196] As part of this study, wild-type C57BL6 mice were fed a high-fat diet for 12 weeks and had their left femoral arteries ligated. Blood flow to the paw was measured by Moor FLPI-2 laser speckle imaging before and after left femoral artery resection, and on days 3, 7, 14, 21, and 28 after surgery. Animals were injected with humanized anti-VEGF-A on days 0, 3, and 7. 165 b (1 mg / kg, ip) or control antibody (human IgG) was administered (see Figure 25 for a schematic diagram). The ratio of ischemic to non-ischemic LDBF was calculated and plotted against time to determine blood flow recovery. Muscles were stained to measure capillary and arteriolar density. Animals that achieved less than 60% inhibition of blood flow were excluded. ZDF diabetic rats were similarly treated and blood flow was measured.

[0197] result The affinity ranking of the variants was compared with that of commercially available VEGF-A 165 b) Biolayer interferometry (BLI) was performed using the protein. The two variants, HC4 LC2 and HC5 LC2, had affinity (KD) within two-fold of the HC0 LC0 chimeric control antibody (KD = 600, 600, and 300 pM, respectively). The new antibodies showed a significant improvement in VEGF activity. 165 b) exhibited over 100-fold higher affinity than the original murine antibody against b, with slower off-rates and comparable on-rates. See Figure 11.

[0198] Furthermore, an exemplary antibody clone, HC4 LC2, inhibits VEGF 165 b to VEGF receptor 2 (VEGFR2) to a degree slightly lower than that of the positive control G6-31, an antibody that binds to the receptor binding site (see Figure 21B).

[0199] These two humanized variants also inhibited VEGF-A in vitro. 165 The humanized variants were able to inhibit monocyte-mediated cell migration inhibition more effectively than the parental murine monoclonal antibody and the chimeric antibody (see Figures 22 and 37, and Figure 23). Furthermore, the humanized variants were also able to reverse human monocyte-mediated endothelial migration inhibition in diabetic peripheral arterial disease (PAD) (see Figure 24B).

[0200] Further studies showed that human monocyte-mediated inhibition of endothelial cell (exemplified by human umbilical vein cells) migration across porous membranes could be significantly reversed with 1 μg / ml of HC4 LC2 (see Figure 33A). Monocytes from seven patients with peripheral vascular disease expressed VEGF-A. 165 a) (see Figure 33B). Migration is increased with 1 μg / ml (see Figure 33C) and 5 μg / ml (see Figure 33D).

[0201] Blood flow recovery in mice administered IgG did not return to normal after 28 days (contralateral: 66±6% on day 14, 29±8.7% on day 21, and 75±8% on day 28, N=6). This impaired revascularization was due to the humanized anti-VEGF-A antibody. 165 The HFHS-obese mice were rescued by the b antibody (78±5% on day 14, 90±4.6% on day 21, and 102±3.5% on day 28, N=7). See Figures 26 and 27. Furthermore, improvements in angiogenesis and arteriogenesis were observed in HFHS-obese mice (see Figures 28A to 28C).

[0202] These effects were also seen in diabetic rats, a schematic of which can be seen in Figure 29, resulting in restored blood flow as shown in Figures 30 and 31, and improved angiogenesis and arteriogenesis as shown in Figures 32A to 32C.

[0203] conclusion Obesity leads to impaired collateral revascularization and insufficient angiogenesis in a PAD model. 165 The reversal of this condition with anti-β antibody suggests that this antibody may be useful in treating revascularization failure in PAD patients.

[0204] Example 5: Humanization of anti-VEGF-A165b mα165b The sequences of the heavy chain (HC) variable region (mVH) and light chain (LC) variable region (mVL) of the parent murine monoclonal antibody (clone 58 / 8 / 31 / 1 / 13) are as follows: Amino acid sequence of clone 58 / 8 / 31 / 1 / 13 mVH (SEQ ID NO: 15) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA Amino acid sequence of clone 58 / 8 / 31 / 1 / 13mVL (SEQ ID NO: 23) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR

[0205] IMGT database of human antibody sequences 6 From the blast analysis, mouse antibody (IGHV4-1 * It was revealed that the heavy chain CDRs (HCDRs, SEQ ID NOs: 1-3) from 02 (see Blast 1 below) were grafted onto the human IGHV3-11 framework (see Blast 2 below).

[0206] Blast 1 (Blast of the HC1 VH region against the IMGT database shows 82% identity to the closest mouse germline): >9323 AJ851868 Mus musculus IGHV4-1* 02 VH Length=98 Score = 179 bits (453), Expected = 5e-47 Identity=81 / 98(82%), Positivity=94 / 98(95%) Query:1 EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTINY 60 EV+LLESGGGLV+PGGSL+LSCAASGFDFSRYWMSW+RQAPGKGLEW+ EI+P SSTINY Subject: 1 EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINY 60 Query: 61 TPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 98 (SEQ ID NO: 83) TPS+KD+F ISRDNAKN+LYLQM+ +R+EDTA+YYCAR (SEQ ID NO: 122) Target: 61 TPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR 98 (SEQ ID NO: 84)

[0207] Blast 2 (Blast of the HC1 VH region against the IMGT database shows 86% identity to the closest human germline): >2883 X92287 Homo sapiens IGHV3-11 * 03 VH Length=98 Score = 169 bits (429), Expected = 3e-44 Identity=85 / 98(86%), Positivity=87 / 98(88%) Query:1 EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTINY 60 +VQLLESGGGLVKPGGSLRLSCAASGF FS Y+MSWIRQAPGKGLEWVS I SS NY Target: 1 QVQLLESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSSSYTNY 60 Query: 61 TPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 98 (SEQ ID NO: 83) SVK RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 123) Target: 61 ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 98 (SEQ ID NO: 85)

[0208] The V kappa light chain of the parent antibody is IGKV1-135 * Belongs to the 01 family. IMGT database of human antibody sequences 7 Blast analysis of revealed that the light chain CDRs from the murine antibody were grafted onto the human IGKV2-30 framework (see Blast 3 below).

[0209] Blast3 (BLAST of the LC1 VH region against the IMGT database) identified the closest human antibody VH sequence (IGKV2-30 * 02) (showing 91% identity): >11075 FM164408 Homo sapiensIGKV2-30 * 02 V-KAPPA Length=100 Score = 186 bits (472), Expected = 3e-49 Identity=91 / 100(91%), Positivity=93 / 100(93%) Query:1 DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLD 60 DVVMTQSPLSLPVTLGQPASISC+SSQSL+SDG TYLNWFQQRPGQSPRRLIY VS D Target: 1 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRD 60 Query: 61 SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHEP 100 (SEQ ID NO: 86) SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QGTH P (SEQ ID NO: 124) Target: 61 SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWP 100 (SEQ ID NO: 87)

[0210] Humanization Check Humanized variants follow the WHO definition of humanized antibodies. 11 The humanized chains were checked for their humanization according to the following criteria: the variable domains of the humanized chains, when analyzed as a whole, have V-region amino acid sequences that are closer to those of humans than to those of other species (assessed using the Immunogenetics Information System® (IMGT®) DomainGapAlign tool). 12 The results are shown in Table 5. All variants can be considered humanized. The approved INN name of the therapeutic candidate is vexobicizumab. [Table 24]

[0211] Antibody characterization Twenty-five humanized full-length antibody variants, designated HCX LCX (X = 1–5), were generated by combining each variant light chain (LC1–5) with each variant heavy chain (HC1–5). Furthermore, chimeric versions of the antibodies (designated HC0 LC0) were generated by grafting the full-length mouse VH and VL domains onto the human IgG1 heavy chain constant region (CH) and human kappa LC constant region (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.

[0212] The antibody yields (mg / L) were comparable to or higher than those of the chimeric antibody HC0 LC0, except for HC1 LC1 and HC3 LC4. The yields of antibodies expressed with LC3, LC4, or LC5 light chains were approximately 16% lower overall than those of the LC1 and LC2 antibodies (n=1). Although some degradation products or contaminants were observed on non-reducing gels for HC1 LC2, HC1 LC4, HC3 LC4, HC4 LC2, and HC4 LC5, all antibodies appeared intact on SDS-PAGE.

[0213] Kinetic analysis of each antibody was performed by biolayer interferometry (BLI) using the ForteBio Octet system. The antibody was captured on an anti-human Fc biosensor and then assayed with commercially available human VEGF-A (purchased from Cyno Biologicals). 165 b. The level of capture of each antibody to the Fc biosensor was comparable. Kinetic analysis showed that the HCX LC1 and HCX LC2 antibodies all exhibited binding properties similar to that of the chimeric control antibody HC0 LC0. Using a 1:1 binding model, it was not possible to determine the k a and k d values ​​of the other antibodies. Under the assay conditions, VEGF-A 165 The affinity of HC0 LC0 (0.3 nM) for binding to b was two-fold higher than that of HC4 LC2 and HC5 LC2 (0.6 nM) and three-fold higher than that of HC4 LC1 (0.9 nM). Antibodies with affinities greater than 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).

[0214] Subsequently, HC0 LC0, HC4 LC2HC5, and LC2 were scaled up. Antibodies were transiently expressed in CHO cells and purified from the culture supernatant by protein A affinity chromatography. The purified antibodies were buffer-exchanged into PBS and analyzed by SDS-PAGE and size-exclusion chromatography. Purity was estimated to be >95% by non-reducing SDS-PAGE. SEC (n=1) detected a single peak corresponding to the monomer fraction (>98%) at a similar retention time for all antibodies. Endotoxin content was measured at <1.0 EU / mg, meeting the criteria for in vivo administration.

[0215] In vitro and in vivo testing In the endothelial cell migration assay, VEGF-A, when tested at 1 μg / ml, 165 The rank order of potency in neutralizing the antiangiogenic effect of mAb b was HC4 LC2 > HC5 LC2 > HC0 LC0 > mAb58 / 6. 165 The IC50 values ​​(% activity) for the inhibition of b were 442 ng / ml, 137 ng / ml and 114 ng / ml for HC0 LC0, HC4 LC2 and HC5 LC2, respectively.

[0216] The chimeric antibody HC0 LC0, administered ip at 1 mg / kg on days 1, 3, and 7 after ischemia, can reverse obesity-dependent revascularization impairment for up to 30 days in a murine femoral artery ligation ischemia model. Humanized antibodies have not yet been tested in this model. Proof of concept was recently reported by Navarro et al. 14 VEGF-A 165 In vivo blockade of b promoted neovascularization, reduced infarct size, and enhanced contractile function in a reperfused, but not non-reperfused, myocardial infarction model.

[0217] References 1.Cebe-Suarez,S.,Zehnder-Fjaellman,A.&Ballmer-Hofer,K.The role of VEGF receptors in angiogenesis;complex partnerships.Cell.Mol.Life Sci.63,601-615(2006)。 2.Kikuchi,R.et al.An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease.Nat.Med.20,1464-1471(2014)。 3.Carter,J.G.et al.The carboxyl terminus of VEGF-A is a potential target for anti-angiogenic therapy.Angiogenesis 18,23-30(2015)。 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)。 5.Beck,A.&Liu,H.Macro-and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies.(2019)。doi:10.3390 / antib8010018 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)。 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)。 8.Jefferis,R.Recombinant antibody therapeutics:the impact of glycosylation on mechanisms of action.Trends Pharmacol.Sci.30,356-362(2009)。 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)。 10.Jarasch,A.et al.Developability assessment during the selection of novel therapeutic antibodies.J.Pharm.Sci.104,1885-1898(2015)。 11.Jones,T.D.et al.The INNs and outs of antibody nonproprietary names.MAbs 8,1-9(2016)。 12.Ehrenmann,F.,Kaas,Q.&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)。 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)。 14.Rios-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)。 15.Haberger,M.et al.Functional assessment of antibody oxidation by native mass spectrometry.MAbs 7,891-900(2015)。 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)。 17.Igawa,T.et al.Engineering the variable region of therapeutic IgG antibodies.MAbs 3,243-252(2011). 18.Liu,H.,Saxena,A.,Sidhu,S.S.&Wu,D.Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds.Front Immunol 8,38(2017)。 19.Temel,D.B.,Landsman,P.&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)。 20.Chai,Q.,Shih,J.,Weldon,C.,Phan,S.&Jones,B.E.Development of a high-throughput solubility screening assay for use in antibody discovery.MAbs 11,747-756(2019). 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)。 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). 23.Schlothauer,T.et al.Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies.MAbs 5,576-586(2013). 24.Kraft,T.E.et al.Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis.MAbs 12,1683432(2020)。 25.Nugent,P.,Duncan,J.N.&Colagiovanni,D.B.The Preparation of a Preclinical Dossier to Support an Investigational New Drug(IND)Application and First-in-Human Clinical Trial.in A Comprehensive Guide to Toxicology in Preclinical Drug Development 309-334(Elsevier Inc.,2013).doi:10.1016 / B978-0-12-387815-1.00012-5 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). 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.

[0218] Example 6: Sequences for site-directed mutagenesis Overview Anti-VEGF-A containing the VH and VL of the original murine antibody (Ab 1126) 165 b Chimeric versions of the mAb, HC0 LC0, were created. 25 humanized variants were created, of which HC4 LC2 and HC5 LC2 had binding affinities within 2-fold of the chimeric antibody. Numerous sequence imperfections were detected in both the VH and VL of the humanized variants. The next step in the development of humanized antibodies was to attempt to remove potential sequence imperfections through site-directed mutagenesis of the VH and VL regions without significant loss of affinity or potency compared to the chimeric antibody.

[0219] Additionally, mutations in the Fc region of IgG1 are also tested: LALA = L234A L235A * eliminates Fc receptor binding, so YTE=M252Y S254T Y256 * is for half-life extension. The chimeric antibody HC0 LC0 was tested alongside the YTE-only and LALA+YTE variants to ensure that the Fc changes did not affect the antibody's binding to its target. Before creating a further set of antibody variants that combined HC and LC mutations, sequence-disordered mutants were first tested in the wild-type IgG1 format to identify tolerated mutations (no loss of binding or functional activity). * Canonical numbering.)

[0220] List of expressed antibodies: Heavy chain Light chain HC0 YTE LC0 ·HC0 LALA+YTE LC0 HC4 YTE LC2 HC5 YTE LC2 HC4 N59Q LC1 HC5 N59Q LC2 HC4 N59Q LC2 HC4 T61A LC2 HC5 T61A LC2 HC4 LC1 S32A HC4 LC2 S32A HC5 LC2 S32A HC4 LC1 D33E HC4 LC2 D33E HC5 LC2 D33E HC4 LC1 G34A HC4 LC2 G34A HC5 LC2 G34A HC0 LC0 HC4 LC1 HC4 LC2 HC5 LC2

[0221] The antibodies were cloned, expressed, and purified at mg scale. Parameters measured included yield, SDS-PAGE (reduced and non-reduced), analytical SEC, and VEGF-A activity compared to the parental antibodies (HC0 LC0, HC4 LC2, HC5 LC2, and HC4 LC1). 165 b.

[0222] Antibody sequence without signal peptide Residues to be mutated are underlined (Fc domain) or double underlined (variable domain). [Table 25] [Table 26] [Table 27] [Table 28] [Table 29]

[0223] Example 7: Anti-VEGF 165 bAntibody is VEGF 165 inhibits the binding of b to VEGFR2 method A 96-well clear microplate (High Sensitivity ThermoImmunon or Costar 9018) was coated with 1 μg / μl rh-Fc-VEGFR2 (357-KD-050 / CF, R&D Systems) at 100 ml per well and shaken overnight at room temperature. 165 bAntibody / VEGF 165 b The mixture contains 4 ng / ml VEGF-A 165 b and increasing concentrations of each antibody were prepared and incubated for 2 hours at 4°C. VEGFR2-coated plates were washed three times with 0.05% Tween 20 in PBS (PBST). Plates were blocked with Suber block buffer and washed three times. 100 μl of anti-VEGF 165 bAntibody / VEGF 165 The b mixture was added and incubated for 1 hour, followed by three washes. 100 μl of biotinylated anti-VEGF-A (BAF293, R&D Systems) in 1% BSA / PBS was added and incubated for 2 hours at room temperature. After three washes, the plate was incubated with 100 μl of filtered streptavidin-HRP in 1% BSA / PBS. The plate was covered with foil and incubated with shaking at room temperature for 30 minutes. After three washes, tetramethylbenzidine (TMB) substrate was added until leaf color developed. The reaction was stopped with 50 μl of 1M HCl. The optical density was measured using a microplate reader set at 450 nm with a reference wavelength of 620 nm.

[0224] result To measure the neutralizing effect of the antibodies, VEGFR2 was bound to an ELISA plate and anti-VEGF-A165 bIncreasing the concentration of anti-VEGF-A antibody 165 bAntibody and rhVEGF-A 165 b. Humanized (HC4LC2) and chimeric (HC0LC0) anti-VEGF 165 b Both antibodies are VEGF 165 b for VEGF receptor 2 (VEGFR2), but to a slightly lesser extent than the positive control G6-31 (a pan-VEGF antibody that binds to the receptor binding site of VEGF-A) (Figure 36). These results support the findings described in Example 4.

[0225] Example 8: Additional sequences for site-directed mutagenesis Background technology Induction of the complement cascade is associated with adverse reactions at the antibody injection site and off-target cell lysis, also known as complement-dependent cytotoxicity (CDC). Interaction of the Fc domain of an antibody that recognizes a cell surface target can lead to binding of the complement factor C1q, activating the complement cascade and mediating complement-dependent cytotoxicity (CDC), which results in the destruction of the antibody-bound cell. C1q binding is the first step in initiating the complement cascade. C1q forms a complex with the serine proteases C1r and C1s to form the C1 complex. Although C1q can bind to six antibodies, binding to only two IgGs is sufficient to activate the complement cascade. Therefore, eliminating C1q binding to Fc, an early event in the activation of antibody-dependent complement cytotoxicity, can be achieved by site-directed mutagenesis of specific residues in the Fc domain of an antibody.

[0226] context Vexobicizumab is a humanized IgG1 antibody. IgG1 is known to activate complement. However, this activation requires the antibody to cluster on the cell surface through interaction with specific targets (receptors) expressed on the cell surface. Theoretically, vexobicizumab inhibits its target, VEGF-A. 165Since VEGF-A is a soluble molecule, it should not fix complement. However, nonspecific binding of VEGF-A may occur through binding to heparin sulfate proteoglycans (HSPGs) expressed on the surface of non-immune cells, such as endothelial cells, epithelial cells, and fibroblasts. The pro-angiogenic form of VEGF-A (VEGF-A 165 Unlike a), VEGF-A 165 The splice variant of VEGF-A b has been reported to have low or no heparin binding (data not shown) (despite the fact that it is predicted to contain a heparin-binding domain) and therefore does not bind to cell surface heparin sulfate proteoglycans, making it susceptible to complement-mediated lysis. 165 It is unclear whether binding of b to its receptor VEGFR2 mediates receptor internalization, and thus receptor-ligand antibody complexes (transiently) present on the cell surface may trigger Fc-mediated complement activation.

[0227] Surface-bound VEGF-A 165 Mutations could be introduced to reduce the risk of cytopathic off-target effects in specialized epithelial cells of the eye and kidney due to the presence of β. These cells may not be a problem because they are located beyond a semipermeable barrier that normally excludes antibody-sized molecules. Mutations could include single amino acid mutations in the Fc domain, which have been reported to disrupt C1q binding and therefore limit complement activation without affecting binding to the neonatal Fc receptor (FcRn).

[0228] array The following single amino acid mutations in the human IgG1 heavy chain of HC4 LC2 are proposed: · 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). · 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). · 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).

[0229]

Table 30

Table 31

Claims

1. An antibody or antigen-binding fragment thereof having specificity 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 (VEGF-Ax and VEGF １８９ b, etc.).

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

3. The antibody or antigen-binding fragment thereof has 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 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising or consisting of CDR3: WQGTHFPYT (SEQ ID NO: 6).

4. The antibody or antigen-binding fragment thereof has 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. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising or consisting of VL CDR3: WQGTHFPYT (SEQ ID NO: 13).

5. The antibody or antigen-binding fragment thereof has a variable heavy chain comprising: 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 EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSA (SEQ ID NO: 20).

6. The antibody or antigen-binding fragment thereof has a variable light chain comprising: DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 23), 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. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising or consisting of: DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 28).

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

8. The antibody or antigen-binding fragment thereof is １６５ 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, which has no specificity for other VEGF splice variants that do not contain exon 8b, such as exon 8b.

9. further comprising a moiety, such as an amino acid sequence motif, to increase the in vivo half-life of the antibody or antigen-binding fragment thereof; Optionally, the moiety is selected from the group of post-translational modifications consisting of polyethylene glycol (PEG), glycosylation, fatty acids, and dextran, or is a gene fusion protein such as, but not limited to, human serum albumin and a cytokine (i.e., an immunocytokine).

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

11. further comprising a cytotoxic moiety, optionally wherein said cytotoxic moiety is 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 d. A chemotherapeutic agent, such as an antimetabolite.

12. further comprising a detectable moiety, optionally wherein said detectable moiety is 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-12, thallium-201, and / or b. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising or consisting 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.

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

14. 14. The pharmaceutical composition of claim 13 adapted for subcutaneous, intravenous, intramuscular, intracranial, or intraocular delivery.

15. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

16. 15. 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 as a medicament.

17. VEGF-related diseases (preferably VEGF-A, more preferably VEGF-A １６５ b), ischemia (peripheral, intestinal / mesenteric, coronary / cardiac, cerebral / brain, retinal, limb, or renal), peripheral arterial disease (PAD), atherosclerosis, conditions related to diabetes (such as diabetic retinopathy, diabetic nephropathy, diabetic neuropathic pain, 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 disease (such as rhegmatogenous retinal detachment and proliferative vitreoretinopathy, retinal ischemia, central vein occlusion, branch vein occlusion, non-proliferative diabetic retinopathy), pulmonary conditions associated with altered angiogenesis (e.g., asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease), inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), neuroischemic 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, inherited renal conditions such as Dennis-Drash or Fraser syndrome, chronic kidney disease, acute kidney disease, glomerular nephropathy), angiogenesis / revascularization (e.g., tumor-associated angiogenesis and VEGF-A １６５ 15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 12, or the pharmaceutical composition of 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: inflammatory bowel disease (including inflammatory bowel disease, including ...

18. VEGF-related diseases (preferably VEGF-A, more preferably VEGF-A １６５ b), ischemia (peripheral, intestinal / mesenteric, coronary / cardiac, cerebral / brain, retinal, limb, or renal), peripheral arterial disease (PAD), atherosclerosis, conditions related to diabetes (such as diabetic retinopathy, diabetic nephropathy, diabetic neuropathic pain, 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 disease (such as rhegmatogenous retinal detachment and proliferative vitreoretinopathy, retinal ischemia, central vein occlusion, branch vein occlusion, non-proliferative diabetic retinopathy), pulmonary conditions associated with altered angiogenesis (e.g., asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease), inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), neuroischemic 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, inherited renal conditions such as Dennis-Drash or Fraser syndrome, chronic kidney disease, acute kidney disease, glomerular nephropathy), angiogenesis / revascularization (e.g., tumor-associated angiogenesis and VEGF-A １６５ 15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, or the pharmaceutical composition of claim 13 or 14, for use in the treatment or prevention of a disease, syndrome, or condition selected from the group consisting of: inflammatory bowel disease (including inflammatory bowel disease (IG)-mediated angiogenesis), cancer (e.g., solid tumors), deep vein thrombosis (DVT), refractory angina, and myocardial infarction (MI) or post-MI symptoms.

19. VEGF-related diseases (preferably VEGF-A, more preferably VEGF-A １６５ b), ischemia (peripheral, intestinal / mesenteric, coronary / cardiac, cerebral / brain, retinal, limb, or renal), peripheral arterial disease (PAD), atherosclerosis, conditions related to diabetes (such as diabetic retinopathy, diabetic nephropathy, diabetic neuropathic pain, 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 disease (such as rhegmatogenous retinal detachment and proliferative vitreoretinopathy, retinal ischemia, central vein occlusion, branch vein occlusion, non-proliferative diabetic retinopathy), pulmonary conditions associated with altered angiogenesis (e.g., asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease), inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), neuroischemic 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, inherited renal conditions such as Dennis-Drash or Fraser syndrome, chronic kidney disease, acute kidney disease, glomerular nephropathy), angiogenesis / revascularization (e.g., tumor-associated angiogenesis and VEGF-A １６５ 15. A method for the treatment or diagnosis of a disease, syndrome, or condition selected from the group consisting of: inflammatory bowel disease (including inflammatory bowel disease, including ...

20. A method for in vitro / ex vivo diagnosis, comprising subjecting an isolated sample to an antibody or antigen-binding fragment thereof described in any one of claims 1 to 12, or a pharmaceutical composition described in claim 13 or 14.

21. 10. An antibody or antigen-binding fragment thereof for use in medicine substantially as hereinbefore described with reference to the specification and claims.

22. 10. A pharmaceutical composition substantially as hereinbefore described with reference to the specification and claims.

23. 10. Use of an antibody or antigen-binding fragment thereof substantially as hereinbefore described with reference to the specification.

24. A method of treatment or diagnosis as claimed in any of the following claims:

25. 10. A kit substantially as hereinbefore described with reference to the specification and claims.

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