WO2024079482A1 - Anticorps anti-vegf - Google Patents

Anticorps anti-vegf Download PDF

Info

Publication number
WO2024079482A1
WO2024079482A1 PCT/GB2023/052666 GB2023052666W WO2024079482A1 WO 2024079482 A1 WO2024079482 A1 WO 2024079482A1 GB 2023052666 W GB2023052666 W GB 2023052666W WO 2024079482 A1 WO2024079482 A1 WO 2024079482A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
antigen
binding fragment
vegf
Prior art date
Application number
PCT/GB2023/052666
Other languages
English (en)
Inventor
David Bates
Original Assignee
The University Of Nottingham
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of Nottingham filed Critical The University Of Nottingham
Publication of WO2024079482A1 publication Critical patent/WO2024079482A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • VEGF ⁇ antibodies ⁇ FIELD OF THE INVENTION The present invention relates to targeting of VEGF-A 165 b and monoclonal antibodies, such as monoclonal antibodies (mAbs), which bind VEGF-A 165 b.
  • monoclonal antibodies such as monoclonal antibodies (mAbs)
  • mAbs monoclonal antibodies
  • VEGF vascular endothelial growth factor
  • VEGF-A 165 b A splice variant of VEGF-A called VEGF-A 165 b is expressed as a protein in normal cells and tissues and circulates in human plasma (see Figure 34).
  • VEGF- A 165 b binds VEGF receptor 2 with the same affinity as VEGF-A 165 a but does not activate it or stimulate the same downstream signalling pathways.
  • VEGF-A165a impairs VEGF-A165a- mediated VEGF receptor 2 phosphorylation and signalling in cultured cells.
  • VEGF-A165b is not angiogenic and it inhibits VEGF-A165a-mediated angiogenesis in rabbit cornea, rat mesentery, mouse mammary glands, dorsal chambers and ovaries, as well as in proliferative oxygen-induced retinopathy, choroidal neovascularisation, and cancer growth.
  • VEGF-A 165 a expressing tumours grow significantly more slowly than VEGF-A 165 a- expressing tumours, indicating that a switch in splice variant expression from VEGF-A 165 a to VEGF-A165b can inhibit tumour growth.
  • an anti-VEGF-A165b mouse monoclonal antibody stimulates revascularisation in a model of hindlimb ischemia in mice who are genetically obese (ob/ob) or who have been fed a high fat high sucrose diet for 12 weeks (Kikuchi et al 2014. Nat Med.; 20(12): 1464-1471, which is incorporated herein by reference), and that an anti-VEGF-A165b mouse monoclonal antibody stimulates revascularisation in a model of impaired hindlimb ischemia in mice who are genetically eNOS deficient, or who overexpress myoglobin (Kuppuswamy et al., Cells 2022, 11, 2676).
  • a first aspect of the invention relates to an antibody or antigen-binding fragment thereof with 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 (such as VEGF-Ax and VEGF 189 b).
  • VEGF vascular endothelial growth factor
  • the antibody is an intact antibody.
  • the antigen-binding fragment is selected from the group consisting of Fv fragments and Fab- like fragments (e.g. Fab fragments, Fab’ fragments and F(ab)2 fragments).
  • Fv fragments we include single chain Fv, disulphide-bonded Fv and domain antibodies.
  • Fab-like fragments we include Fab fragments, Fab’ fragments and F(ab) 2 fragments.
  • the antibody or antigen-binding fragment thereof is recombinant.
  • the antibody or antigen-binding fragment thereof is monoclonal.
  • the antibody or antigen-binding fragment thereof is polyclonal.
  • 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 humanised. In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the following Complementarity determining regions (CDRs), as determined by the IMGT determination method: 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.
  • CDRs Complementarity determining regions
  • VL CDR1 QSLLDSDGKTY (SEQ ID NO: 4); e. VL CDR2: LVS (SEQ ID NO: 5); and/or f. VL CDR3: WQGTHFPYT (SEQ ID NO: 6).
  • the antibody or antigen-binding fragment thereof comprises or consists of the following CDRs, as determined by the Kabat determination method: a. V H CDR1: RYWMSW (SEQ ID NO: 7); b. V H CDR2: EIHPYSSTINYTPSVKD (SEQ ID NO: 8); c. V H 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. V L CDR3: WQGTHFPYT (SEQ ID NO: 13).
  • the V L CDR1, as determined by the Kabat determination method is *SSQSLLDSDGKTYLN (SEQ ID NO: 14), wherein * is R or K.
  • the CDR sequences are determined by the Kabat determination method.
  • the CDR sequences are determined by the IMGT determination method.
  • the antibody or antigen-binding fragment thereof comprises or consists of the following variable heavy chains: a. EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFI ISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA (SEQ ID NO: 15; also referred to herein as the V H chain of the parental mouse mAb clone “58/8/31/1/13”); b.
  • EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFI ISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA (SEQ ID NO: 15); also referred to herein as “VH0”); c. EVQLLESGGGLVKPGGSLRLSCAASGFDFSRYWMSWIRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO: 16; also referred to herein as “VH1”); d.
  • EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFI ISRDNAKNSVYLQLNSLRAEDTAVYYCARAFAYWGQGTLVTVSS (SEQ ID NO: 17; also referred to herein as “VH2”); e. EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWISEIHPYSSTINYTPSVKDRFT ISRDNAKNSLYLQMNSLRDEDTALYYCARAFAYWGQGTLVTVSS (SEQ ID NO: 18; also referred to herein as “VH3”); f.
  • EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFT ISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSS SEQ ID NO: 19; also referred to herein as “VH4”
  • EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARAFAYWGQGTLVTVSA SEQ ID NO: 20; also referred to herein as “VH5”.
  • the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable heavy chain sequence, wherein substitutions or deletions in particular positions are indicated in parenthesis, deletions being marked as ‘X’: x EVKLLESGGGLVQPGGS(L/P)KLSCAASGFDFSRYW(M/R)SWVRQAPGK(G/E)LEWIGEIHPYSST INYTPSVKDK(F/L)IISRD(N/S)AKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA (SEQ ID NO: 21)
  • the antibody or antigen-binding fragment thereof comprises or consists of the following consensus heavy chain sequence in which the most common amino acids are indicated at each position (SEQ ID NO: 22) (variable domain is highlighted in bold, and CDRs double underlined): x EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTP
  • the antibody or antigen-binding fragment thereof comprises or consists of the following variable light chains: a. DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTG SGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 23; also referred to herein as the VL chain of the parental mouse mAb clone “58/8/31/1/13”); b.
  • DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTG SGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 23; also referred to herein as “VL0”); c. DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 24; also referred to herein as “VL1”); d.
  • DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWELQRPGQSPRRLIYLVSKLDSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKVEIKR (SEQ ID NO: 25; also referred to herein as “VL2”); e. DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSG to f.
  • DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGKTYLNWLQQRPGQPPRLLIYLVSKLDSGVPDRFSG SGAGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR (SEQ ID NO: 27; also referred to herein as “VL4”); or g. DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKVEIKR (SEQ ID NO: 28; also referred to herein as “VL5”).
  • the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable light chain sequence, wherein substitutions or deletions are particular positions are indicated in parenthesis, deletions being marked as ‘X’: x D(I/V)VMTQ(T/S)PL(T/S)(L/S)(S/P)VT(I/L/P)GQPASISC(K/R)SSQSLLDSDGKTYLN W(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)
  • the antibody or antigen-binding fragment thereof comprises or consists of the following consensus variable light chain sequence, wherein
  • variable heavy chain sequence and/or the variable light chain sequence further comprises a signal peptide sequence, for example MGWTLVFLFLLSVTAGVHS (SEQ ID NO: 32). Accordingly, antibodies or antigen-binding fragments thereof can be formed by combining any VH specified above with any VL specified above.
  • VH4 or VH5 may be combined with VL2, as follows (which may optionally comprise a linker, for example (G4S)3, between the two domains): VH4-VL2 (SEQ ID NO: 33) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVAEIHPYSSTINYTPSVKDRFT ISRDNAKNSLYLQMNSLRSEDTAVYYCARAFAYWGQGTLVTVSS[linker]DVVMTQSPLSLPVTLGQ PASISCRSSQSLLDSDGKTYLNWELQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHFPYTFGQGTKVEIKR VH5-VL2 (SEQ ID NO: 34) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWVSEIHPYSSTINYTPSVKDRFT ISR
  • VH0 is combined with VL0, VL1, VL2, VL3, VL4 or VL5.
  • VH1 is combined with VL0, VL1, VL2, VL3, VL4 or VL5.
  • VH2 is combined with VL0, VL1, VL2, VL3, VL4 or VL5.
  • VH3 is combined with VL0, VL1, VL2, VL3, VL4 or VL5.
  • VH4 is combined with VL0, VL1, VL2, VL3, VL4 or VL5.
  • VH5 is combined with VL0, VL1, VL2, VL3, VL4 or VL5.
  • the antibody or antigen-binding fragment thereof further comprises a constant domain.
  • the constant domain may correspond to the following sequence: Heavy chain constant domain: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK (SEQ ID NO: 35) Light chain constant domain: TVAAPSVFIFPPSD
  • the antibody or antigen-binding fragment thereof comprises or consists of the VH4-VL2 CDR sequences, and/or comprises or consists of the VH4-VL2 VH and/or VL sequences, optionally further comprising a heavy chain constant domain and/or light chain constant domain as specified herein (i.e.
  • the antibody or antigen-binding fragment thereof may comprise or consist of the HC4-LC2 sequences).
  • the antibody or antigen-binding fragment thereof comprises or consists of the VH5-VL2 CDR sequences, and/or comprises or consists of the VH5-VL2 VH and/or VL sequences, optionally further comprising a heavy chain constant domain and/or light chain constant domain as specified herein (i.e. the antibody or antigen-binding fragment thereof may comprise or consist of the HC5-LC2 sequences).
  • the antibody or antigen-binding fragment thereof does not have specificity for any other VEGF splice variants not containing exon 8b.
  • VEGF-A165a An example of a VEGF splice variant that does not contain exon 8b is VEGF-A165a. Therefore, in some embodiments, the antibody or antigen-binding fragment thereof does not have specificity for VEGF-A 165 a. 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.
  • PEG polyethylene glycol
  • glycosylation glycosylation
  • fatty acids and dextran
  • the genetic fusion protein may be to human serum albumin. Additionally, or alternatively, the genetic fusion protein may be to a cytokine, which may form an immunocytokine.
  • the moiety is a half-life extension motif, optionally wherein the half-life extension motif is contained in the Fc domain and not the variable domain.
  • the antibody or antigen-binding fragment thereof is PEGylated. In some embodiments, the moiety is a cytotoxic moiety.
  • the cytotoxic moiety may comprise or consist of a radioisotope, such as a radioisotope selected from the group consisting of Astatine-211, Bismuth-212, Bismuth-213, Iodine-131, Yttrium-90, Lutetium-177, Samarium-153 and Palladium-109.
  • the cytotoxic moiety may comprise or consist of a toxin, such as, but not limited to, saporin or calicheamicin.
  • the cytotoxic moiety may comprise or consist of a chemotherapeutic agent, such as an antimetabolite.
  • the moiety is a detectable moiety.
  • 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; Thallium-201.
  • 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; Iron-56.
  • a second aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof according to the first aspect of the invention, and a pharmaceutically-acceptable diluent, carrier or excipient.
  • the pharmaceutical composition is adapted for a particular delivery route.
  • the delivery may be subcutaneous, intravenous, intramuscular, intracranial, or intraocular delivery.
  • a third aspect of the invention relates to a kit comprising the antibody or antigen-binding fragment thereof according to the first or second aspects of the invention.
  • the kit further comprises instructions for use. Any one or more part of the kit may be stored within a vial in a form in which reconstitution and/or mixing with other components is required prior to use.
  • a fourth aspect of the invention relates to the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the pharmaceutical composition according to the second aspect of the invention, for use as a medicament.
  • a fifth aspect of the invention relates to a use of the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the 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 specified herein.
  • a sixth aspect of the invention relates to the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the pharmaceutical composition according to the second aspect of the invention, for use in treating or preventing a disease, syndrome or condition as specified herein.
  • a seventh aspect of the invention relates to a method of treatment or diagnosis, wherein the method comprises administering an effective amount of the antibody or antigen- binding fragment thereof according to the first aspect of the invention, or the pharmaceutical composition according to the second aspect of the invention.
  • An eighth aspect of the invention relates to a method of diagnosis, wherein the method comprises subjecting a sample to the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the pharmaceutical composition according to the second aspect of the invention.
  • the method is an in vitro or ex vivo method.
  • the sample is an isolated sample, such as a sample isolated from a subject.
  • the sample is selected from the group consisting of a bodily fluid, cell, population of cells, tissue, organ, plasma and serum.
  • the disease, syndrome or condition is selected from the group consisting of: - VEGF-associated diseases (preferably VEGF-A, even more preferably VEGF-A 165 b); - Ischemia (peripheral, intestinal/mesenteric, coronary/cardiac, cerebral/brain, retinal, limb, or renal); - Peripheral arterial disease (PAD); - Atherosclerosis; - Conditions associated with 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-associated cutaneous conditions such as cyanosis and gangrene
  • Retinal ischemic diseases such as rhegmatogenous retinal detachment and proliferative vitreoretinopathy, retinal ischemia, central vein occlusion, branch vein occlusion, nonproliferative diabetic retinopathy
  • - Lung conditions associated with altered angiogenesis e.g.
  • Pulmonary hypertension Pulmonary hypertension, Idiopathic Pulmonary Fibrosis, Chronic Obstructive Pulmonary Disease); - Inflammatory bowel disease (such as ulcerative colitis and Crohn’s disease); - Neuronal ischemic conditions (e.g. chronic pain, peripheral neuropathy, traumatic neuropathy, chemotherapy induced peripheral neuropathy); - Stroke; - Pre-eclampsia; - Hypertension; - Obesity; - Hair loss; - Kidney failure (e.g. IgA nephropathy, hereditary kidney conditions, e.g.
  • Denys- Drash or Frasier Syndromes chronic kidney disease, acute kidney disease, glomerular nephropathy); - Angiogenesis/revascularisation (including tumour-associated angiogenesis and VEGF-A165a-mediated angiogenesis); - Cancer (e.g. solid tumours); - Deep vein thrombosis (DVT); - Refractory angina; and - Myocardial infarction (MI), or post-MI conditions.
  • MI Myocardial infarction
  • VEGF vascular endothelial growth factor
  • the splice variant is VEGF-A165b or any VEGF sequence containing the sequence encoded by Exon 8b of the VEGF gene (such as VEGF-Ax, isoform 15 of VEGF-A, and VEGF 189 b).
  • splice variant we include the meaning that the gene that encodes VEGF may be alternatively spliced to result in different isoforms or versions that translate into alternative VEGF molecules.
  • the described anti-VEGF-A165b antibodies are effective in reversing anti- angiogenic effects of monocytes from patients with peripheral arterial disease in vitro and in vivo.
  • the present invention includes a chimeric antibody (mouse variable domains VH and VL and human heavy and light chain constant domains, HC0LC0) and humanised versions of the antibody to VEGF-A165b.
  • the chimeric antibody has an affinity of 300 pM and the humanised versions have affinities of 600 pM (2 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).
  • the antibody or antigen-binding fragment thereof has an affinity of 100 to 1000 pM, for example, 200 to 800 pM, preferably 300 to 600 pM.
  • 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.
  • affinity is increased.
  • 1 pM is a higher (i.e. increased, improved, better, greater) affinity than 1 nM.
  • the antibody or antigen-binding fragment thereof may be specific for VEGF-A 165 b.
  • the antibody or antigen-binding fragment thereof may not bind, or may not significantly bind, VEGF-A165a.
  • the antibody or antigen-binding fragment thereof binds VEGF- A165b and does not bind, or does not significantly bind, VEGF-A165a or any other splice variant of VEGF that uses the proximal splice site in exon 8 encoding CDKPRR.
  • the antibody may bind VEGF-A 165 b with at least 50x or 100x greater affinity than VEGF-A 165 a or any other exon 8 proximal splice variant of VEGF.
  • the antibody or antigen-binding fragment thereof does not bind if the splice variant excludes exon 8b.
  • the antibody or antigen-binding fragment thereof may have an affinity/dissociation constant (kD) for VEGF-A165b binding that is less than 1 nanomolar.
  • the antibody or antigen-binding fragment thereof may have an affinity/dissociation constant (k D ) for VEGF-A 165 b binding that is less than about 0.8 nM.
  • the antibody or antigen-binding fragment thereof may have an affinity/dissociation constant (kD) for VEGF-A165b binding that is about 0.6 nM (600 pM) or less.
  • the antibody or antigen-binding fragment thereof may have an affinity/dissociation constant (kD) for VEGF-A165b binding that is between about 0.3 nM and about 0.8 nM.
  • the antibody or antigen-binding fragment thereof may have an affinity/dissociation constant (k D ) for VEGF-A 165 b binding that is between about 0.5 nM and about 0.7 nM.
  • the antibody or antigen-binding fragment thereof may have an affinity/dissociation constant (k D ) for VEGF-A 165 b binding that is between about 0.55 nM and about 0.65 nM.
  • the antibody or antigen-binding fragment thereof has one or more of the following properties: a.
  • VEGF-A165b binding to VEGF receptor 2 VAGFR2
  • VEGF receptor 2 VAGFR2
  • PAD diabetic peripheral arterial disease
  • the present invention advantageously provides an antibody or antigen-binding fragment thereof that has a significantly high specificity for VEGF-A165b in the picomolar range, and it is humanized for efficacy and safety in human treatment.
  • the VEGF-A 165 b target of the antibody or antigen-binding fragment thereof is human VEGF-A 165 b.
  • the sequence of VEGF-A 165 b may comprise of 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 coupled to keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • the sequence TCRSLTRKD (SEQ ID NO: 37) corresponds to the final 9 amino acids of human VEGF165b, which was used as an immunogen to generate the antibodies.
  • VEGFA_HUMAN Isoform 15 of Vascular endothelial growth factor A and vascular endothelial growth factor A isoform VEGF-Ax precursor as follows: >sp
  • an antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain is comprised 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 a binding domain that interacts with an antigen.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • the constant regions of the antibodies may 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 (Clq) of the classical complement system.
  • 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 chains and lambda chains.
  • variable regions (VH and VL) of antibodies of the present invention may further comprise (e.g. be linked to) antibody constant regions or parts thereof.
  • the variable regions of antibodies may be attached at their C- terminal end to antibody light chain constant domains including human Clj or CNJ chains.
  • the variable regions of antibodies may be attached at their C-terminal end to all, or part of, an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, particularly IgG1, IgG2 and IgG4.
  • antibodies and their antigen-binding fragments that have been “isolated” so as to exist in a physical milieu distinct from that in which it may occur in nature or that have been modified so as to differ from a naturally occurring antibody in amino acid sequence.
  • isolated refers to the state in which specific antibodies or antigen-binding fragments of the invention, or nucleic acid encoding such, will preferably be, in accordance with the present invention.
  • Antibodies and antigen-binding fragments thereof, and nucleic acids will generally be free or substantially free of material with which they are naturally associated such as other polypeptides (e.g.
  • nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g., cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo.
  • Specific monoclonal antibodies and nucleic acid may be formulated with diluents or adjuvants or excipients and still for practical purposes be isolated – for example, the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers, diluents and/or excipients when used in diagnosis or therapy.
  • Specific binding antibodies or antigen-binding fragments thereof may be glycosylated, either naturally or by systems of heterologous eukaryotic cells, or they may be aglycosylated (for example if produced by expression in a prokaryotic cell or mammalian cells deficient in specific glycosylation pathways or if the antibody or antigen binding fragment has been specifically engineered to remove glycosylation sites).
  • An antibody or antigen-binding portion thereof may be a polyclonal antibody or a monoclonal antibody. The antibody or antigen-binding portion thereof may be produced by any suitable method.
  • Suitable methods for producing monoclonal antibodies are disclosed in “Monoclonal Antibodies; A manual of techniques”, H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Application”, SGR Hurrell (CRC Press, 1982). Recombinant techniques may also be used.
  • the term “antigen-binding portion” or “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, such as VEGF-A 165 b. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include a Fab fragment, a F(ab')2 fragment, a Fab’ fragment, a Fd fragment, a Fv fragment, and a dAb fragment.
  • Single chain antibodies such as scFv and heavy chain antibodies such as VHH and camelid antibodies termed nanobodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.
  • These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact (i.e. full length) antibodies.
  • An antibody for use in the methods of the invention may be a human antibody.
  • human antibody 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 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).
  • human antibody 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 – such antibodies are typically referred to as chimeric or humanised.
  • Methods for humanising non-human antibodies are well known in the art.
  • the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et al., 1986, Nature 321:522- 525; Reichmann et al., 1988.
  • humanised antibodies may be 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. mice) antibodies.
  • a chimeric antibody can be one in which the VH and VL of the rodent antibody are fused to a human IgG constant region (CH and CL or CK).
  • a humanised antibody is one in which the CDRs from the rodent antibody replace the CDRs in a human antibody framework which has a high level of sequence identity/homology to the parental 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 from other non-human parental antibody species, for example including rabbits, llamas, monkeys, etc.
  • a human antibody for use the methods of the invention is typically a human monoclonal antibody. Such a human monoclonal antibody may be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g.
  • Human antibodies may also be prepared by in vitro immunisation of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.
  • human antibody derivatives refers to any modified form of the human antibody, e.g. a conjugate of the antibody and another agent or antibody.
  • An antibody or antigen-binding portion thereof according to the invention may alternatively be a humanised antibody.
  • humanised refers to an antibody molecule, generally prepared using recombinant techniques, having an antigen binding site derived from an immunoglobulin from a non-human species and a remaining immunoglobulin structure based upon the structure and/or sequence of a human immunoglobulin.
  • the antigen-binding site may comprise complete non-human antibody variable domains fused to human constant domains, 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).
  • fully humanised antibodies described as “humanised” herein only the complementarity determining regions (CDRs) of such variable domains are grafted into the appropriate human framework regions of human variable domains.
  • the framework residues of such humanised 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 has served as the basis for humanisation. Humanisation lessens or eliminates the likelihood that the ‘humanised antibody’ will act as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A.F. et al. (1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224).
  • human or humanised antibodies are preferably used.
  • Humanised forms of non-human (e.g. murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having preferably minimal amino acid residues derived from non-human antibodies.
  • Humanised antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementary determining region of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired functionality.
  • donor antibody such as mouse, rat or rabbit having the desired functionality.
  • Fv framework residues of the human antibody are replaced by corresponding non-human residues.
  • Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences.
  • the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non-human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence.
  • Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, 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). Methods for humanising non-human antibodies are well known in the art.
  • the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et al., 1986, Nature 321:522- 525; Reichmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536l; US 4,816,567) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions.
  • humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be identified using various techniques known in the art, including from phage display libraries (see, for example, 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).
  • variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the antigens in question and determine binding affinity, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffold for the CDRs.
  • CDRs complementarity-determining regions
  • FRs framework regions
  • the variable regions can be “reshaped” or “humanised” by grafting CDRs derived from nonhuman antibody into the FRs present in the human antibody to be modified.
  • humanised antibodies preserve all six CDR sequences (for example, a humanised mouse antibody which contains all six CDRs from the mouse antibodies).
  • humanised antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.
  • CDRs one, two, three, four, five, six
  • the ability to humanise an antibody is well known (see, e.g., US Patents No. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).
  • Any antibody referred to herein may be provided in isolated form or may optionally be provided linked (directly or indirectly) to another moiety.
  • the other moiety may be a therapeutic molecule such as a cytotoxic moiety, antibiotic or a drug.
  • the therapeutic molecule may be directly attached, for example by chemical conjugation, to an antibody or antigen-binding fragment of the invention.
  • Methods for conjugating molecules to such are known in the art.
  • carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159) may be used to conjugate a variety of agents, including doxorubicin, to antibodies or peptides.
  • the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is particularly useful for conjugating a functional moiety to a binding moiety.
  • Other methods for conjugating a moiety to antibodies can also be used.
  • a cytotoxic moiety may be directly and/or indirectly cytotoxic.
  • directly cytotoxic it is meant that the moiety is one which on its own is cytotoxic.
  • indirectly cytotoxic it is meant that the moiety is one which, although is not itself cytotoxic, can induce cytotoxicity, for example by its action on a further molecule or by further action on it.
  • the cytotoxic moiety may be cytotoxic only when intracellular and is preferably not cytotoxic when extracellular.
  • the antibody or antigen-binding portion thereof may be linked to a cytotoxic moiety which is a directly cytotoxic chemotherapeutic agent.
  • the cytotoxic moiety is a directly cytotoxic polypeptide.
  • Cytotoxic chemotherapeutic agents are well known in the art.
  • the antibody or antigen-binding portion thereof with a cytotoxic chemotherapeutic agent is for use in combination with a distinct chemotherapy.
  • the antibody or antigen-binding portion thereof is not conjugated to a cytotoxic chemotherapeutic agent.
  • Cytotoxic chemotherapeutic agents include: alkylating agents including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethyleneimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfane; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogue
  • VLB vinblastine
  • epipodophyllotoxins such as etoposide and teniposide
  • antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C)
  • enzymes such as L-asparaginase
  • Miscellaneous agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o,p’-DDD) and aminoglutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.
  • the cytotoxic moiety may be a cytotoxic peptide or polypeptide moiety which leads to cell death.
  • Cytotoxic peptide and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor and the like. 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 may also be used as the cytotoxic polypeptide. Certain cytokines, such as TNF ⁇ and IL-2, may also be useful as cytotoxic agents. Certain radioactive atoms may also be cytotoxic if delivered in sufficient doses.
  • the cytotoxic moiety may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as 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 which emits enough energy to destroy neighbouring cells, organelles or nucleic acid.
  • the isotopes and density of radioactive atoms in the agents of the 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 and, preferably, to the cells at the target site and their organelles, particularly the nucleus.
  • the radioactive atom may be attached to the antibody, antigen-binding fragment, variant, fusion or derivative thereof in known ways.
  • EDTA or another chelating agent may be attached to the binding moiety and used to attach 111In or 90Y.
  • Tyrosine residues may be directly labelled with 125I or 131I.
  • the cytotoxic moiety may be a suitable indirectly cytotoxic polypeptide.
  • the indirectly cytotoxic polypeptide may be a polypeptide which has enzymatic activity and can convert a non-toxic and/or relatively non-toxic prodrug into a cytotoxic drug.
  • this type of system is often referred to as ADEPT (Antibody-Directed Enzyme Prodrug Therapy).
  • ADEPT Antibody-Directed Enzyme Prodrug Therapy
  • the system requires that the antibody locates the enzymatic portion to the desired site in the body of the patient and after allowing time for the enzyme to localise at the site, administering a prodrug which is a substrate for the enzyme, the end product of the catalysis being a cytotoxic compound.
  • the object of the approach is to maximise the concentration of drug at the desired site and to minimise the concentration of drug in normal tissues.
  • the cytotoxic moiety may be capable of converting a non-cytotoxic prodrug into a cytotoxic drug.
  • the enzyme and prodrug of the system using a targeted enzyme as described herein may be any of those previously proposed.
  • the cytotoxic substance may be any existing anti- cancer drug such as an alkylating agent; an agent which intercalates in DNA; an agent which inhibits any key enzymes such as dihydrofolate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinases or topoisomerase; or an agent which effects cell death by interacting with any other cellular constituent.
  • Etoposide is an example of a topoisomerase inhibitor.
  • Reported prodrug systems include those listed in Table A.
  • Table A Reported prodrug systems.
  • Enzyme Prodrug Derivatives of L-glutamic acid and benzoic acid such as carboxypeptidases G, G1 and G2 (for glutamylated mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs) and aminopeptidases (for 2- ⁇ - aminocyl MTC prodrugs); endopeptidases, such as e.g. thrombolysin (for thrombin prodrugs); hydrolases, such as phosphatases (e.g. alkaline phosphatase) or sulphatases (e.g.
  • aryl sulphatases (for phosphylated or sulphated prodrugs); amidases, such as penicillin amidases and arylacyl amidase; lactamases, such as ⁇ -lactamases; glycosidases, such as ⁇ -glucuronidase (for ⁇ -glucuronomide anthracyclines), ⁇ -galactosidase (for amygdalin) and ⁇ -galactosidase (for ⁇ -galactose anthracycline); deaminases, such as cytosine deaminase (for 5FC); kinases, such as urokinase and thymidine kinase (for gancyclovir); reductases, such as nitroreductase (for CB1954 and analogues), azoreductase (for azobenzene mustards) and DT-diaphorase (for CB
  • the prodrug is relatively non-toxic compared to the cytotoxic drug. Typically, it has less than 10% of the toxicity, preferably less than 1% of the toxicity as measured in a suitable in vitro cytotoxicity test. It is likely that the moiety which is able to convert a prodrug to a cytotoxic drug will be active in isolation from the rest of the agent of the invention but it is necessary only for it to be active when (a) it is in combination with the rest of the agent of the invention and (b) the agent of the invention is attached to, adjacent to or internalised in target cells. When each moiety is a polypeptide, the two portions may be linked together by any of the conventional ways of cross-linking polypeptides.
  • the antibody or antigen- binding portion thereof may be enriched with thiol groups and the further moiety reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N- hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP).
  • a bifunctional agent capable of reacting with those thiol groups
  • Amide and thioether bonds for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulphide bonds.
  • the cytotoxic moiety may be a radiosensitiser.
  • Radiosensitisers include fluoropyrimidines, thymidine analogues, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3-aminobenzodiamide, etanixadole, pimonidazole and misonidazole.
  • delivery of genes into cells can radiosensitise them, for example delivery of the p53 gene or cyclin D.
  • the further moiety may be one which becomes cytotoxic, or releases a cytotoxic moiety, upon irradiation.
  • the boron-10 isotope when appropriately irradiated, releases ⁇ particles which are cytotoxic.
  • the cytotoxic moiety may be one which is useful in photodynamic therapy such as photofrin.
  • therapeutically effective amount By “therapeutically effective amount”, “effective amount” or “therapeutically effective”, it is meant 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 severity of disease symptoms, or an increase in frequency or duration of symptom-free periods.
  • Effective amounts 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 state of the subject. This may be a predetermined quantity of active antibody calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle.
  • a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host.
  • the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent.
  • a therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
  • the optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.
  • a serum concentration of polypeptides and antibodies that permits saturation of receptors is desirable.
  • a concentration in excess of approximately 0.1 nM is normally sufficient.
  • a dose of 100 mg/kg of antibody provides a serum concentration of approximately 20 nM for approximately eight days.
  • doses of antibodies may be given weekly in amounts of 10 – 300 mg/kg.
  • the dose of the composition will be dependent upon the properties of the monoclonal antibody, e.g.
  • doses of 300 ⁇ g of antibody per patient per administration may be provided, although dosages may range from about 10 ⁇ g to 6 g per dose. Different dosages are utilised during a series of sequential inoculations; the practitioner may administer an initial inoculation and then boost with relatively smaller doses of antibody.
  • the term “subject”, includes any animal, including a human, that is in need of treatment with an antibody or antigen-binding portion thereof of the present invention.
  • the subject or patient may be mammalian or non-mammalian.
  • the subject is mammalian, such as a horse, or a cow, or a sheep, or a pig, or a camel, or a dog, or a cat.
  • the mammalian patient is a human.
  • the antibody or antigen-binding portion thereof is formulated and/or adapted for delivery by a route selected from the group comprising: intravenous; intramuscular; subcutaneous; intracranial; and intraocular.
  • the antibody or antigen-binding portion thereof may be formulated and/or adapted for intravenous (i.e. “i.v” or “IV”) delivery.
  • the antibody or antigen-binding portion thereof is delivered to the subject by a route selected from the group comprising: intravenous; intramuscular; subcutaneous; intracranial; and intraocular.
  • the antibody or antigen-binding portion thereof may be delivered intravenously. It is envisaged that injections will be the primary route for therapeutic administration of the compositions although delivery through a catheter or other surgical tubing is also used.
  • Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal, intradermal intramuscular, intracranial and intraocular administration.
  • Liquid formulations may be utilised after reconstitution from powder or lyophilized formulations.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, Lactated Ringer’s Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required, and as further described herein.
  • the composition may be administered in a localised manner or systemically.
  • the antibody or antigen-binding portion thereof may be coated in a material to protect the agent(s) from the action of acids and other natural conditions that may inactivate or denature the antibody or antigen-binding portion thereof.
  • Preferred pharmaceutically acceptable carriers comprise aqueous carriers or diluents.
  • suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline.
  • other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), 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, using coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and using surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Methods and formulations for various routes of administration are well known in the art.
  • the antibody or antigen-binding fragment thereof may be defined by binding affinity.
  • binding activity is intended to refer to the tendency of an antibody molecule to bind or not to bind to a target. Binding affinity may be quantified by determining the dissociation constant (Kd) for an antibody and its target.
  • the specificity of binding of an antibody to its target may be defined in terms of the comparative dissociation constants (Kd) of the antibody for its target as compared to the dissociation constant with respect to the antibody and another, non-target molecule.
  • Kd comparative dissociation constants
  • the Kd for the antibody with respect to the target will be 2-fold, preferably 5- fold, more preferably 10-fold lower than Kd with respect to the other, non-target molecule such as unrelated material or accompanying material in the environment. More preferably, the Kd will be 50-fold less, even more preferably 100-fold less, for example, 200-fold less, and yet more preferably 1000-fold less.
  • this dissociation constant can be determined directly by well-known methods and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al. (Byte 9:340-362, 1984).
  • the Kd may 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 to evaluate the binding ability of ligands such as antibodies towards targets are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis.
  • the binding kinetics (e.g., binding affinity) of the antibody also can be assessed by standard assays known in the art, such as by BIAcoreTM (SPR) system analysis or biolayer interferometry (BLI) using an Octet system.
  • SPR BIAcoreTM
  • BLI biolayer interferometry
  • a competitive binding assay can be conducted in which the binding of the antibody to the target is compared to the 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 the Ki. Under ideal conditions, the Ki is equivalent to Kd. The Ki value will never be less than the Kd, so measurement of Ki can conveniently be substituted to provide an upper limit for Kd.
  • the antibodies or antigen-binding fragments thereof described herein are preferably capable of binding to its target with an affinity that is at least two-fold, 10-fold, 50-fold, 100-fold or greater than its affinity for binding to another non-target molecule.
  • the antibody may be or may comprise a variant or a fragment of one of the specific anti- VEGF-A 165 b antibodies disclosed herein, provided that said variant or fragment retains specificity for VEGF-A 165 b.
  • a fragment is preferably an antigen-binding portion of an antibody as described herein.
  • a fragment may be made by truncation, e.g. by removal of one or more amino acids from the N and/or C-terminal ends of a polypeptide.
  • a variant may comprise one or more substitutions, deletions or additions with respect to the sequences of a specific anti-VEGF-A165b antibody disclosed herein.
  • a variant may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and/or deletions from the specific sequences disclosed herein.
  • “Deletion” variants may comprise the deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions, such as the deletion of specific amino acid domains or other features.
  • substitution variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions.
  • an amino acid may be substituted with an alternative amino acid having similar properties, for example, 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.
  • 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.
  • 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.
  • 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.
  • the variant may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 substitutions in SEQ ID NOs: 19, 20 and/or 25.
  • the variant may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 deletions in SEQ ID NOs: 19, 20 and/or 25.
  • the variant comprises or consists of a percentage of sequence identity with the sequences (for example, the variable heavy chain sequences, the variable light chain sequences, and/or the CDR sequences) disclosed herein.
  • the variant comprises or consists of 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65% or 60% sequence identity to the sequences (for example, the variable heavy chain sequences, the variable light chain sequences, and/or the CDR sequences) disclosed herein.
  • the variant comprises or consists of a percentage of sequence identity with the variable heavy chain sequence and/or variable light chain sequence while retaining identical CDR sequences with the corresponding variable heavy chain sequence and/or variable light chain sequence, respectively.
  • the antibody or antigen-binding fragment thereof may comprise a variable heavy chain sequence selected from SEQ ID NOs: 15-22 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the antibody or antigen-binding fragment thereof may comprise a variable light chain sequence selected from SEQ ID NOs: 23-31 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the antibody or antigen-binding fragment thereof may comprise a CDR sequence selected from SEQ ID NOs: 1-6 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the antibody or antigen-binding fragment thereof may comprise a CDR sequence selected from SEQ ID NOs: 7-13 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the antibody or antigen-binding fragment thereof may comprise a V L CDR1 sequence selected from SEQ ID NOs: 14 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the VH CDR1 sequence comprises or consists of 1, 2, 3, 4, 5 or 6 substitutions.
  • the VH 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.
  • the V H CDR3 sequence comprises or consists of 1, 2, 3 or 4 substitutions.
  • the V L CDR1 sequence comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 substitutions.
  • the VL CDR2 sequence comprises or consists of 1, 2, 3, 4, 5, 6 or 7 substitutions.
  • the VL CDR3 sequence comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8 or 9 substitutions.
  • the antibody or antigen-binding fragment thereof comprises VH4- VL2 (SEQ ID NO: 33) or an amino acid sequence having at least 60% sequence identity therewith, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the antibody or antigen-binding fragment thereof comprises VH5-VL2 (SEQ ID NO: 34) or an amino acid sequence having at least 60% sequence identity therewith, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the antibody or antigen-binding fragment thereof may comprise the CDRs of any one or more of the variable heavy chain sequences selected from SEQ ID NOs: 15-22.
  • the antibody or antigen-binding fragment thereof may comprise the CDRs of any one or more of the variable light chain sequences selected from SEQ ID NOs: 23-31.
  • Some properties of the 20 main amino acids which can be used to select suitable substituents are as follows: Ala (A) aliphatic, hydrophobic, neutral Met (M) hydrophobic, neutral l h h i l A l h hili l ) l l Pre o acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, providing the function of the antibody is not significantly adversely affected.
  • Variants may be prepared during synthesis of the antibody or by post-production modification, or when the antibody is in recombinant form using the known techniques of site-directed mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.
  • the invention also includes within its scope polypeptides and polynucleotides described herein and sequences having substantial identity thereto, for example, at least 70%, 80%, 85%, 90%, 95% or 99% identity thereto.
  • the CDR sequences of the sequences may be the same, but the variable regions outside of the CDR (for example, any of the VH and/or VL sequences specified herein) may be varied for example by at least 70%, 80%, 85%, 90%, 95% or 99% identity for a given variable sequence.
  • the CDR sequences of the sequences may be the same, but the framework regions may be varied for example by at least 70%, 80%, 85%, 90%, 95% or 99% identity for a given framework sequence.
  • the framework regions may be varied by 1, 2, 3, 4 or 5 amino acid modifications, such as substitutions, deletions or insertions. The variants may retain their target binding function.
  • the percent identity of two amino acid sequences or of two nucleic acid sequences is generally determined by aligning the sequences for optimal comparison purposes (e.g. gaps can be introduced in the first sequence for best alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions.
  • the “best alignment” is an alignment of two sequences that results in the highest percent identity.
  • Preferably variant antibodies have an amino acid sequence which has more than 60%, or more than 70%, e.g. 75% or 80%, preferably more than 85%, e.g.
  • amino acid identity to the VL or VH domain of an antibody disclosed herein.
  • This level of amino acid identity may be seen across the full length of the relevant SEQ ID NO sequence or over a part of the sequence, such as across 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art.
  • An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993).
  • the NBLAST and XBLAST programs of Altschul et al. (1990) have incorporated such an algorithm.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997).
  • PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.).
  • 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.
  • modifying the antibodies and antigen-binding fragments thereof for example to remove one or more sites of N- linked glycosylation (HCDR2), Met oxidation (HCDR1), Trp oxidation or Asp isomerization, may avoid liabilities arising from such post-translation modifications that can decrease antigen binding affinity, lead to product heterogeneity and aggregation, or negatively impact their expression level during production or manufacturing.
  • the residue numbering herein may be determined by the Kabat numbering system.
  • the heavy chains HC4 or HC5 of the antibodies described herein may comprise the mutations N59Q and/or T61A. Equivalent residues may be mutated in any of the heavy chains described herein.
  • the light chains LC1 or LC2 of the antibodies described herein may comprise one or more of the mutations selected from S32A, D33E, G34A and D60E. Equivalent residues may be mutated in any of the light chains described herein.
  • Mutant chain SEQ ID Mutant chain SEQ ID HC4 129 LC1 S32A 111 LC1 132 LC1 D60E 117 LC2 133 LC2 D60E 118 in may be selected from one or more of the following in which a specified mutation of the heavy chain or light chain is indicated in parenthesis 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).
  • 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).
  • 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).
  • the antibody or antigen-binding fragment thereof comprises a combination of any of the aforementioned mutations.
  • 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).
  • 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).
  • a linker is present between the HC and LC domains.
  • the antibody or antigen-binding fragment thereof comprises the domain combinations selected from the list consisting of: SEQ ID NO: 129 + linker + SEQ ID NO: 132; SEQ ID NO: 130 + linker + SEQ ID NO: 132; SEQ ID NO: 104 + linker + SEQ ID NO: 132; SEQ ID NO: 105 + linker + SEQ ID NO: 132; SEQ ID NO: 106 + linker + SEQ ID NO: 132; SEQ ID NO: 107 + linker + SEQ ID NO: 132; SEQ ID NO: 129 + linker + SEQ ID NO: 133; SEQ ID NO: 130 + linker + SEQ ID NO: 133; SEQ ID NO: 104 + linker + SEQ ID NO: 133; SEQ ID NO: 105 + linker + SEQ ID NO: 133; SEQ ID NO: 106 + linker + SEQ ID NO:
  • the heavy chain is selected from one of SEQ ID NOs: 99-107.
  • the light chain is selected from one of SEQ ID NOs: 108-118. Any combination of heavy chain and light chain may be used to create an antibody or antigen- binding fragment thereof. It can be beneficial to eliminate antibody Fc function for example, in the case of using a receptor agonist to crosslink receptors and induce signalling, or a receptor antagonist to block receptor:ligand interactions to prevent signalling. Fc engagement of FcD ⁇ receptors on effector cells or engagement of C1q may not be wanted, as it can lead to undesired killing of biologically important cells expressing the receptor through ADCC or CDC.
  • a single mutation of Leu235Glu in the CH2 domain of the Fc portion of human IgG1 was found to be sufficient for knocking 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 fcD ⁇ ri and fcD ⁇ riia bind to a region in the fc distinct from that recognized by neonatal fcr and protein a. J Immunol, May 15, 2000, 164 (10) 5313-5318).
  • the antibody or antigen-binding fragment thereof comprises an Fc portion
  • the antibody or antigen-binding fragment thereof according to the invention may comprise LALA (SEQ ID NO: 121) modifications in the CH2 domain of the Fc portion.
  • the antibodies according to 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 which may not be numbered 234 or 235 respectively).
  • the antibody or antigen-binding fragment thereof comprises an Fc portion
  • the antibody or antigen-binding fragment thereof according to the invention may comprise an Fc portion mutated to reduce cytopathic off-target effects on specialized epithelial cells in eye and kidney due to presence of surface bound VEGF-A165b.
  • the Fc region may be selected from the group consisting of SEQ ID NOs: 164- 166.
  • Exemplary HC4 sequences with such Fc mutations may be selected from the group consisting of SEQ ID NOs: 161-163.
  • sequence identity refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters: Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10; Multiple alignment parameters -Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR (SEQ ID NO: 120).
  • An anti-VEGF-A 165 b antibody of the invention may bind to the same epitope as a specific antibody as disclosed herein, since such an antibody is likely to mimic the action of the disclosed antibody. Whether or not an antibody binds to the same epitope as another antibody may be determined by routine methods. For example, the binding of each antibody to a target may be using a competitive binding assay. Methods for carrying out competitive binding assays are well known in the art. For example, they may involve contacting together an antibody and a target molecule under conditions under which the antibody can 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 is able to displace the first antibody from antibody/target complexes may be assessed.
  • Such assessment may use any suitable technique, including, for example, Surface Plasmon Resonance, ELISA, or flow cytometry.
  • the ability of a test antibody to inhibit the binding of a first antibody to the target demonstrates that the test antibody can compete with said first antibody for binding to the target and thus that the test antibody binds to the same epitope or region on the target as the first antibody, and may therefore mimic the action of the first antibody.
  • the invention also provides a kit comprising the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the pharmaceutical composition according to the second aspect of the invention.
  • the kit may comprise (a) a therapeutically effective amount of an antibody or antigen-binding portion thereof that specifically binds to VEGF-A 165 b as described herein.
  • the kits of the invention may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above to be carried out.
  • Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions) and means to administer the anti-VEGF-A 165 b antibody (such as a vessel or an instrument comprising a needle).
  • the kit may include instructions for performing a combination therapy or method as described herein.
  • the anti-VEGF-A165b antibody described herein, or provided in the kits of the invention, may be provided as a pharmaceutical composition formulated together with a pharmaceutically acceptable carrier.
  • 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 are also compatible with the required routes of administration.
  • a pharmaceutical composition may include a pharmaceutically acceptable antioxidant. These compositions may also contain excipients and adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the active agent (e.g. antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • active agent e.g. antibody
  • dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active agent plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Pharmaceutical compositions may comprise additional active ingredients as well as those mentioned above.
  • buffer is intended to include an aqueous solution containing an acid-base mixture with the purpose of stabilising pH.
  • 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, imidazolelacetic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.
  • diluent is intended to include an aqueous or non-aqueous solution with the purpose of diluting the agent in the pharmaceutical preparation.
  • the diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil, olive oil or sesame oil).
  • adjuvant is intended to include any compound added to the formulation to increase the biological effect of the agent of the invention.
  • the adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition.
  • anions for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition.
  • the adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.
  • the excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrins, which are added to the composition, e.g., for facilitating lyophilisation.
  • polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carrageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation.
  • lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers.
  • minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.
  • the active antibody-based agents of the invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for the delivery thereof.
  • the pharmaceutical compositions of the invention may be in the form of a liposome, in which the agent is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
  • Suitable lipids also include the lipids above modified by poly(ethylene glycol) in the polar headgroup for prolonging bloodstream circulation time. Preparation of such liposomal formulations can be found in for example US 4,235,871.
  • the pharmaceutical compositions of the invention may also be in the form of biodegradable microspheres.
  • Aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(caprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres. Preparations of such microspheres can be found in US 5,851,451 and in EP 0213303.
  • the pharmaceutical compositions of the invention are 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.
  • the pharmaceutical compositions of the invention are provided in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carrageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the agent.
  • polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carrageenans, hyaluronic
  • the polymers may also comprise gelatin or collagen.
  • the agents 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.
  • the pharmaceutical compositions of the invention may include ions and a defined pH for potentiation of action of the active agent.
  • the compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc.
  • the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the pharmaceutical composition according to the second aspect of the invention for use in the treatment or prevention of ischemia in a subject.
  • the use of the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the 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.
  • ischemia comprising administering the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the pharmaceutical composition according to the second aspect of the invention, to a subject in need thereof.
  • the ischemia may be the result of peripheral artery disease (PAD) (also called peripheral arterial disease).
  • PAD peripheral artery disease
  • the disease to be treated or prevented is a condition associated with ischemia.
  • the disease to be treated or prevented is peripheral artery disease (PAD) and/or conditions associated with PAD.
  • the ischemia may be selected from cardiac or coronary ischemia, intestinal ischemia, brain ischemia, limb ischemia, retinal ischemia, renal ischemia and associated cutaneous conditions such as cyanosis and gangrene.
  • the monoclonal antibody of the invention for use in the treatment or prevention of diseases in which VEGF- A165b is raised relative to VEGF-A165a, including but not limited to myocardial infarction (MI) including ST Elevated MI, angina, systemic sclerosis/scleroderma, Raynaud’s syndrome, ulcerative colitis, Crohn’s disease, inflammatory bowel disease, diabetic retinopathy, diabetic nephropathy, diabetic neuropathic pain, diabetic neuropathy, stroke, pre-eclampsia, hypertension, obesity or hair loss.
  • MI myocardial infarction
  • VEGF- A 165 b is raised relative to VEGF-A 165 a
  • diseases in which VEGF- A 165 b is raised relative to VEGF-A 165 a including but not limited to myocardial infarction (MI) including ST Elevated MI, angina, systemic sclerosis/scleroderma, Raynaud’s syndrome, ulcerative colitis, Crohn’s disease, inflammatory bowel disease, diabetic retinopathy, diabetic nephropathy, diabetic neuropathic pain, diabetic neuropathy, stroke, pre-eclampsia, hypertension, obesity or hair loss.
  • MI myocardial infarction
  • a method of treating or preventing diseases in which VEGF-A 165 b is raised relative to VEGF-A 165 a including but not limited to myocardial infarction (MI) including ST Elevated MI, angina, systemic sclerosis/scleroderma, Raynaud’s syndrome, ulcerative colitis, Crohn’s disease, inflammatory bowel disease, diabetic retinopathy, diabetic nephropathy, diabetic neuropathic pain, diabetic neuropathy, stroke, pre-eclampsia, hypertension, obesity or hair loss, comprising administering the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the pharmaceutical composition according to the second aspect of the invention, to a subject in need thereof.
  • MI myocardial infarction
  • a convenient way of producing an antibody or antigen-binding portion according to the present invention is to express the nucleic acid encoding it, by use of nucleic acid in an expression system. Therefore, in further aspects, the invention provides nucleic acid which comprises a sequence encoding the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the heavy chain and/or light chain thereof. The present invention further provides an isolated nucleic acid encoding a specific monoclonal antibody of the present invention. Nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid which codes for a specific antibody or antigen-binding portion of the invention as defined above.
  • the skilled person will be able to determine substitutions, deletions and/or additions to such nucleic acids which will still provide an antibody or antigen-binding portion thereof in accordance with the present invention.
  • the present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one nucleic acid as described above.
  • the present invention also provides a recombinant host cell which comprises one or more constructs as above.
  • a nucleic acid encoding an antibody or antigen-binding portion of the invention forms an aspect of the present invention, as does a method of production of such, which method comprises expression from encoding nucleic acid therefor.
  • Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, a specific monoclonal antibody may be isolated and/or purified using any suitable technique, then used as appropriate.
  • Systems for cloning and expression of a polypeptide 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 a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells and many others.
  • 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 as appropriate.
  • Vectors may be plasmids, viral, e.g. phage, or phagemid, as appropriate.
  • a cell comprising the aforementioned nucleic acid.
  • 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 of the cells selected from CHO, NS0, Sp2/0, HEK293, and PER.C6.
  • a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein.
  • the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
  • the invention provides methods of preparing specific antibodies or antigen-binding fragments of the invention which comprise expressing said nucleic acids under conditions to bring about expression of said antibody or antigen-binding fragment in a cell, and recovering the antibody or antigen-binding fragment.
  • Specific antibodies or antigen-binding fragments according to the invention may be used in a method of treatment of the human or animal body, such as a method of treatment of ischemia in a patient (preferably human) which comprises administering to said patient an effective amount of a specific antibody or antigen-binding fragment of the invention.
  • Figure 1 PCR using several combinations of Ig variable domain primers.
  • Figure 2 Sequence alignment of heavy chains using a Clustal W 2.1 multiple sequence alignment tool (www.expasy.ch).
  • Figure 3 2D representation or Collier de Perles of the V-REGION of the heavy chain CDR loops (Lefranc, M.-P. et al., Dev. Comp.
  • Figure 5 2D representation or Collier de Perles of the V-REGION of the light chain CDR loops (Lefranc, M.-P. eta/., Dev. Comp. lmmunol., 27, 55-77 (2003) PMID: 12477501). Amino acids are shown in the one-letter abbreviation.
  • the CDRs assigned using the IMGT numbering system are limited by amino acids shown in squares (anchor positions), which belong to the neighbouring FR-IMGT. Hatched circles correspond to missing positions according to the IMGT unique numbering. Arrows indicate the direction of the beta strands and their different designations in 3D structures (from IMGT repertoire, http://imgt.cines.fr).
  • Figure 6 4-20% denaturing, reducing (A) and non-reducing (B), SDS-PAGE analysis of Ab 1126 HC0 LC0 to HC1 LC5 #250719. Molecular weight marker shown in kilodaltons. Lanes are as follows: Lane number Sample Lot Amount ( ⁇ g) Conditions* 1 PageRule (Thermo Fisher) NA NA R/NR Figure 7: 4-20% denaturing, reducing (A) and non-reducing (B), SDS-PAGE analysis of Ab 1126 HC2 LC1 to HC2 LC5 #250719 and #080819. Molecular weight marker shown in kilodaltons.
  • Lanes are as follows: Lane number Sample Lot Amount ( ⁇ g) Conditions* 1 PageRule (Thermo Fisher) NA NA R/NR 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 marker shown in kilodaltons. Lanes are as follows: Lane number Sample Lot Amount ( ⁇ g) Conditions* 1 PageRule (Thermo Fisher) NA NA R/NR Figure 9: 4-20% denaturing, reducing (A) and non-reducing (B), SDS-PAGE analysis of Ab 1126 HC4 LC1 to HC4 LC5 #250719.
  • Lanes are as follows: Lane number Sample Lot Amount ( ⁇ g) Conditions* 1 PageRule (Thermo Fisher) NA NA R/NR Figure 10: 4-20% denaturing, reducing (A) and non-reducing (B), SDS-PAGE analysis of Ab 1126 HC5 LC1 to HC5 LC5 #250719. Molecular weight marker shown in kilodaltons. Lanes are as follows: Lane number Sample Lot Amount ( ⁇ g) Conditions* 1 PageRule (Thermo Fisher) NA NA R/NR Figure 11: Characterization of IgG:antigen interactions.
  • Lanes are as follows: Lane number Sample Lot Amount ( ⁇ g) Conditions 1 PageRule (Thermo Fisher) NA NA Reducing , , 1126 HC4 LC2 lot #061020. Molecular weight marker shown in kilodaltons. Lanes are as follows: Lane number Sample Lot Amount ( ⁇ g) Conditions 1 PageRule (Thermo Fisher) NA NA Reducing , , 1126 HC5 LC2 lot #061020. Molecular weight marker shown in kilodaltons. Lanes are as follows: Lane number Sample Lot Amount ( ⁇ g) Conditions 1 PageRule (Thermo Fisher) NA NA Reducing .
  • FIG. 21 (A) A pictographic portrayal of anti-VEGF-A, anti-VEGF 165 b, VEGF 165 b and rh- Fc-VEGFR2. (B) The inhibitory effect of anti-VEGF 165 b (HC4 LC2) on VEGF 165 b binding to fc-VEGF receptor 2.
  • Figure 22 Cell migration assay for chimeric anti-VEGF165b (HC0 LC0) and humanised anti-VEGF165b (HC4 LC2 Vexobicizumab) and mouse anti-VEGF165b antibody (mD165b) in the presence of VEGF 165 a.
  • Figure 23 Cell migration assay for mD165b, chimeric anti-VEGF165b (HC0 LC0) and humanised anti-VEGF 165 b (HC4 LC2 Vexobicizumab) and previously published mouse anti- VEGF 165 b antibody (MRVL56/1, AbCam) in the presence of VEGF 165 a.
  • B. IC50 for each of the antibodies (Vex HC4 LC2).
  • Figure 24 (A) Pictographic representation of a cell migration assay in diabetic PAD human monocyte mediated inhibition of endothelial migration. (B) Results of the cell migration assay.
  • Figure 25 A schematic of the mouse experiment described in Example 4.
  • Figure 26 Murine analysis of revascularisation in hind limb ischemia. Examples of mice fed a high fat high sucrose (HFHS) diet for 12 weeks followed by blood flow imaging by laser speckle of mice before (Pre-Op) and after (Post-Op) femoral artery ligation, and on subsequent days.
  • Figure 27 Graphical quantification of the murine analysis of blood flow in hind limb ischemia. Speckle intensity was calculated as the ipsilateral (ischemic) relative to the contralateral flow.
  • HFHS high fat high sucrose
  • Figure 29 A schematic of the rat experiment described in Example 4.
  • Figure 30 Rat analysis of revascularisation in hind limb ischemia. Examples of paw blood flow imaging by laser speckle of rats before (Pre-Op) and after (Post-Op) femoral artery ligation, and on subsequent days. Speckle intensity was calculated as the ipsilateral (ischemic) relative to contralateral flow.
  • Figure 33 A. Human monocytes inhibit migration of endothelial cells across a porous membrane. 1 Njg/ml HC4 LC2 significantly reverses this inhibitory effect on migration of human umbilical vein cells.
  • N 3, cells from a single subject.
  • B Monocytes from 7 patients with peripheral vascular disease inhibit migration towards VEGF-A 165 a.
  • Figure 34 A single gene can result in opposing isoforms of VEGF – proangiogenic VEGF- A 165 a and antiangiogenic VEGF-A 165 b.
  • Figure 35 A key to show the antibody derivation from parental hybridomas.
  • FIG. 36 To determine the neutralizing effect of the antibodies, VEGFR2 was bound to an ELISA plate and the anti-VEGF-A165b antibodies mixed with rhVEGF-A165b at increasing concentrations of VEGF-A165b antibody.
  • Humanised (HC4LC2) and chimeric (HC0LC0) anti-VEGF 165 b were both able to significantly reduce the affinity of VEGF 165 b to VEGF receptor 2 (VEGFR2), again, to a slightly lesser extent than the positive control of G6-31 (a pan VEGF antibody that binds to the receptor binding site of VEGF-A).
  • Figure 37 Cell migration assay for humanised anti-VEGF165b (HC5 LC2) and mouse anti- VEGF165b antibody (mD165b – referred to as “56/8” in the figure) in the presence of VEGF 165 a.
  • Example 1 Monoclonal sequencing mRNA was extracted from hybridoma cell pellets. Total RNA was extracted from the pellets using Fusion Antibodies Pie in-house RNA extraction protocol. cDNA was reverse transcribed from the RNA with an oligo (dT) primer. 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 are detailed in Figure 1. The VH and VL products were cloned into the Thermo Fisher sequencing vector pCRTM2.1. TOP 10 cells were transformed with the cloned plasmid and screened by PCR for positive transformants.
  • Heavy chain x VH2.2 protein (SEQ ID NO: 38) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFIISR DNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAAKTTPPPVYP x VH2.2 nucleic acid (SEQ ID NO: 39) GAGGTGAAGCTTCTCGAATCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCC TCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGATTCATCCATATAGCAGTACGATAAACTATACGCCATCTGTAAAGGATAAATTCATCATCTCCAGA GACAACG
  • Light chain x VL2.1 protein (SEQ ID NO: 137) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGS GTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF x VL2.1 nucleic acid (SEQ ID NO: 149) GATATTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAG TCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCT CCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCA GGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGG
  • Example 2 Transient expression, purification and affinity screening of Ab 1126 humanised anti-VEGF-A165b clone mD165b (also referred to herein as 56/8/31/1/16) variants (25 + 1) Summary Ab 1126 humanized variants are 150 kilodalton (kDa) antibodies composed of two heavy chains and two light chains, complexed together via disulphide bonds.
  • a mammalian expression vector encoding each variant was transfected into CHO cells and batch cultures of each variant grown for up to seven days.
  • the expressed antibodies were purified from cell culture supernatant via affinity chromatography. The concentration and purity were determined for the purified antibody products. Results from these quality control experiments are detailed below.
  • Expression and purification of Ab 1126 DNA coding for the amino acid sequence of the Ab 1126 variants were synthesised and cloned into the mammalian transient expression plasmid pETE V2 (property of Fusion antibodies).
  • Ab 1126 variants were expressed using a CHO based transient expression system and the resulting antibody containing cell culture supernatants were clarified by centrifugation and filtration.
  • Ab 1126 variant sequences (the signal peptides used for expression of the antibody heavy and light chains are underlined, which may be excluded from the heavy and light chain sequences): >Ab 1126 HC0 (SEQ ID NO: 71, with signal peptide) MGWTLVFLFLLSVTAGVHSEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGE IHPYSSTINYTPSVKDKFIISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSAASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVDV
  • ND not (mg/ml) (ml) (mg) yield IgG antibodies were immobilized on biosensors using suitable capture surfaces and binding of soluble antigen to immobilized antibodies was monitored by BLI (Octet). The resulting sensorgrams were analyzed using the manufacturer’s supplied software (Fortebio).
  • Kinetic analysis Materials and methods Antigen: Human VEGF165b, Sino Biological, Catalog No.
  • Kinetic analysis Experimental parameters Kinetic assays were performed by first capturing IgG using anti-human Fc biosensors. The mAb capture biosensors were then submerged in wells containing different concentrations of antigen (association stage) followed by a dissociation step in running buffer. To allow for reference correction, IgG-captured sensors were dipped into wells containing only buffer. This referencing provided a means of compensating for the natural dissociation of the capture IgG. Steps were performed at 25°C at a constant flow-rate of 1000 rpm. New sensors were used for each sample. Dissociation rate constants (KD) were calculated using the ForteBio Data Analysis software. All consumables used were those recommended by ForteBio.
  • Table 2 Kinetic parameters (fit to 1:1 interaction model) for Ab 1126 variants and antigen interaction.
  • Antibody Capture level k a k d K D R 2 X 2 Mean (nm) (m -1 s -1 ) (s -1 ) (nM) R max HC4 LC3 0.688 NF NF NF NF NF NF NF HC4 LC4 0.669 NF NF NF NF NF NF NF NF . re considered a good fit;
  • X 2 is the sum of the squared deviation should be generally below 3 (ideally ⁇ 0.5).
  • X 2 is the measure of error between the experimental data and the fitted line. The smaller the X 2 indicates a better fit.
  • Mean Rmax should be similar ( ⁇ 20%) to that of the chimeric control.
  • K D should be within 2-fold of the chimeric control antibody, HC0LC0.
  • Abbreviations are as follows; NF, does not fit to 1:1 binding model.
  • All Ab 1126 variants have been successfully expressed and purified. From the SDS-PAGE analysis, all antibodies exhibit adequate levels of purity. Under denaturing and reducing conditions, both heavy and the light chains of the antibody are visible and are observed at the expected molecular weight of ⁇ 50 and 25kDa, respectively. Under denaturing and non-reducing conditions, a single major band and several minor bands are observed. The additional bands (impurities) are likely the result of non- glycosylated IgG and IgG degradation products (e.g.
  • HCX LC1 and HCX LC2 all exhibit binding to the antigen and in most cases, the experimental data fits well to a 1:1 binding model. Under the experimental conditions used here, HC5 LC2 and HC4 LC2 exhibit binding characteristics similar to that of the chimeric control antibody HC0 LC0, exhibiting dissociation constants within 2-fold of the chimeric HC0 LC0 control antibody.
  • Example 3 Transient expression of Ab 1126 variants, HC0 LC0, HC4 LC2 and HC5
  • Ab 1126 variants HC0 LC0, HC4 LC2 and HC5 LC2 are 150 kilodalton (kDa) antibodies composed of two heavy chains and two light chains, complexed together via disulphide bonds.
  • a mammalian expression vector encoding each antibody was transfected into CHO cells.
  • the expressed antibodies were subsequently purified from cell culture supernatant using standard chromatography techniques. The concentration, purity and endotoxin were determined for the purified antibody products.
  • DNA coding for the amino acid sequence of each antibody was synthesised and cloned into the mammalian transient expression plasmid pETE V2 (property of Fusion antibodies).
  • Antibodies were expressed using a CHO based transient expression system and the resulting antibody containing cell culture supernatants were clarified by centrifugation and filtration.
  • Antibodies were purified (using state-of-the-art AKTA chromatography equipment) from cell culture supernatants via affinity chromatography and preparative grade size exclusion chromatography (SEC column was pre-equilibrated in phosphate buffered saline solution). Purified antibodies were buffer exchanged into phosphate buffered saline solution.
  • the purity of the antibodies was determined to be >95%, as judged by reducing and denaturing Sodium Dodecyl Sulfate Polyacrylamide gels (Figure 12-14).
  • Antibody samples were analysed via size exclusion chromatography (SEC). The chromatograms each show one major peak (>95% total area [Figure 15-17]).
  • Bacterial endotoxin levels were determined using the Endosafe®-PTS system and Endosafe® PTS cartridges (Charles River Laboratories).
  • Table 3 Purification summary: Ab 1126 variants. Sample Concentration Volume Total *Purity Monomer Endotoxin (mg/ml) (ml) (mg) (%) level (EU/mg) d . Conclusion Antibodies were successfully expressed and purified. SEC analyses were performed successfully for all antibodies. The chromatogram shows one major peak (>98% total area). From the SDS-PAGE analysis, all antibodies exhibit adequate levels of purity, under reducing and denaturing conditions. Under reducing conditions, both heavy and the light chains of the antibody are visible and are observed at the expected molecular weight of ⁇ 50 and 25kDa, respectively. Under non-reducing conditions, a single major band and several minor bands are observed.
  • Example 4 Evaluation of the suitability of a novel humanised monoclonal A acronyms Abbreviation Definition aSEC Analytical size exclusion chromatography DSC Differential scanning calorimetry DSF Differential scanning fluorimetry s Back Peripheral Artery Disease (PAD) is a major unmet clinical need afflicting approximately 10 million people in the United States. Vascular insufficiency in PAD results in tissue ischemia.
  • PAD Peripheral Artery Disease
  • VEGF-A vascular endothelial growth factor
  • sfrp5 soluble frizzled related protein 5
  • Wnt5a activity resulting in overexpression of the anti-angiogenic isoform, VEGF-A 165 b.
  • sfrp5 soluble frizzled related protein 5
  • VEGF-A 165 b antibodies to antibodies to VEGF-A 165 b can reverse impaired revascularisation in mouse models of hindlimb ischemia.
  • Lower-extremity ischemia in PAD is painful and disabling, causing non-healing ulcers and results in 200,000 amputations per year in the United States alone.
  • VEGF-A 165b is a competitive inhibitor of VEGF-A165a mediated angiogenesis. Circulating VEGF-A in patients with PAD is predominantly comprised of the VEGF-A165b isoform. In mouse experimental models, mVEGF-A165b expression is upregulated under conditions of systemic metabolic dysfunction.
  • VEGF-A 165 b impairs revascularization in an experimental model of PAD 2
  • acute antibody-mediated neutralization of mVEGF-A 165 b isoform promotes revascularization of ischemic tissue under conditions in which the process of regenerative angiogenesis is impaired.
  • VEGF-A165b may serve as a new pharmacological target to treat limb ischemia in patients with PAD and that its neutralization may augment the activities of pro-angiogenic growth factors 3 .
  • a mouse monoclonal antibody specific for VEGF-A165b was generated by immunisation of BALB/c mice with synthetic peptide fragments of the nine amino acid carboxy-terminal sequence of VEGF-A165b, the final six of which (CDKPRR) are specific to VEGF-A 165 b, coupled to keyhole limpet haemocyanin (KLH) 4 .
  • CDKPRR keyhole limpet haemocyanin
  • One of the resultant hybridomas specifically detected VEGF-A 165 b and not VEGF-A165a.
  • Twenty-five humanised variants of one mouse anti-VEGF-A165b neutralizing mAb were generated. The variants were transiently expressed in CHO cells and purified from culture supernatants by single column protein A affinity chromatography.
  • an exemplary antibody clone, HC4 LC2 was able to reduce the affinity of VEGF 165 b to VEGF receptor 2 (VEGFR2), to a slightly lesser extent than the positive control of G6-31, an antibody that binds the receptor binding site (see Figure 21B).
  • VEGFR2 VEGF receptor 2
  • FIG 21B an exemplary antibody clone, HC4 LC2
  • PAD diabetic peripheral arterial disease
  • Example 5 Humanisation of anti-VEGF-A165b mD165b
  • the sequences of the heavy chain (HC) variable region (mVH) and light chain (LC) variable region (mVL) of the parental mouse 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) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIHPYSSTINYTPSVKDKFI ISRDNAKNTLYLQMSEVRSEDTALYYCARAFAYWGQGTLVTVSA Amino acid sequence of Clone 58/8/31/1/13 mVL (SEQ ID NO: 23) DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRP
  • Blast analysis against the IMGT database 7 of human antibody sequences reveals that the light chain CDRs from the mouse antibody have been grafted into a human IGKV2-30 framework (see Blast 3 below).
  • Results are shown in Table 5. All variants can be considered humanized.
  • the approved INN name for the therapeutic candidate is Vexobicizumab.
  • HC0 LC0 a chimeric version of the antibody was made (designated HC0 LC0) by grafting the full-length mouse VH and VL domains onto 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 culture supernatants by Protein A affinity chromatography and dialysed/buffer exchanged into PBS for in vitro testing.
  • the yields of antibody (in mg/L) were comparable or higher than for the chimeric antibody HC0 LC0 except for HC1 LC1 and HC3 LC4.
  • Kinetic analysis of each of the antibodies was performed by biolayer interferometry (BLI) using the Fortebio Octet system. Antibodies were captured on anti-human Fc biosensors then incubated with commercially available human VEGF-A165b purchased from Sino Biological. Capture levels of each antibody to the Fc biosensor were comparable.
  • Antibodies which had affinities >1 nM were HC1 LC1 (5.0 nM), HC1 LC2 (2.1 nM), HC2 LC1 (4.4 nM) HC2 LC2 (3.6 nM), HC3 LC1 (1.5 nM), HC3 LC2 (1.17 nM) and HC5 LC1 (3.2 nM). Scale up of HC0 LC0, HC4 LC2 and HC5 LC2 was subsequently performed. Antibodies were transiently expressed in CHO cells and purified by Protein A affinity chromatography from culture supernatants. Purified mAbs were buffer exchanged into PBS and analysed by SDS-PAGE and size exclusion chromatography.
  • the IC50 values for inhibition of VEGF-A 165 b were 442 ng/ml, 137 ng/ml, and 114 ng/ml for HC0 LC0, HC4 LC2, and HC5 LC2 respectively.
  • the chimeric antibody HC0 LC0 is able to reverse obesity-dependent, impaired revascularisation in a mouse femoral artery ligation ischemia model for up to 30 days when administered i.p. at 1 mg/kg on Day 1, Day 3 and Day 7 after ischemia.
  • the humanized antibodies have not yet been tested in this model. Proof of concept has also recently been demonstrated by Navarro et al 14 .
  • 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.
  • 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. R ⁇ os-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).
  • the chimeric antibody HC0 LC0 was tested alongside YTE only and LALA + YTE variants to ensure that changes in the Fc have no impact on antibody binding to target.
  • Sequence liability mutants were tested in wild type IgG1 format in the first instance to identify mutations which are tolerated (no loss of binding or functional activity) before making a further set of antibody variants in which HC and LC mutations will be combined.
  • Parameters measured include: yield, SDS-PAGE (reducing and non-reducing), analytical SEC, binding affinity to VEGF-A 165 b compared with parental antibodies (HC0 LC0, HC4 LC2, HC5 LC2 and HC4 LC1). Antibody sequences without signal peptide Residues to be mutated are shown in underlined (Fc domain) or double-underlined (variable domain).
  • Anti-VEGF165b antibody/VEGF165b mixtures was made up with 4 ng/ml VEGF-A 165 b and increasing concentrations of each antibody and incubate for 2 hours at 4°C.
  • the VEGFR2 coated plate was washed with 0.05% tween20 in PBS (PBST) three times. The plate was blocked with Suberblock buffer and washed 3 times.
  • 100 Njl of the anti-VEGF165b antibody/VEGF165b mixture was added and incubated for 1 hour followed by a 3 x wash.
  • 100 Njl Biotinylated anti-VEGF-A (BAF293, R&D Systems) in 1% BSA/PBS was added and incubated for 2 hours at room temp.
  • HC4LC2 and chimeric (HC0LC0) anti-VEGF165b antibodies were both able to significantly reduce the affinity of VEGF 165 b to VEGF receptor 2 (VEGFR2), again, to a slightly lesser extent than the positive control of G6-31 (a pan VEGF antibody that binds to the receptor binding site of VEGF-A) ( Figure 36).
  • G6-31 a pan VEGF antibody that binds to the receptor binding site of VEGF-A
  • C1q binding is the first step in the initiation of the complement cascade.
  • C1q forms a complex with the serine proteases C1r and C1s to form the C1 complex.
  • C1q is capable of binding six antibodies, although binding to two IgGs is sufficient to activate the complement cascade. Therefore, eliminating C1q binding to Fc – the initial event in the activation of antibody-dependent complement cytotoxicity – can be achieved through site-directed mutagenesis of specific residues in the Fc domain of antibodies.
  • Vexobicizumab is a humanised IgG1 antibody. IgG1 is known to activate complement. However, the activation requires clustering of the antibody on the cell surface via interaction with its specific target (receptor) expressed on the cell surface. In theory, Vexobicizumab should not fix complement as its target, VEGF-A165b, is a soluble molecule. However, non-specific binding of VEGF-A may occur through binding to heparin sulphate proteoglycans (HSPGs) expressed on the surface of non-immune cells, e.g. endothelial cells, epithelial cells and fibroblasts.
  • HSPGs heparin sulphate proteoglycans
  • VEGF-A165b Unlike the pro-angiogenic form of VEGF-A (VEGF- A165a), the VEGF-A165b splice variant has been reported (data not shown) to have low or no binding to heparin (despite the fact that it is predicted to contain a heparin binding domain), so, therefore, it should not bind to heparin sulphate proteoglycans on the cell surface, making it susceptible to complement mediated lysis. On the other hand, it is unknown if binding of VEGF-A165b to its receptor VEGFR2 mediates internalization of the receptor, thus it is possible that the receptor-ligand antibody complex (transiently) present on the cell surface may elicit Fc-mediated complement activation.
  • Mutations may be introduced to reduce risk of possible cytopathic off-target effects on specialized epithelial cells in eye and kidney due to presence of surface bound VEGF-A 165 b. These cells are on the far side of the semi-permeable barriers which normally exclude antibody-sized molecules so it may not be a problem.
  • the mutations may contain single amino acid mutations in the Fc domain that have been reported to abrogate C1q binding and thus limit complement activation, without impacting binding to the neonatal FC receptor (FcRn).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des anticorps anti-VEGF-A165b. L'invention concerne également des compositions pharmaceutiques, des kits et des méthodes d'utilisation thérapeutiques.
PCT/GB2023/052666 2022-10-13 2023-10-13 Anticorps anti-vegf WO2024079482A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB2215115.3 2022-10-13
GBGB2215115.3A GB202215115D0 (en) 2022-10-13 2022-10-13 VEGF antibodies

Publications (1)

Publication Number Publication Date
WO2024079482A1 true WO2024079482A1 (fr) 2024-04-18

Family

ID=84818408

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2023/052666 WO2024079482A1 (fr) 2022-10-13 2023-10-13 Anticorps anti-vegf

Country Status (3)

Country Link
GB (1) GB202215115D0 (fr)
TW (1) TW202417493A (fr)
WO (1) WO2024079482A1 (fr)

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235871A (en) 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
EP0213303A2 (fr) 1985-07-12 1987-03-11 Bo Magnus Ekman Procédé pour préparer de petites particules sphériques de polymères
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
WO1996006641A1 (fr) 1994-08-29 1996-03-07 Prizm Pharmaceuticals, Inc. Conjugues du facteur de croissance endothelial vasculaire avec des agents cibles
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5851451A (en) 1995-12-15 1998-12-22 Takeda Chemical Industries, Ltd. Production of microspheres
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
US6407213B1 (en) 1991-06-14 2002-06-18 Genentech, Inc. Method for making humanized antibodies
US6881557B2 (en) 2001-07-12 2005-04-19 Arrowsmith Technologies Llp Super humanized antibodies
WO2011128642A1 (fr) 2010-04-13 2011-10-20 Alligator Bioscience Ab Compositions comprenant des nanoparticules d'acide polyglutamique et des polypeptides tels que des agonistes de cd40
WO2014117053A1 (fr) * 2013-01-27 2014-07-31 The Cleveland Clinic Foundation Isoforme anti-angiogénique du polypeptide vegf-ax

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235871A (en) 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0213303A2 (fr) 1985-07-12 1987-03-11 Bo Magnus Ekman Procédé pour préparer de petites particules sphériques de polymères
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
US6407213B1 (en) 1991-06-14 2002-06-18 Genentech, Inc. Method for making humanized antibodies
WO1996006641A1 (fr) 1994-08-29 1996-03-07 Prizm Pharmaceuticals, Inc. Conjugues du facteur de croissance endothelial vasculaire avec des agents cibles
US5851451A (en) 1995-12-15 1998-12-22 Takeda Chemical Industries, Ltd. Production of microspheres
US6881557B2 (en) 2001-07-12 2005-04-19 Arrowsmith Technologies Llp Super humanized antibodies
WO2011128642A1 (fr) 2010-04-13 2011-10-20 Alligator Bioscience Ab Compositions comprenant des nanoparticules d'acide polyglutamique et des polypeptides tels que des agonistes de cd40
WO2014117053A1 (fr) * 2013-01-27 2014-07-31 The Cleveland Clinic Foundation Isoforme anti-angiogénique du polypeptide vegf-ax

Non-Patent Citations (65)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING COMPANY
"SGR Hurrell", 1982, CRC PRESS, article "Monoclonal Hybridoma Antibodies: Techniques and Application"
AL KAWAS HIVIN ET AL: "How VEGF-A and its splice variants affect breast cancer development - clinical implications", CELLULAR ONCOLOGY, SPRINGER, DORDRECHT, vol. 45, no. 2, 18 March 2022 (2022-03-18), pages 227 - 239, XP037810137, ISSN: 2211-3428, [retrieved on 20220318], DOI: 10.1007/S13402-022-00665-W *
AVERY, L. B. ET AL.: "Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics", MABS, vol. 10, 2018, pages 244 - 255
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, vol. 8, 2016, pages 1064 - 1078, XP055882632, DOI: 10.1080/19420862.2016.1193660
BAUMINGERWILCHEK, METHODS ENZYMOL., vol. 70, 1980, pages 151 - 159
BECK, A.LIU, H., MACRO-AND MICRO-HETEROGENEITY OF NATURAL AND RECOMBINANT IGG ANTIBODIES, 2019
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., vol. 109, 2020, pages 3330 - 3339
BOERNER ET AL.: "Human FcyRI and FcyRII interact with distinct but overlapping sites on human IgG", J. IMMUNOL., vol. 147, 1991, pages 2657 - 95
BRADER, M. L. ET AL.: "Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies", MOL. PHARM., vol. 12, 2015, pages 1005 - 1017, XP055894975, DOI: 10.1021/mp400666b
CACECI ET AL., BYTE, vol. 9, 1984, pages 340 - 362
CARTER, J. G. ET AL.: "The carboxyl terminus of VEGF-A is a potential target for anti-angiogenic therapy", ANGIOGENESIS, vol. 18, 2015, pages 23 - 30, XP035416196, DOI: 10.1007/s10456-014-9444-3
CARTER, P. ET AL.: "Humanization Of An Anti-pl85her2 Antibody ForHuman Cancer Therapy", PROC. NATL. ACAD. SCI. (U.S.A., vol. 89, 1992, pages 4285 - 4289
CEBE-SUAREZ, S.ZEHNDER-FJÄLLMAN, A.BALLMER-HOFER, K.: "The role of VEGF receptors in angiogenesis; complex partnerships", CELL. MOL. LIFE SCI., vol. 63, 2006, pages 601 - 615, XP019201039, DOI: 10.1007/s00018-005-5426-3
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, vol. 11, 2019, pages 747 - 756, XP055907038, DOI: 10.1080/19420862.2019.1589851
CO, M. S. ET AL.: "Humanized Antibodies For Antiviral Therapy", PROC. NATL. ACAD. SCI. (U.S.A., vol. 88, 1991, pages 2869 - 2873, XP000200583, DOI: 10.1073/pnas.88.7.2869
CO, M.S. ET AL.: "Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen", J. IMMUNOL., vol. 148, 1992, pages 1149 - 1154
COLE ET AL.: "Monoclonal antibodies and Cancer Therapy", 1985, ALAN R. LISS, pages: 77
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, vol. 38, 2010, pages D301 - 7, XP055247165, DOI: 10.1093/nar/gkp946
FRENZEL AHUST MSCHIRRMANN T: "Expression of recombinant antibodies", FRONT IMMUNOL., vol. 4, 29 July 2013 (2013-07-29), pages 217, XP055283974, DOI: 10.3389/fimmu.2013.00217
GANTA VIJAY CHAITANYA ET AL: "Antiangiogenic VEGF 165 b Regulates Macrophage Polarization via S100A8/S100A9 in Peripheral Artery Disease", CIRCULATION, vol. 139, no. 2, 8 January 2019 (2019-01-08), US, pages 226 - 242, XP093104172, ISSN: 0009-7322, DOI: 10.1161/CIRCULATIONAHA.118.034165 *
GANTA VIJAY CHAITANYA ET AL: "VEGF 165 b Modulates Endothelial VEGFR1-STAT3 Signaling Pathway and Angiogenesis in Human and Experimental Peripheral Arterial Disease", CIRCULATION RESEARCH, vol. 120, no. 2, 20 January 2017 (2017-01-20), US, pages 282 - 295, XP093101673, ISSN: 0009-7330, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453503/pdf/nihms837443.pdf> DOI: 10.1161/CIRCRESAHA.116.309516 *
GORMAN, S. D. ET AL.: "Reshaping A Therapeutic CD4 Antibody", PROC. NATL. ACAD. SCI. (U.S.A., vol. 88, 1991, pages 4181 - 4185, XP000368701, DOI: 10.1073/pnas.88.10.4181
HABERGER, M ET AL.: "Functional assessment of antibody oxidation by native mass spectrometry", MABS, vol. 7, 2015, pages 891 - 900
HOOGENBOOMWINTER, J. MOL. BIOL., vol. 222, 1991, pages 581
IDUSOGIE EEPRESTA LGGAZZANO-SANTORO HTOTPAL KWONG PYULTSCH M ET AL.: "Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc", J IMMUNOL., vol. 164, 2000, pages 4178 - 84, XP002965858
IGAWA, T. ET AL.: "Engineering the variable region of therapeutic IgG antibodies", MABS, vol. 3, 2011, pages 243 - 252, XP055532830, DOI: 10.4161/mabs.3.3.15234
JARASCH, A ET AL.: "Developability assessment during the selection of novel therapeutic antibodies", J. PHARM. SCI., vol. 104, 2015, pages 1885 - 1898, XP055217440, DOI: 10.1002/jps.24430
JEFFERIS, R.: "Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action", TRENDS PHARMACOL. SCI., vol. 30, 2009, pages 356 - 362, XP026250779, DOI: 10.1016/j.tips.2009.04.007
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
JONES, T. D. ET AL.: "The INNs and outs of antibody nonproprietary names", MABS, vol. 8, 2016, pages 1 - 9, XP055813029, DOI: 10.1080/19420862.2015.1114320
KETTLEBOROUGH, C. A. ET AL.: "Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation", PROTEIN ENGINEERING, vol. 4, 1991, pages 773 - 3783
KIKUCHI ET AL., NAT MED, 2014
KIKUCHI ET AL., NAT MED., vol. 20, no. 12, 2014, pages 1464 - 1471
KIKUCHI, R. ET AL.: "An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease", NAT. MED., vol. 20, 2014, pages 1464 - 1471, XP055442124, DOI: 10.1038/nm.3703
KRAFT, T. E. ET AL.: "Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis", MABS, vol. 12, 2020, pages 1683432, XP055718036, DOI: 10.1080/19420862.2019.1683432
KUPPUSWAMY ET AL.: "Targeting Anti-Angiogenic VEGF165b-VEGFR1 Signaling Promotes Nitric Oxide Independent Therapeutic Angiogenesis in Preclinical Peripheral Artery Disease Models", CELLS, vol. 11, 2022, pages 2676
KUPPUSWAMY SIVARAMAN ET AL: "Targeting Anti-Angiogenic VEGF165b-VEGFR1 Signaling Promotes Nitric Oxide Independent Therapeutic Angiogenesis in Preclinical Peripheral Artery Disease Models", CELLS, vol. 11, no. 17, 28 August 2022 (2022-08-28), pages 2676, XP093101671, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9454804/pdf/cells-11-02676.pdf> DOI: 10.3390/cells11172676 *
LAZAR GADANG WKARKI SVAFA OPENG JSHYUN L ET AL.: "Engineered antibody Fc variants with enhanced effector function", PROC NATL ACAD SCI USA., vol. 103, 2006, pages 4005 - 10
LEFRANC, M.-P. ET AL., NUCLEIC ACIDS RESEARCH, vol. 27, 1999, pages 209 - 272
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., vol. 27, 2003, pages 55 - 77, XP055585227, DOI: 10.1016/S0145-305X(02)00039-3
LEFRANC, M.-P.: "IMGT, the international ImMunoGeneTics information system, http://imgt.cines.fr", NOVARTIS FOUND. SYMP., vol. 254, 2003, pages 126 - 142
LIU, H.SAXENA, A.SIDHU, S. S.WU, D.: "Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds", FRONT IMMUNOL, vol. 8, 2017, pages 38
LOBUGLIO, A. F. ET AL.: "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response", PROC. NATL. ACAD. SCI. (U.S.A., vol. 86, 1989, pages 4220 - 4224, XP002100301, DOI: 10.1073/pnas.86.11.4220
LUND ET AL.: "Multiple binding sites on the CH2 domain of IgG for mouse FcyRII", MOL. IMMUNOL., vol. 29, 1992, pages 53, XP023682519, DOI: 10.1016/0161-5890(92)90156-R
MAEDA, H. ET AL.: "Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity", HUMAN ANTIBODIES HYBRIDOMA, vol. 2, 1991, pages 124 - 134
NEUBERGER ET AL., 8TH INTERNATIONAL BIOTECHNOLOGY SYMPOSIUM PART 2, 1998, pages 792 - 799
NUGENT, P.DUNCAN, J. N.COLAGIOVANNI, D. B.: "A Comprehensive Guide to Toxicology in Preclinical Drug Development", 2013, ELSEVIER INC., article "The Preparation of a Preclinical Dossier to Support an Investigational New Drug (IND) Application and First-inHuman Clinical Trial", pages: 309 - 334
PAUL, S ET AL.: "Development and validation of a broad scheme for prediction of HLA class II restricted T cell epitopes", J. IMMUNOL. METHODS, vol. 422, 2015, pages 28 - 34, XP029239932, DOI: 10.1016/j.jim.2015.03.022
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
RIECHMANN, L. ET AL.: "Reshaping Human Antibodies for Therapy", NATURE, vol. 332, 1988, pages 323 - 327, XP002007067, DOI: 10.1038/332323a0
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., vol. 74, 2021, pages 131 - 139, XP086466662, DOI: 10.1016/j.rec.2020.03.013
RYOSUKE KIKUCHI ET AL: "An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease", NATURE MEDICINE, vol. 20, no. 12, 1 December 2014 (2014-12-01), New York, pages 1464 - 1471, XP055442124, ISSN: 1078-8956, DOI: 10.1038/nm.3703 *
SARMAY ET AL.: "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 Fcy receptor", MOL. IMMUNOL., vol. 29, 1992, pages 633, XP000915240, DOI: 10.1016/0161-5890(92)90200-H
SATO, K. ET AL., CANCER RES, vol. 53, 1993, pages 851 - 856
SAUNDERS K: "O Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life 2019 Front", IMMUNOL., 7 June 2019 (2019-06-07)
SCHLOTHAUER, T ET AL.: "Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies", MABS, vol. 5, 2013, pages 576 - 586, XP055132983, DOI: 10.4161/mabs.24981
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., vol. 567, 2016, pages 359 - 389
TEMPEST, P.R. ET AL.: "Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo", BIO/TECHNOLOGY, vol. 9, 1991, pages 266 - 271, XP000986147, DOI: 10.1038/nbt0391-266
VAN DE BOVENKAMP, F. S.: "Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability", FRONT. IMMUNOL., vol. 9, 2018, pages 740
VERHOEYEN, M. ET AL.: "Reshaping Human Antibodies: Grafting An Antilysozyme Activity", SCIENCE, vol. 239, 1988, pages 1534 - 15361
WINES ET AL.: "The IgG fc contains distinct fc receptor (fcr) binding si es: the leukocyte receptors feyri and fcyriia bind to a region in the fc distinct from that recognized by neonatal fcr and protein a", J IMMUNOL, vol. 164, no. 10, 15 May 2000 (2000-05-15), pages 5313 - 5318
WONGLOHMAN, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5428 - 5432
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., vol. 64, 2004, pages 7822 - 7835
XU YOOMEN RKLEIN 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., vol. 269, 1994, pages 3469 - 74

Also Published As

Publication number Publication date
GB202215115D0 (en) 2022-11-30
TW202417493A (zh) 2024-05-01

Similar Documents

Publication Publication Date Title
US20210301028A1 (en) Composition and methods for anti-tnfr2 antibodies
US20220119543A1 (en) ANTI-TfR ANTIBODIES AND THEIR USE IN TREATING PROLIFERATIVE AND INFLAMMATORY DISORDERS
JP6286532B2 (ja) 抗ｃｄ３８抗体及び弱毒化インターフェロンアルファ−２ｂとの融合物
US20170233481A1 (en) Anti-cd25 antibodies and their uses
KR101852915B1 (ko) 과립구-대식세포 콜로니 자극 인자에 대한 항체
EP3262075B1 (fr) Nouvel anticorps se liant à la tfpi et composition le comprenant
CA2681530A1 (fr) Proteines liantes, y compris des anticorps, derives d&#39;anticorps et fragments d&#39;anticorps qui lient specifiquement cd154
US20150010538A1 (en) Anti-cd25 antibodies and their uses
TW202003570A (zh) 抗trem-1抗體及其用途
KR102602564B1 (ko) 인터루킨-6의 인간 수용체에 결합가능한 항체 또는 항원 결합성 단편
JP6522585B2 (ja) Ｃｘｃｒ５に対するモノクローナル抗体
KR101804988B1 (ko) 신규 항-tfpi 항체 및 이를 포함하는 조성물
KR20230042030A (ko) 항-IgE 조작된 항체 및 이의 적용
WO2024079482A1 (fr) Anticorps anti-vegf
JP6853392B2 (ja) 抗ｃｄ３８抗体及び弱毒化インターフェロンアルファ−２ｂとの融合物
CN113166264B (zh) 一种分离的抗原结合蛋白及其用途
CN113164601B (zh) 一种分离的抗原结合蛋白及其用途
JP2023551907A (ja) 腫瘍関連抗原及びｃｄ３結合タンパク質、関連する組成物、及び方法
JP2018087218A (ja) 抗ｃｄ３８抗体及び弱毒化インターフェロンアルファ−２ｂとの融合物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23797852

Country of ref document: EP

Kind code of ref document: A1