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WO2014108854A1 - Anticorps anti-hgf et anti-ang2 monospécifiques, et anticorps anti-hgf/anti-ang2 bispécifiques - Google Patents

Anticorps anti-hgf et anti-ang2 monospécifiques, et anticorps anti-hgf/anti-ang2 bispécifiques Download PDF

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Publication number
WO2014108854A1
WO2014108854A1 PCT/IB2014/058159 IB2014058159W WO2014108854A1 WO 2014108854 A1 WO2014108854 A1 WO 2014108854A1 IB 2014058159 W IB2014058159 W IB 2014058159W WO 2014108854 A1 WO2014108854 A1 WO 2014108854A1
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Prior art keywords
antibody
seq
heavy chain
nos
antigen binding
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PCT/IB2014/058159
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English (en)
Inventor
Itai Benhar
Rahel HAKIM
Silvia Noiman
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Fusimab Ltd.
Ramot At Tel-Aviv University Ltd.
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Publication of WO2014108854A1 publication Critical patent/WO2014108854A1/fr

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    • 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
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention in some embodiments thereof, relates to monospecific and bispecific antibodies and, more particularly, but not exclusively, to bispecific antibodies which target hepatocyte growth factor (HGF) and angiopoietin-2 (ANG2).
  • HGF hepatocyte growth factor
  • ANG2 angiopoietin-2
  • Angiogenesis is implicated in the pathogenesis of a variety of disorders which include solid tumors, intraocular neovascular syndromes such as proliferative retinopathies or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis.
  • solid tumors intraocular neovascular syndromes such as proliferative retinopathies or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis.
  • the neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells. Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors.
  • ANG2 Human angiopoietin-2 (ANG2) (alternatively abbreviated with ANGPT2 or ANG2) is described in Maisonpierre, P. C, et al, Science 277 (1997) 55-60 and Cheung, A. H., et al., Genomics 48 (1998) 389-91.
  • the angiopoietins- 1 and -2 (ANG-1 and ANG2) were discovered as ligands for the Tie2 Receptor, a tyrosine kinase receptor that is selectively expressed within the vascular endothelium.
  • Ang-1 was shown to support EC survival and to promote endothelium integrity
  • ANG2 was shown to promote angiogenesis in tumors by destabilizing the blood vessels (e.g. by decreasing pericyte coverage) and sensitizing ECs to proliferation signals mediated by other proangiogenic factors, namely VEGF.
  • VEGF vascular endothelial growth factor
  • ANG2 mediated blood vessel destabilization facilitates the infiltration of proteases, cytokines and angiogenic myeloid cells—which primes the vasculature for a robust angiogenic response in the presence of growth factors such as VEGFA.
  • the endothelial cells of unstable vessels die in the absence of VEGFA.
  • ANG2 is also often expressed at the invasive fronts of human tumours, such as metastatic melanoma [Helfrich, I. et al. Angiopoietin-2 levels are associated with disease progression in metastatic malignant melanoma. Clin. Cancer Res. 15, 1384-1392 (2009)], invasive ductal breast carcinoma [Tsutsui, S. et al. Angiopoietin 2 expression in invasive ductal carcinoma of the breast: its relationship to the VeGF expression and microvessel density. Breast Cancer Res. Treat 98, 261-266 (2006)], and glioma [Hu, B. et al.
  • Angiopoietin-2 induces human glioma invasion through the activation of matrix metalloprotease-2. Proc. Natl Acad. Sci. USA 100, 8904-8909 (2003)].
  • treatment with recombinant ANG2 disrupted pulmonary vascular integrity and led to increased vascular permeability and extravasation of circulating tumour cells [Huang, Y. et al. Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis. Cancer Res. 69, 7529-7537 (2009)].
  • Angiopoietin- 1 Angiopoietin-2 and/or Tie-2 have been proposed as possible anti-cancer therapeutic targets.
  • Various strategies to target Tie-2 and block Tie-2 signaling pathways have been reported.
  • Small molecule tyrosine kinase inhibitors ARRY-614 (Array BioPharma) and CEP- 11981 (Cephalon) are found in Phase 1 clinical development. However, both are not Tie-2 selective, ARRY-614 inhibits also p38 and CEP- 11981 inhibits VEGFR.
  • Monoclonal antibodies which target the extracellular region of Tie-2 or ANG2, block ligand binding and thereby inhibit downstream events.
  • Monoclonal antibodies to the extracellular domain of Tie-2 will inhibit ligand binding of both ANG2 and Ang-1.
  • U.S. Pat. No. 6,166,185, U.S. Pat. No. 5,650,490 and U.S. Pat. No. 5,814,464 each discloses anti-Tie-2 ligand and receptor antibodies.
  • AMG386 Amgen
  • AMG386 is an anti-Angl/2 peptide fused to the Fc portion of an antibody which is found in Phase 3 clinical development.
  • PF-04856884 (Pfizer) is an engineered protein which targets ANG2 and is found in Phase 2 clinical development. See, for example, U.S. Pat. No. 6,166,185 and US 2003/10124129.
  • WO 03/030833, WO 2006/068953, WO 03/057134 or US 2006/0122370 See, for example, U.S. Pat. No. 6,166,185 and US 2003/10124129.
  • Hepatocyte growth factor is a multifunctional heterodimeric polypeptide produced by mesenchymal cells.
  • HGF is composed of an alpha-chain containing an N- terminal domain and four kringle domains (NK1-4) covalently linked to a serine protease-like beta-chain C-terminal domain.
  • Mammalian HGF is synthesized as a biologically inactive single chain precursor consisting of 728 amino acids with a 29 amino acid signal peptide which is not present in the mature protein.
  • Biologically active HGF is achieved through cleavage at the R494 residue by a specific, extracellular serum serine protease.
  • the active HGF thus achieved is a fully active heterodimer which is composed of disulfide linked 69 kDa alpha-chain and 34 kDa beta-chain.
  • HGF tyrosine kinase receptor
  • Met was originally isolated as a product of a human oncogene, trp-met, which encodes a constitutively active altered protein kinase with transforming activity. Met activation has also been shown to remarkably enhance the metastatic spread of cancer stemming from its stimulatory influence of processes such as angiogenesis, cell motility, and cell surface protease regulation (Wielenga et al., American Journal of Pathology 157(5): 1563-1573, 2000). Since Met was reported to be over-expressed in various human cancers of liver, prostate, colon, breast, brain and skin (Maulik et al., Cytokine & Growth Factor Reviews 13(1): 1-59, 2002), it has been regarded as an important target factor for the prevention and treatment of cancer.
  • HGF/c-Met are a recognized target for cancer (Yap et. al. Molecular Cancer Therapeutics 9: 1077-1079, 2012).
  • Antibodies that recognize HGF for the treatment of cancer have been disclosed for example in U.S. Patent Application Numbers 20090155284, 20090023894 and 20070036789.
  • Three anti-HGF antibodies were found in clinical development and have reached Phase 2.
  • AV-299 (Aveo Pharma) and TAK- 701 (Takeda) have failed to show efficacy in Phase 2 studies and their development has ceased.
  • AMG102 Amgen has shown efficacy in Phase 2 only in a subset of patients which show high expression of HGF in the tumor.
  • U.S. Patent application No. 20050118643 teaches the use of an antibody which recognizes HGF in combination with an inhibitor of ANG2.
  • U.S. Patent Application No. 20120065380 teaches bispecific antibodies where one arm of the antibody recognizes VEGFR-1, VEGFR-2, VEGFR-3, FLT3, c- FMS/CSF1R, RET, c-Met, EGFR, Her2/neu, HER3, HER4, FGFRs, IGFR, PDGFR, c- KIT, BCR, integrin or an MMP, whereas the other arm of the antibody recognizes VEGF, EGF, P1GF, FGF, PDGF, HGF, Tie-2 or angiopoietin.
  • U.S. Patent 8,129,331 teaches anti-ang 2 peptibodies in combination with HGF inhibitors.
  • U.S. Patent Application No. 20110311546 teaches antibodies which recognize ANG2 in combination with an antagonist of HGF.
  • a bispecific antibody comprising a first antigen-binding site that specifically binds to human hepatocyte growth factor (HGF) and a second antigen-binding site that specifically binds to human angiopoietin-2 (ANG2).
  • HGF hepatocyte growth factor
  • ANG2 human angiopoietin-2
  • an antibody comprising an ANG2 recognition region which comprises CDR amino acid sequences as set forth:
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one of the antibodies described herein.
  • a pharmaceutical composition comprising the antibody described herein as the active agent and a pharmaceutically acceptable carrier.
  • an isolated nucleic acid encoding a heavy chain of the first antigen binding site of the antibody described herein. According to an aspect of some embodiments of the present invention there is provided an isolated nucleic acid encoding a light chain of the first antigen binding site of any one of the antibodies described herein.
  • an isolated nucleic acid encoding a heavy chain of the second antigen binding site of any one of the antibodies described herein.
  • an isolated nucleic acid encoding a light chain of the second antigen binding site of any one of the antibodies described herein.
  • an expression vector comprising the nucleic acid described herein.
  • a prokaryotic or eukaryotic host cell comprising the nucleic acid described herein.
  • the first antigen binding site comprises CDR amino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; and
  • the second antigen binding site comprises CDR amino acid sequences as set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
  • the antibody is monospecific. According to some embodiments of the invention, the antibody is bispecific. According to some embodiments of the invention, the VH region of the first antigen binding domain comprises a sequence as set forth in SEQ ID NO: 135. According to some embodiments of the invention, the VL region of the first antigen binding domain comprises a sequence as set forth in SEQ ID NO: 136.
  • the VH region of the second antigen binding domain comprises a sequence as set forth in SEQ ID NO: 157.
  • the VL region of the second antigen binding domain comprises a sequence as set forth in SEQ ID NO: 158.
  • the first heavy chain of the first antigen binding domain is non-identical to a second heavy chain of the second antigen binding domain in the Fc region of the antibody, wherein at least one of the heavy chains comprises an amino acid modification so as to form complementation between two non-identical heavy chains thereby increasing the probability of forming heterodimers of the non-identical heavy chains and decreasing the probability of forming homodimers of identical heavy chains.
  • the bispecific antibody comprises a first disulfide bond between a first heavy chain and a first light chain of an Fab region of the first antigen binding site and a second disulfide bond between a second heavy chain and a second light chain of an Fab region of the second antigen binding site, wherein a position of the first disulfide bond relative to the first heavy chain is different to a position of the second disulfide bond relative to the second heavy chain.
  • the complementation comprises a steric complementation.
  • the complementation comprises a charge complementation
  • the Fc region comprises a protuberance of one heavy chain of the Fc region and a sterically compensatory cavity on a second heavy chain of the Fc region, the protuberance protruding into the compensatory cavity.
  • the protuberance is generated by substituting an amino acid at one position on a CH3 domain of the one heavy chain with another amino acid having a larger side chain volume than the original amino acid.
  • the compensatory cavity is generated by substituting an amino acid at one position on a CH3 domain of the second heavy chain with another amino acid having a smaller side chain volume than the original amino acid.
  • the first disulfide bond is between a V H domain of the first heavy chain and a V L domain of the first light chain and the second disulfide bond is between a CHI domain of the second heavy chain and a CL domain of the second light chain.
  • the at least one antibody comprises a combination of the antibody comprising an ANG2 recognition region which comprises CDR amino acid sequences as set forth:
  • HGF recognition region which comprises CDR sequences selected from the group consisting of:
  • the amino acid having a larger side chain volume than the original amino acid is selected from the group consisting of tyrosine, arginine, phenylalanine, isoleucine and tryptophan.
  • the amino acid having a smaller side chain volume than the original amino acid is selected from the group consisting of alanine, glycine, valine and threonine.
  • the bispecific antibody is selected from the group consisting of a chimeric antibody, a humanized antibody and a fully human antibody.
  • the CH3 domain of a first heavy chain of the first antigen binding site is covalently linked to a CH3 domain of a second heavy chain of the second antigen binding site.
  • each light chain of the first and second antigen binding site is linked to its cognate heavy chain via a single disulfide bond.
  • the bispecific antibody is an intact antibody.
  • the antibody is selected from the group consisting of IgA, IgD, IgE and IgG.
  • the IgG comprises IgGl, IgG2, IgG3 or IgG4.
  • the first heavy chain comprises a T366W mutation; and the second heavy chain comprises T366S, L368A, Y407V mutations.
  • the first heavy chain comprises an S354C mutation and the second heavy chain comprises a Y349C mutation.
  • At least one heavy chain of the antibody is attached to a therapeutic moiety.
  • At least one heavy chain of the antibody is attached to an identifiable moiety.
  • the bispecific antibody is selected from the group consisting of a primate antibody, a porcine antibody, a murine antibody, a bovine antibody, a goat antibody and an equine antibody.
  • the cancer is a solid tumor or lymphoid tumor.
  • the cancer is selected from the group consisting of colorectal cancer, lung cancer, breast cancer, renal cancer, ovarian cancer, gastric cancer, bladder cancer, liver cancer, ovarian cancer, fallopian cancer, glioblastoma, Mesothelioma and leukemia.
  • the host cells comprise bacterial cells.
  • the host cells comprise mammalian cells.
  • the expression takes place in inclusion bodies of the bacterial cells.
  • each of the nucleic acid molecules are transfected into different host cells.
  • each of the nucleic acid molecules are transfected into the same host cell.
  • the bacterial cells comprise gram negative bacterial cells.
  • FIG. 1 is an example of an anti-HGF heavy chain DNA sequence (SEQ ID NO: 165).
  • the VH domain coding sequence is highlighted in yellow.
  • the codon of the engineered cysteine for disulfide stabilization (Cys 44 according to the Kabat numbering scheme) is highlighted in red.
  • the codon for elimination of the native disulfide bond between CHI and CL (Ala 222 according to the EU index, Kabat numbering scheme) is highlighted in grey.
  • the "knob" mutation (Trp 366 according to the EU index, Kabat numbering scheme) is highlighted in blue.
  • the mutation to generate the additional disulfide bond Cys 354 is highlighted in green.
  • FIG. 2 is an example of an anti-HGF light chain DNA sequence (SEQ ID NO:
  • VL (lambda) domain is highlighted in green.
  • the engineered cysteine for disulfide stabilization (Cys 100 according to the Kabat numbering scheme) is highlighted in red.
  • the codon for elimination of the native disulfide bond between CHI and CL (Ala 214 according to the Kabat numbering scheme) is highlighted in grey.
  • FIG. 3 is an example of an anti-ANG2 heavy chain DNA sequence (SEQ ID NO: 1
  • VH domain coding sequence is highlighted in yellow.
  • the "hole” mutations (Ser366, Ala 368 and Val 407 according to the EU index, Kabat numbering scheme) are highlighted in blue. Cys 349, the mutation which generates the additional disulfide bond is highlighted in green.
  • FIG. 4 is an example of an anti-ANG2 light chain DNA sequence (SEQ ID NO:
  • VL (lambda) domain is highlighted in green.
  • FIGs. 5A-B are bar graphs illustrating specific binding by phage ELISA.
  • ELISA plates were coated with HGF (A), Ang2 (B) and control antigens (Pseudomonas exotoxin A (PE), Erbitux and BSA) at 5 ⁇ g/ml.
  • HGF HGF
  • Ang2 B
  • PE Pseudomonas exotoxin A
  • Erbitux and BSA control antigens
  • FIGs. 6A-C are graphs illustrating SDS/PAGE analysis of E.coli produced 2C3 monospecific (A), 1A2 monospecific (B) and BIC104 bispecific (C) antibodies.
  • Lanes Al-5, B l-5 and Cl-6 were analyzed under reducing conditions.
  • Lane Al purified inclusion bodies of 2C3 heavy chain knob.
  • Lane A2 purified inclusion bodies of 2C3 heavy chain hole.
  • Lane A4 commercial Avastin.
  • Lane A5 Protein-A purified 2C3 monospecific IgG.
  • Lane A8 commercial Avastin.
  • Lane B l purified inclusion bodies of 1A2 heavy chain knob.
  • Lane B2 purified inclusion bodies of 1A2 heavy chain hole. Lane B3, purified inclusion bodies of 1A2 light chain. Lane B4, Protein-A purified 1A2 monospecific IgG. Lane B5, commercial Avastin. Lane B6, MW marker. Lane B7, commercial Avastin. Lane B8, Protein-A purified 1A2 monospecific IgG. Lane CI, purified inclusion bodies of mutated disulfide stabilized 2C3 heavy chain knob. Lane C2, purified inclusion bodies of 2C3 mutated disulfide stabilized light chain. Lane C3, purified inclusion bodies of 1A2 heavy chain hole. Lane C4, purified inclusion bodies of 1A2 light chain. Lane C5, Protein-A purified BIC 104 IgG. Lane C6, commercial Avastin. Lane C7, MW marker. Lane C8, commercial Avastin. Lane C9, Protein-A purified BIC 104 bispecific IgG.
  • FIG. 7 is a graph illustrating ELISA binding analysis of anti-HGF 2C3 monospecific IgG, anti-Ang2 monospecific IgG and bispecific BIC 104 IgG.
  • ELISA plates were coated with human HGF or human Ang2 at 2.5 ⁇ g/ml. serial dilutions of E. coli produced IgG antibodies were added. Binding was detected with HRP-conjugated anti-human antibody.
  • the EC 50 values (determined as the half maximal binding signal) are 0.2 nM for 2C3 IgG on HGF, 0.1 nM for 1A2 IgG on Ang2 and 2 nM and 0.25 nM for BIC 104 IgG on HGF and Ang2 respectively.
  • FIGs. 8A-C are graphs illustrating kinetics of HGF binding to bispecific and monospecific antibodies by SPR analysis.
  • C The (monovalent) affinities for HGF of the bispecific compound BIC 104 and the parental antibody 2C3 determined by surface plasmon resonance.
  • FIG. 9 is a graph illustrating binding of 2C3 IgG to mouse HGF.
  • ELISA plates were coated with human HGF or mouse HGF at 2.5 ⁇ g/ml. Serial dilutions of 2C3 monospecific IgG antibody were added. Binding was detected with HRP-conjugated anti-human antibody. The EC 50 values (determined as the half maximal binding signal) are 0.2 nM and 0.7 nM for 2C3 IgG on human HGF and mouse HGF respectively.
  • FIG. 10 is a graph illustrating binding of BIC 104 IgG to human and to mouse Ang2.
  • ELISA plates were coated with human Ang2 or mouse Ang2 at 2.5 ⁇ g/ml.
  • Serial dilutions of BIC 104 bispecific IgG antibody were added. Binding was detected with HRP-conjugated anti-human antibody.
  • FIG. 11 is a graph illustrating competition of c-Met binding to HGF by antibody
  • 2C3 as evaluated by ELISA.
  • ELISA plates were coated with human HGF at 2.5 ⁇ g/ml.
  • Serial dilutions of 2C3 IgG antibody mixed with 1 nM of c-Met Fc were added.
  • c-MET binding was detected with biotinylated anti-human c-Met mixed with HRP-conjugated streptavidin.
  • FIG. 12 is a graph illustrating competition with Tie2 by ELISA.
  • ELISA plates were coated with 4 ⁇ g/ml human Tie2 Fc.
  • Serial dilutions of BIC 104 antibody mixed with 100 ng/ml of human Ang2 were added.
  • Ang2 binding was detected with HRP- conjugated streptavidin.
  • FIGs. 13A-B are photographs of purified BIC 104 and BIC204. SDS/PAGE analysis (A) and Immunoblot (B) of BIC104 and BIC204.
  • A Samples in lanes Al-3 were analyzed under reducing conditions. Lane Al, commercial Avastin. Lane A2, BIC 204. Lane A3, BIC 104. Lane A4, MW marker. Lane A5, commercial Avastin. Lane A6, BIC 204. Lane A7, BIC 104.
  • FIGs. 14A-B are graphs illustrating binding of E. coli produced BIC 104 IgG or CHO produced BIC 204.
  • ELISA plates were coated with human HGF or human Ang2 at 2.5 ⁇ g/ml.
  • Serial dilutions of E. co/z-produced BIC 104 IgG or CHO-produced BIC 204 IgG antibodies were added. Binding was detected with HRP-conjugated anti-human antibody.
  • the EC 50 values (determined as the half maximal binding signal) are 3 nM and 0.4 nM for BIC 104 IgG and 2 nM and 0.6 nM for BIC 204 IgG on HGF and Ang2 respectively.
  • FIGs. 15A-B are graphs illustrating bispecific binding of BIC104 and BIC204 by ELISA.
  • ELISA plates were coated with human HGF or human Ang2 at 2.5 ⁇ g/ml.
  • serial dilutions of BIC 104, BIC204 antibodies and 2C3 monospecific (as a control) mixed with 1 ⁇ g/ml of biotinylated Ang2 were added.
  • Detection was with HRP- conjugated streptavidin.
  • FIGs. 16A-B provide the amino acid sequence alignment of selected anti- human HGF - SEQ ID NOs: 125-140 (A) and anti Ang2 - SEQ ID NOs: 157-160 (B) scFv binding clones.
  • FIGs. 17A-B are graphs illustrating the anti-tumor growth inhibition activity of c-Met expressing Colo205 colorectal tumors grown as xenografts in nude mice by bispecific anti-ANG2/anti-HGF antibody molecules (BIC 104 and BIC 204) and their parent monospecific antibodies.
  • FIG. 18 is a graph illustrating the anti-tumor growth inhibition activity of SKOV-3 human ovary tumors grown as xenografts in nude mice cells by anti-ANG2 1A2 and a control antibody directed against CD20 (Rituximab).
  • the present invention in some embodiments thereof, relates to monospecific and bispecific antibodies and, more particularly, but not exclusively, to antibodies that target hepatocyte growth factor (HGF) and/or angiopoietin-2 (ANG2).
  • HGF hepatocyte growth factor
  • ANG2 angiopoietin-2
  • mice injected with c-Met expressing Colo205 colorectal cells demonstrated an enhanced decrease in tumor size following antibody treatment directed against both ANG2 and HGF as compared with antibody treatment directed against only one of these proteins.
  • mice injected with c-Met expressing Colo205 colorectal tumor cells survived longer following antibody treatment directed against both ANG2 and HGF as compared with antibody treatment directed against only one of these proteins and surprisingly survival showed a synergistic activity as it is more than just an additive effect of the two inhibitory pathways.
  • the present inventors contemplate inhibition of the activity of two proteins using a single bifunctional antibody or alternatively by simultaneous administration of one antibody directed against HGF and another directed against ANG2.
  • a human synthetic combinatorial library of arrayable single-chain antibodies was screened. After their isolation by antibody phage display, the coding sequences for the VH and VL domain were prepared as synthetic genes with optimization for expression in E. coli or in mammalian CHO cells.
  • the sequence of the bispecific antibody was modified to ensure correct assembly of the bispecific antibody.
  • This modification involved heterodimerization of the two heavy chains by applying the knobs into holes approach, combined with facilitation of pairing of each heavy chain with only its cognate light chain by addition of an engineered disulfide bond on one arm of the antibody (between the VL and VH domain) and elimination of a naturally occurring disulfide bond on the same arm (between the CHI and CL domains).
  • the generated recombinant bispecific antibodies were capable of specifically binding to both HGF and ANG2.
  • a bispecific antibody comprising a first antigen-binding site that specifically binds to human hepatocyte growth factor (HGF) and a second antigen-binding site that specifically binds to human angiopoietin-2 (ANG2).
  • HGF hepatocyte growth factor
  • ANG2 human angiopoietin-2
  • bispecific antibody refers to an antibody which comprises two antigen binding sites, each binding to a different epitope of a different antigen.
  • the bispecific antibodies of this aspect of the present invention do not share common light chains or common heavy chains.
  • Bispecific antibodies can be produced by many methods known in the art including for example chemical cross-linkage, genetic engineering, or somatic hybridization, as further described herein below.
  • bispecific antibodies according to the invention may be "bivalent” - denoting the presence of two binding sites.
  • the antibodies of this aspect of the present invention are trivalent or tetravalent.
  • hepatocyte growth factor or "HGF” refers to a human polypeptide
  • the antibody of this aspect of the present invention also recognizes the mouse HGF.
  • an HGF polypeptide includes terminal residues, such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues.
  • terminal residues such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues.
  • Other synonyms for HGF include hepapoietin A and scatter factor (SF).
  • ANG2 refers to human angiopoietin-2 (ANG2) (alternatively abbreviated with ANGPT2 or ANG-2) (SEQ ID NO: 14) which is described e.g. in Maisonpierre, P. C, et al, Science 277 (1997) 55-60 and Cheung, A. H., et al., Genomics 48 (1998) 389-91.
  • the angiopoietins-1 and -2 were discovered as ligands for the Ties, a family of tyrosine kinases that is selectively expressed within the vascular endothelium. Yancopoulos, G. D., et al., Nature 407 (2000) 242-48.
  • Angiopoietin-3 and -4 may represent widely diverged counterparts of the same gene locus in mouse and man.
  • ANGl and ANG2 were originally identified in tissue culture experiments as agonist and antagonist, respectively (see for ANG-1: Davis, S., et al., Cell 87 (1996) 1161-69; and for ANG2: Maisonpierre, P.
  • the antibody of this aspect of the present invention also recognizes mouse ANG2.
  • ANG2 also refers to fragments thereof, as well as related polypeptides, which include, but are not limited to, allelic variants, splice variants, derivative variants, substitution variants, deletion variants, and/or insertion variants, fusion polypeptides, and interspecies homologs.
  • an ANG2 polypeptide includes terminal residues, such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues.
  • binding or specifically binding refers to the binding of the antibody to an epitope of the antigen (either HGF or ANG2) in an in-vitro assay, e.g. in an ELISA assay or in an plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) with purified wild-type antigen.
  • the affinity of the binding is defined by the terms Ka (rate constant for the association of the antibody from the antibody/antigen complex), kD (dissociation constant), and KD (kD/ka).
  • binding or specifically binding means a binding affinity (KD) of 1(P -8 mol/1 or less, preferably 10 "9 M to 10 "13 mol/1.
  • the affinity of each of the antigen binding sites of the antibody for its target is not substantially reduced as compared with one arm of its corresponding monoclonal antibody for the identical target.
  • the affinity is not reduced more than 100 fold, more preferably is not reduced more than 50 fold, more preferably is not reduced more than 20 fold, more preferably is not reduced more than 10 fold and even more preferably is not reduced more than 5 fold.
  • epitope includes any polypeptide determinant capable of specific binding to an antibody.
  • epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody.
  • an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • the antibody is an intact antibody i.e. full length antibody.
  • full length antibody denotes an antibody consisting of two “full length antibody heavy chains” and two “full length antibody light chains”.
  • a “full length antibody heavy chain” is a polypeptide consisting in N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CHI), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR— CH2-CH 3 ; and optionally an antibody heavy chain constant domain 4 (CH4) in case of an antibody of the subclass IgE.
  • VH antibody heavy chain variable domain
  • CHI antibody constant heavy chain domain 1
  • HR antibody hinge region
  • CH2 antibody heavy chain constant domain 2
  • CH3 antibody heavy chain constant domain 3
  • VH-CH1-HR— CH2-CH 3 optionally an antibody heavy chain constant domain 4 (CH4) in case of an antibody of the subclass
  • the "full length antibody heavy chain” is a polypeptide consisting in N-terminal to C-terminal direction of VH, CHI, HR, CH2 and CH3.
  • a “full length antibody light chain” is a polypeptide consisting in N-terminal to C-terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated as VL-CL.
  • VL antibody light chain variable domain
  • CL antibody light chain constant domain
  • full length antibodies are natural antibodies like IgG, IgM, IgA, IgD, and IgE.
  • the full length antibodies according to the invention can be from a single species e.g. human, or they can be chimerized or humanized antibodies.
  • the full length antibodies according to the invention comprise two antigen binding sites each formed by a pair of VH and VL, which both specifically bind to the same antigen.
  • the C-terminus of the heavy or light chain of the full length antibody denotes the last amino acid at the C-terminus of the heavy or light chain.
  • the N-terminus of the heavy or light chain of the full length antibody denotes the first amino acid at the N-terminus of the heavy or light chain.
  • variable domain denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen.
  • the domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions” (or complementarity determining regions, CDRs).
  • the framework regions adopt a ⁇ -sheet conformation and the CDRs may form loops connecting the ⁇ -sheet structure.
  • the CDRs in each chain are held in their three- dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.
  • the antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.
  • the antigen-binding sites of the bispecific antibody of the invention contain six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity of the binding site for the particular antigen.
  • CDRs complementarity determining regions
  • the extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences.
  • functional antigen binding sites comprised of fewer CDRs (i.e., where binding specificity is determined by one, two, three, four or five CDRs). For example, less than a complete set of 6 CDRs may be sufficient for binding. In some cases, a VH or a VL domain will be sufficient.
  • CDRs which may be present in the arm of the bispecific antibody that recognizes HGF are set forth in Table 1, herein below.
  • Exemplary CDRs which may be present in the arm of the bispecific antibody that recognizes ANG2 are set forth in Table 2, herein below.
  • variable domains which may be present in the arm of the bispecific antibody that recognizes HGF are set forth in Table 3, herein below.
  • variable domains which may be present in the arm of the bispecific antibody that recognizes ANG2 are set forth in Table 4, herein below.
  • the contemplated bifunctional antibody may comprise any combination of the above disclosed CDRs, or and VL sequences so long as the first antigen binding site recognizes HGF and the second antigen binding site recognizes ANG2.
  • the first antigen binding site comprises the CDR sequences as set forth in SEQ ID NOs: 1-6 and the second antigen binding site comprises the CDR sequences as set forth in SEQ ID NOs: 7-12.
  • the VH region of the arm that recognizes the first antigen binding site is set forth in SEQ ID NO: 135, the VL region of the arm that recognizes the first antigen binding site is set forth in SEQ ID NO: 136, the VH region of the arm that recognizes the second antigen binding site is set forth in SEQ ID NO: 157, and the VL region of the arm that recognizes the first antigen binding site is set forth in SEQ ID NO: 158.
  • the antibodies of the invention further comprise immunoglobulin constant regions of one or more immunoglobulin classes. Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE isotypes and, in the case of IgG and IgA, their subtypes.
  • the Fc region is an IgG Fc region.
  • the Fc region of the antibodies described herein comprises two non-identical heavy chains (e.g. that differ in the sequence of the variable domains), wherein at least one of the two non-identical heavy chains comprises an amino acid modification so as to increase the probability of forming a stable heterodimer of the non-identical heavy chains and decrease the probability of forming a stable homodimer of identical heavy chains.
  • At least one heavy chain is genetically modified such that an altered charge polarity across the interface is created.
  • an altered charge polarity across the interface is created.
  • the amino acid modifications are effected at the rim of the interface between the two heavy chains and not in structurally conserved buried residues at the hydrophobic core of the interface.
  • At least one heavy chain is genetically modified, to generate a heavy chain with a 3D structure which binds more efficiently to the non-identical heavy chain (i.e. a heterodimer) as opposed to an identical heavy chain (i.e. a homodimer).
  • the generation of heterodimers is encouraged due to steric complementation and the generation of homodimers is discouraged due to steric hindrance.
  • one heavy chain is genetically modified to generate a protuberance and the second heavy chain is genetically modified to generate a sterically compensatory cavity, the protuberance protruding into the compensatory cavity.
  • Proturbances are constructed by replacing small amino acid side chains from the interface of the first heavy chains with larger side chains (e.g. tyrosine, arginine, phenylalanine, isoleucine, leucine or tryptophan).
  • Compensatory "cavities” of identical or similar size to the protuberances are optionally created on the interface of the second heavy chain by replacing large amino acid side chains with smaller ones (e.g. alanine, glycine, serine, valine, or threonine).
  • the protuberance or cavity can be "introduced" into the interface of the first or second heavy chain by synthetic means, e.g. by recombinant techniques, in vitro peptide synthesis, those techniques for introducing non-naturally occurring amino acid residues previously described, by enzymatic or chemical coupling of peptides or some combination of these techniques.
  • the protuberance, or cavity which is "introduced” is "non-naturally occurring” or “non-native", which means that it does not exist in nature or in the original polypeptide (e.g. a humanized monoclonal antibody).
  • the import amino acid residue for forming the protuberance has a relatively small number of “rotamers” (e.g. about 3 6).
  • a “rotamer” is an energetically favorable conformation of an amino acid side chain. The number of rotamers of the various amino acid residues are reviewed in Ponders and Richards, J. Mol. Biol. 193:775 791 (1987).
  • the three-dimensional structure of the antibodies are obtained using techniques which are well known in the art such as X-ray crystallography or NMR. Based on the three-dimensional structure, those skilled in the art will be able to identify the interface residues.
  • the preferred interface is the C H3 domain of an immunoglobulin constant domain. It is preferable to select "buried" residues to be replaced.
  • the interface residues of the CH3 domains of IgG, IgA, IgD, IgE and IgM have been identified (see, for example, PCT/US96/01598, herein incorporated by reference in its entirety), including those which are optimal for replacing with import residues; as were the interface residues of various IgG subtypes and "buried" residues.
  • the preferred C H3 domain is derived from an IgG antibody, such as an human IgGi.
  • the C H3 C H3 interface of human IgGi involves sixteen residues on each domain located on four anti-parallel ⁇ -strands which buries 1090 ANG from each surface. Mutations are preferably targeted to residues located on the two central anti-parallel ⁇ - strands. The aim is to minimize the risk that the protuberances which are created can be accommodated by protruding into surrounding solvent rather than by compensatory cavities in the partner C R3 domain. Methods of selection particular sites on the heavy chains have been disclosed in U.S. Patent No. 7,183,076, incorporated herein by reference.
  • the first heavy chain comprises a T366W mutation (i.e. threonine to tryptophan); and the second heavy chain comprises T366S, L368A, Y407V mutations (i.e. threonine to serine; leucine to alanine; and tyrosine to valine).
  • the heavy chain which comprises the T366W mutation is the one that forms the HGF arm of the bispecific antibody - see for example Figure 1.
  • the heavy chain which comprises the T366S, L368A, Y407V mutation is the one that forms the ANG2 arm of the bispecific antibody - see for example Figure 3.
  • the amino acid modifications are effected at structurally conserved buried residues at the hydrophobic core of the interface, and not in at the rim of the interface between the two heavy chains.
  • amino acid replacements may be introduced into the heavy chains using techniques which are well known in the art.
  • Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution variants of the DNA encoding the first or second heavy chain. This technique is well known in the art as described by Adelman et al., DNA, 2: 183 (1983). Briefly, first or second polypeptide-coding DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single- stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of heteromultimer. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the heteromultimer DNA.
  • Cassette mutagenesis can be performed as described Wells et al. Gene 34:315 (1985) by replacing a region of the DNA of interest with a synthetic mutant fragment generated by annealing complimentary oligonucleotides.
  • PCR mutagenesis is also suitable for making variants of the first or second polypeptide DNA. While the following discussion refers to DNA, it is understood that the technique also finds application with RNA. The PCR technique generally refers to the following procedure (see Erlich, Science, 252: 1643 1650 (1991), the chapter by R. Higuchi, p. 61 70).
  • a gene encoding the antibody of this aspect of the present invention can be synthesized by total gene synthesis (as illustrated in the Examples section below) by transferring the variable domains of the anti HGF and anti ANG2 antibodies from the phage clones into the IgG expression vectors.
  • gene synthesis can involve gene optimization for expression in a particular host (for example for E. coli or for CHO cells).
  • the present invention incorporates a covalent link between the two heavy chains (e.g. on the CH3 domains).
  • Examples of covalent links contemplated by the present invention include amide links and disulfide links.
  • the present invention contemplates introduction of a free thiol which forms an intermolecular disulfide bond between the two heavy chains of the antibody.
  • the free thiol may be introduced into the interface of one of the heavy chains by substituting a naturally occurring residue of the heavy chain with, for example, a cysteine at a position allowing for the formation of a disulfide bond between the heavy chains.
  • free thiol-containing compound refers to a compound that can be incorporated into or reacted with an amino acid of a polypeptide interface of the invention such that the free thiol moiety of the compound is positioned to interact with a free thiol of moiety at the interface of additional polypeptide of the invention to form a disulfide bond.
  • the free thiol-containing compound is cysteine.
  • the first heavy chain comprises a S354C mutation (i.e. serine to cysteine), according to the EU index, Kabat numbering scheme; and the second heavy chain comprises a Y349C mutation (tyrosine to cysteine).
  • the heavy chain which comprises the S354C mutation is the one that forms the HGF arm of the bispecific antibody - see for example Figure 1.
  • the heavy chain which comprises the Y349C mutation is the one that forms the ANG2 arm of the bispecific antibody - see for example Figure 3.
  • At least one light chain of the antibodies described herein may also be modified such that there is a first covalent link between a first heavy chain and a first light chain and a second covalent link between a second heavy chain and a second light chain, wherein a position of the first covalent link relative to the first heavy chain is different to a position of the second covalent link relative to the second heavy chain.
  • the positioning of the first and second covalent link is selected such that pairing between a heavy chain with its cognate light chain is facilitated, whilst the specificity and stability of the antibody is not reduced by more than 20 % or preferably by more than 10 % or even more preferably by more than 5 % as compared to the individual antibodies from which it is generated.
  • the covalent link between the first heavy chain (e.g. the HGF arm of the bispecific antibody) to its cognate light chain is positioned between the V H and the V L region and the covalent link between the second heavy chain (e.g. the ANG2 arm of the bispecific antibody) to its cognate light chain is positioned between the C H I and the C L region.
  • covalent links contemplated by the present invention include for example amide links, disulfide links and additional forms of covalent bonds occurring between site-specifically inserted amino acid residues, including non-natural amino acids (see Wu, X., Schultz, P.G. "Synthesis at the Interface of Chemistry and Biology.” J. Am. Chem. Soc, 131(35): 12497-515, 2009; Hutchins BM, Kazane SA, Staflin K, Forsyth JS, Felding-Habermann B, Schultz PG, Smider VV. Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids J Mol Biol. 2011 Mar 4;406(4):595-603. Epub 2011 Jan 13; Liu CC, Schultz PG. Adding new chemistries to the genetic code. Annu Rev Biochem. 2010;79:413-44. Review, all of which are incorporated herein by reference).
  • the present invention contemplates mutating at least one of the heavy chains and its cognate light chain such that at least one naturally occurring (i.e. native) disulfide bond that connects the two molecules can no longer be generated. Typically, this is effected by deleting (or substituting) the cysteines at the positions described herein above.
  • the phrase "native disulfide bond” refers to the interchain disulfide bond that connects a heavy chain to its cognate light chain (typically between the constant region of the light chain and the CHI region of the heavy chain) encoded in a naturally occurring germline antibody gene.
  • Substitution of the cysteine is typically effected by replacing the amino acid with one similar in size and charge (i.e. a conservative amino acid, such as cysteine to alanine).
  • the first covalent link is a naturally occurring disulfide bond (e.g. in the anti ANG2 arm of the antibody) and the second covalent link is a non-naturally occurring covalent bond, (e.g. an engineered disulfide bond), wherein at least one cysteine amino acid residue has been inserted into the chain - i.e. an engineered cysteine (in the anti HGF arm of the antibody).
  • a naturally occurring disulfide bond e.g. in the anti ANG2 arm of the antibody
  • the second covalent link is a non-naturally occurring covalent bond, (e.g. an engineered disulfide bond), wherein at least one cysteine amino acid residue has been inserted into the chain - i.e. an engineered cysteine (in the anti HGF arm of the antibody).
  • engineered cysteine refers to a cysteine which has been introduced into the antibody fragment sequence at a position where a cysteine does not occur in the natural germline antibody sequence.
  • both the first and second covalent links may be non-naturally occurring and the cysteines (which in the non-modified antibody serve as amino acid residues to generate disulfide bonds) may be replaced by other amino acids that are not capable of serving as amino acid residues to generate covalent bonds.
  • Information regarding the antibody of interest is required in order to produce proper placement of the disulfide bond.
  • the amino acid sequences of the variable regions that are of interest are compared by alignment with those analogous sequences in the well-known publication by Kabat and Wu [Sequences of Proteins of Immunological Interest," E. Kabat, et al., U.S. Government Printing Office, NIH Publication No.
  • positions 43, 44, 45, 46, and 47 groups 43, 44, 45, 46, and 47 (group 1) and positions 103, 104, 105, and 106 (group 2) of the heavy chain variable region; and positions 42, 43, 44, 45, and 46 (group 3) and positions 98, 99, 100, and 101 (group 4) of the light chain variable region. In some cases, some of these positions may be missing, representing a gap in the alignment.
  • Contemplated pairs of amino acids to be selected are,: V H 44-V L 100, V H 105- V L 43, V H 105-V L 42, V H 44-V L 101 , V H 106-V L 43, V H 104-V L 43, V H 44-V L 99, V H 45-V L 98, V H 46-V L 98, V H 103-V L 43, V H 103-V L 44, V H 103-V L 45.
  • substitutions of cysteine are made at the positions: V R 44- V L 100; or V H 105-V L 43.
  • V H 44-V L 100 refers to a polypeptide with a V H having a cysteine at position 44 and a cysteine in V L at position 100; the positions being in accordance with the numbering given by Kabat and Wu.
  • selection of which amino acid to mutate may be effected according to the rules set out in U.S. Patent No. 5,747,654, incorporated herein by reference.
  • the sites of mutation to the cysteine residues can be identified by review of either the actual antibody or the model antibody of interest as exemplified below.
  • Computer programs to create models of proteins such as antibodies are generally available and well-known to those skilled in the art (see Kabat and Wu; Loew, et al., Int. J. Quant. Chem., Quant. Biol.
  • a pair of suitable amino acid residues should (1) have a C a -C a distance between the two residues less than or equal to 8 ANG, preferably less than or equal to 6.5 ANG (determined from the crystal structure of antibodies which are available such as those from the Brookhaven Protein Data Bank) and (2) be as far away from the CDR region as possible. Once they are identified, they can be substituted with cysteins.
  • Modifications of the genes to encode cysteine at the target point may be readily accomplished by well-known techniques, such as oligonucleotides-directed mutagenesis (as described herein above), site-directed mutagenesis (see, Gillman and Smith, Gene, 8:81-97 (1979) and Roberts, S., et al, Nature, 328:731-734 (1987), both of which are incorporated herein by reference), by the method described in Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), incorporated by reference herein, by total gene synthesis (Hughes, R.A. et al, , Methods in Enzymology, Volume 498 p. 277-309 (2011)) or by any other means known in the art.
  • oligonucleotides-directed mutagenesis as described herein above
  • site-directed mutagenesis see, Gillman and Smith, Gene, 8:81-97 (1979) and Roberts, S.,
  • the present invention contemplates antibodies which recognize either HGF and/or ANG2.
  • the antibodies may be monospecific or bispecific.
  • Monospecific antibodies of this aspect may or may not be modified to comprise mutations.
  • the monospecific antibodies comprise the "knobs into hole” mutation described herein above.
  • an antibody (either monospecific or bispecific) comprising an HGF recognition region which comprises six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 1-6, 17-22, 23-28, 29-34, 35-40, 41-46, 47-52, 53-58.
  • an antibody (either monospecific or bispecific) comprising an ANG2 recognition region which comprises six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 7-12 and 107-112.
  • the antibody which recognizes ANG2 has the CDR sequences as set forth in SEQ ID NOs: 7-12.
  • Bispecific antibodies which comprise a first antigen binding site which recognizes ANG2 may have a second antigen binding site which recognizes a second target which is not directed to HGF, but a different protein.
  • bispecific antibodies which comprise a first antigen binding site which recognizes HGF may have a second antigen binding site which recognizes a second target which is not directed to ANG2, but a different protein.
  • Antibodies of some embodiments of the present invention may be from any mammalian origin including human, porcine, murine, bovine, goat, equine, canine, feline, ovine and the like.
  • the antibody may be a heterologous antibody.
  • a heterologous antibody is defined in relation to a transgenic host such as a plant expressing the antibody.
  • the antibody is an isolated intact antibody (i.e., substantially free of cellular material other antibodies having different antigenic specificities and/or other chemicals).
  • recombinant antibody refers to intact antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., mouse) that is transgenic for immunoglobulin genes (e.g., human immunoglobulin genes) or hybridoma prepared therefrom; (b) antibodies isolated from a host cell (e.g. prokaryotic cells) transformed to express the antibody; (c) antibodies isolated from a recombinant antibody library; and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.
  • an animal e.g., mouse
  • immunoglobulin genes e.g., human immunoglobulin genes
  • hybridoma prepared therefrom e.g., hybridoma prepared therefrom
  • a host cell e.g. prokaryotic cells
  • immunoglobulin of the present invention may have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies comprise sequences that while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • human antibody refers to intact antibodies having variable regions in which both the framework and CDR regions are derived from human immunoglobulin sequences as described, for example, by Kabat et al. (see Kabat 1991, Sequences of proteins of immunological Interest, 5 th Ed. NIH Publication No. 91-3242). The constant region of the human antibody is also described from human immunoglobulin sequences.
  • the human antibodies may include amino residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site directed mutagenesis in vitro or somatic mutation in vivo).
  • the term "human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • a "chimeric antibody” refers to an intact antibody in which the variable regions derive from a first species and the constant regions are derived from a second species.
  • Chimeric immunoglobulins can be constructed by genetic engineering from immunoglobulin gene segments belonging to different species (e.g., VH and VL domains from a mouse antibody with constant domains of human origin).
  • humanized immunoglobulin refers to an intact antibody in which the minimum mouse part from a non-human (e.g., murine) antibody is transplanted onto a human antibody; generally humanized antibodies are 5-10 % mouse and 90-95 % human.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • the antibodies of the present invention may be conjugated to a functional moiety such as a detectable or a therapeutic moiety.
  • detectable or reporter moieties may be conjugated to the antibody of the invention. These include, but not are limited to, a radioactive isotope (such as [125] iodine), a phosphorescent chemical, a chemiluminescent chemical, a fluorescent chemical (fluorophore), an enzyme, a fluorescent polypeptide, an affinity tag, and molecules (contrast agents) detectable by Positron Emission Tomagraphy (PET) or Magnetic Resonance Imaging (MRI).
  • Table 5 provides non-limiting examples of identifiable moieties which can be conjugated to the antibody of the invention.
  • the antibody may be conjugated to a therapeutic moiety.
  • the therapeutic moiety can be, for example, a cytotoxic moiety, a toxic moiety, a cytokine moiety and a second antibody moiety comprising a different specificity to the antibodies of the invention.
  • Non-limiting examples of therapeutic moieties which can be conjugated to the antibody of the invention are provided in Table 6, hereinbelow.
  • NP_000589.2 NP_000580.1 interleukin 4
  • the functional moiety may be conjugated to the V H or the V L sequence at either the N- or C-terminus or be inserted into other protein sequences in a suitable position.
  • V H and V L sequences for application in this invention can be obtained from antibodies produced by any one of a variety of techniques known in the art.
  • antibodies are provided by immunization of a non-human animal, preferably a mouse, with an immunogen comprising a desired antigen or immunogen.
  • antibodies may be provided by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in Ward et al (Nature 341 (1989) 544).
  • the step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)).
  • the non-human animal is a mammal, such as a rodent (e.g., mouse, rat, etc.), bovine, porcine, horse, rabbit, goat, sheep, etc.
  • the non-human mammal may be genetically modified or engineered to produce "human" antibodies.
  • the immunogen is suspended or dissolved in a buffer, optionally with an adjuvant, such as complete Freund's adjuvant.
  • an adjuvant such as complete Freund's adjuvant.
  • the location and frequency of immunization sufficient to stimulate the production of antibodies is also well known in the art.
  • the non-human animals are injected intraperitoneally with antigen on day 1 and again about a week later. This is followed by recall injections of the antigen around day 20, optionally with adjuvant such as incomplete Freund's adjuvant.
  • the recall injections are performed intravenously or intraperitoneally and may be repeated for several consecutive days. This is followed by a booster injection at day 40, either intravenously or intraperitoneally, typically without adjuvant.
  • This protocol results in the production of antigen-specific antibody-producing B cells after about 40 days. Other protocols may also be utilized as long as they result in the production of B cells expressing an antibody directed to the antigen used in immunization.
  • lymphocytes from a non-immunized non-human mammal are isolated, grown in vitro, and then exposed to the immunogen in cell culture. The lymphocytes are then harvested and the fusion step described below is carried out.
  • the next step is the isolation of splenocytes from the immunized non-human mammal and the subsequent fusion of those splenocytes with an immortalized cell in order to form an antibody-producing hybridoma.
  • the isolation of splenocytes from a non-human mammal is well-known in the art and typically involves removing the spleen from an anesthetized non-human mammal, cutting it into small pieces and squeezing the splenocytes from the splenic capsule and through a nylon mesh of a cell strainer into an appropriate buffer so as to produce a single cell suspension.
  • the cells are washed, centrifuged and re-suspended in a buffer that lyses any red blood cells.
  • the solution is again centrifuged and remaining lymphocytes in the pellet are finally re-suspended in fresh buffer.
  • the lymphocytes are fused to an immortal cell line.
  • This is typically a mouse myeloma cell line, although many other immortal cell lines useful for creating hybridomas are known in the art.
  • Preferred murine myeloma lines include, but are not limited to, those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. U.S.A., X63 Ag8653 and SP-2 cells available from the American Type Culture Collection, Rockville, Md. U.S.A.
  • the fusion is effected using polyethylene glycol or the like.
  • the resulting hybridomas are then grown in selective media that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • the hybridomas are typically grown on a feeder layer of macrophages.
  • the macrophages are preferably from littermates of the non-human mammal used to isolate splenocytes and are typically primed with incomplete Freund's adjuvant or the like several days before plating the hybridomas. Fusion methods are described in (Goding, "Monoclonal Antibodies: Principles and Practice,” pp. 59-103, Academic Press, 1986).
  • the cells are allowed to grow in the selection media for sufficient time for colony formation and antibody production. This is usually between 7 and 14 days.
  • the hybridoma colonies are then assayed for the production of antibodies that bind the immunogen/antigen.
  • the assay is typically a colorimetric ELISA-type assay, although any assay may be employed that can be adapted to the wells that the hybridomas are grown in. Other assays include immunoprecipitation and radioimmunoassay.
  • the wells positive for the desired antibody production are examined to determine if one or more distinct colonies are present. If more than one colony is present, the cells may be re- cloned and grown to ensure that only a single cell has given rise to the colony producing the desired antibody. Positive wells with a single apparent colony are typically recloned and re-assayed to insure only one monoclonal antibody is being detected and produced.
  • Hybridomas that are confirmed to be producing a monoclonal antibody are then grown up in larger amounts in an appropriate medium, such as DMEM or RPMI-1640.
  • an appropriate medium such as DMEM or RPMI-1640.
  • the hybridoma cells can be grown in vivo as ascites tumors in an animal.
  • the growth media containing monoclonal antibody (or the ascites fluid) is separated away from the cells and the monoclonal antibody present therein is purified. Purification is typically achieved by gel electrophoresis, dialysis, chromatography using protein A or protein G- Sepharose, or an anti-mouse Ig linked to a solid support such as agarose or Sepharose beads (all described, for example, in the Antibody Purification Handbook, Amersham Biosciences, publication No. 18-1037-46, Edition AC, the disclosure of which is hereby incorporated by reference).
  • the bound antibody is typically eluted from protein A, protein G or protein L columns by using low pH buffers (glycine or acetate buffers of pH 3.0 or less) with immediate neutralization of antibody-containing fractions. These fractions are pooled, dialyzed, and concentrated as needed.
  • DNA encoding the heavy and light chains of the antibody may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of antibodies such as murine or human). Once isolated, the DNA can be ligated into expression vectors, which are then transfected into host cells.
  • the antibodies according to the invention are typically produced by recombinant means.
  • the DNA sequences encoding the immunoglobulin light chain and heavy chain polypeptides may be independently inserted into separate recombinant vectors or one single vector, which may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity.
  • nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods.
  • Expression is performed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or bacterial cells, and the antibody is recovered from the cells (supernatant or cells after lysis).
  • the present invention contemplates expressing each component of the antibody in its own individual host cell, or various combinations of the antibody components in their own host cells.
  • the light chains may be expressed in one host cell and the heavy chains in another host cell.
  • one light chain and one heavy chain is expressed in one host cell and the second light chain and the second heavy chain is expressed in another host cell.
  • both the heavy chains and both the light chains may be expressed in the same host cell.
  • the host cell comprises bacterial cells.
  • the antibodies are generated as inclonals as described in WO2009/107129 incorporated herein by reference.
  • the bacterial host may be selected capable of producing the recombinant proteins (i.e., heavy and light chains) as inclusion bodies (i.e., nuclear or cytoplasmic aggregates of stainable substances).
  • recombinant proteins i.e., heavy and light chains
  • inclusion bodies i.e., nuclear or cytoplasmic aggregates of stainable substances.
  • the host cells e.g., first host cell and second host cell
  • the host cells can be of identical species or different species.
  • the host cells are selected from a Gram-negative bacterium/bacteria.
  • Gram-negative bacteria examples include, but are not limited to, Escherichia coli Pseudomonas, erwinia and Serratia. It should be noted that the use of such Gram-negative bacteria other than E. coli such as Pseudomonas as a host cell would provide great economic value owing to both the metabolic and physiologic properties of pseudomonas. Under certain conditions, pseudomonas, for example, can be grown to higher cell culture densities than
  • bacterial expression vectors suitable for use in accordance with the present teachings include, but are not limited to, pETTM systems, the T7 systems and the pBADTM system, which are well known in the art. Methods of introducing expression vectors into bacterial host cells are well known in the art and mainly depend on the host system used.
  • the host cells can either be co-cultured in the same medium, or cultured separately.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit recombinant protein production.
  • An effective medium refers to any medium in which a bacterium is cultured to produce the recombinant protein of the present invention.
  • Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Bacterial hosts of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates, dependent on the desired amount. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant host. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • the polypeptides are recovered from the inclusion bodies.
  • Methods of recovering recombinant proteins from bacterial inclusion bodies are well known in the art and typically involve cell lysis followed by solubilization in denaturant [e.g., De Bernardez-Clark and Georgiou, "Inclusion bodies and recovery of proteins from the aggregated state” Protein Refolding Chapter 1: 1-20 (1991). See also Examples section which follows, under “Expression of Inclonals in E. coli"].
  • the inclusion bodies can be separated from the bulk of cytoplasmic proteins by simple centrifugation giving an effective purification strategy. They can then be solubilized by strong denaturing agents like urea (e.g., 8 M) or guanidinium hydrochloride and sometimes with extremes of pH or temperature. The denaturant concentration, time and temperature of exposure should be standardized for each protein. Before complete solubilization, inclusion bodies can be washed with diluted solutions of denaturant and detergent to remove some of the contaminating proteins. Finally, the solubilized inclusion bodies can be directly subjected to further purification through chromatographic techniques under denaturing conditions or the heavy and light chains may be refolded to native conformation before purification.
  • strong denaturing agents like urea (e.g., 8 M) or guanidinium hydrochloride and sometimes with extremes of pH or temperature. The denaturant concentration, time and temperature of exposure should be standardized for each protein.
  • inclusion bodies can be washed with diluted solutions of denaturant
  • purification can be affinity-based through the identifiable or therapeutic moiety (e.g., using affinity columns which bind thereto).
  • Further purification of antibodies may be performed in order to eliminate cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g.
  • cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta- mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl- sepharose, aza-arenophilic resins, or m- aminophenylboronic acid), metal chelate affinity chromatography (e.g.
  • Ni(II)- and Cu(II)-affvnity material size exclusion chromatography
  • electrophoretical methods such as gel electrophoresis, capillary electrophoresis
  • Gel electrophoresis capillary electrophoresis
  • Exemplary matrices useful for purifying the antibodies of this aspect of the present invention include Lambda-select (GE Healthcare, USA).
  • the reconstituted heavy chains and reconstituted light chains are provided at a ratio selected to maximize the formation of an intact antibody.
  • a heavy to light chain molar ratio of about 1: 1 to 1:3, 1: 1.5 to 1:3, 1:2 to 1:3 is.
  • the heavy to light chain molar ratio is about 1: 1.
  • the immunoglobulin may be subjected to directed in vitro glycosylation, which can be done according to the method described by Isabelle Meynial-salles and Didier Combes. In vitro glycosylation of proteins: An enzymatic approach. Journal of Biotechnology Volume 46, Issue 1, 18 April 1996, Pages 1-14.
  • One aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention.
  • Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a pharmaceutical composition.
  • a further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising an antibody according to the invention.
  • the present invention provides a composition, e.g. a pharmaceutical composition, containing an antibody according to the present invention, formulated together with a pharmaceutical carrier.
  • Antibodies and compositions comprising same may be used in diagnostic and therapeutic applications and as such may be included in therapeutic or diagnostic kits.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient i.e., antibody.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • a monospecific antibody which is directed against HGF and a monospecific antibody which is directed against ANG2 may be packaged in a single article of manufacture, each one being packaged separately.
  • the monospecific antibody which is directed against HGF and the monospecific antibody which is directed against ANG2 may be combined in a single formulation.
  • a method of treating an angiogenesis related disorder comprises administering to a subject in need thereof a therapeutically effective amount of an antibody (or antibodies) that targets either ANG2 and/or HGF.
  • the antibody may be a bispecific antibody such as described herein or may be a combination of two monospecifics i.e. one targeting ANG2 and the other targeting HGF.
  • the antibody may be a single monospecific antibody that targets ANG2 or HGF, the CDRs of which have been described herein above. Together with administration of such monospecific antibodies, the present invention contemplates administering additional agents that downregulate tumor microenvironment and/or immunomodulatory targets.
  • tumor microenvironment targets include, but are not limited to bone morphogenic protein 9 (BMP9) or its receptor activin receptor- like kinase 1 (Alkl), Delta like ligand 4 (DLL4) (one of the Notch ligands), TNF- related wea inducer of apoptosis (TWEAK), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) or epidermal growth factor receptor (EGFR), ErbB2, ErbB3, integrin-alpha-9-betal, integerin-alpha-5-betal, Tumor endothelial marker 8 protein (TEM8), Tumor endothelial marker 1 protein (TEM1), Galectin3, CD24
  • immunomodulatory targets include, but are not limited to Programmed cell death I ligand (PD-L1) or its receptor PD-1, CD47, Macrophage colony-stimulating factor 1 (CSF1) or its receptor CSF1R, CD137, CT
  • agents that may be used to downregulate these additional targets include antibodies, polynucleotide agents (e.g. siRNAs), receptor antagonists, small molecules etc.
  • the antibodies of the present invention are useful for treating angiogenesis related diseases.
  • angiogenesis related diseases include diabetic retinopathy, ischemic chronic wounds, age-related macular degeneration, cardiovascular diseases and cancer.
  • cancer examples include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancerous diseases include but are not limited to: Myeloid leukemia such as Chronic myelogenous leukemia. Acute myelogenous leukemia with maturation. Acute promyelocytic leukemia, Acute nonlymphocytic leukemia with increased basophils, Acute monocytic leukemia. Acute myelomonocytic leukemia with eosinophilia; Malignant lymphoma, such as Birkitt's Non- Hodgkin's; Lymphoctyic leukemia, such as Acute lymphoblastic leukemia.
  • Chronic lymphocytic leukemia Myeloproliferative diseases, such as Solid tumors Benign Meningioma, Mixed tumors of salivary gland, Colonic adenomas; Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovial sarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoid chonodro sarcoma, Ewing's tumor; other include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, breast, skin, prostate, and ovarian.
  • Adenocarcinomas such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas
  • the cancer is colorectal cancer, lung cancer, breast cancer, renal cancer, ovarian cancer, gastric cancer, bladder cancer, liver cancer, ovarian cancer, fallopian cancer, glioblastoma, leukemia, including acute myeloid leukemia and lymphoma.
  • Treatment of diseases may be effected by administering the antibody alone, or together with a carrier as a pharmaceutical composition.
  • pharmaceutical 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.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
  • a composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra- articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical art.
  • composition must be sterile and fluid to the extent that the composition is deliverable by syringe.
  • carrier preferably is an isotonic buffered saline solution.
  • Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
  • bispecific antibodies of the present invention include purification of analytes; in immunohistochemistry and enzyme immunoassays; for radioimaging and radioimmunotherapy and for drug delivery.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • HGF human Angiopoietin 2
  • ANG2 human Angiopoietin 2
  • the infected cells were collected by centrifugation and resuspended in 2xYT medium supplemented with 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin.
  • the culture was incubated with shaking (250 RPM) at 30°C overnight for phage proliferation.
  • the culture was spun down and the supernatant was filtered through 0.45 ⁇ filter to eliminate remaining bacteria.
  • PEG/NaCl (20% PEG6000, 2.5 M NaCl) was added (fifth of the volume) to the supernatant, mixed well and incubated for >1 hr on ice. Phage- precipitates were collected by centrifugation at 10,800 x g for 30 min at 4°C and the supernatant was carefully aspirated off.
  • the phages were resuspended in sterile and filtered PBS.
  • phage -particles were used at the first round.
  • 24 wells plate were coated with human HGF or human Ang2 (R&D systems) in PBS overnight at 4°C.
  • the wells were blocked with 3% milk in PBS (in the first and the second panning cycles) or 1% BSA in PBS (in the third and the fourth panning cycles) for 1 hour at room temperature.
  • Phages were pre-incubated for 1 hour at 37°C with 3% milk or 1% BSA in PBS to deplete and avoid anti-milk/BSA binders.
  • the phages were added to the blocked wells and incubated for 1 hour at room temperature on a rotator.
  • phages were eluted from wells by the addition of 1 ml of 100 mM Triethylamine, followed by 20 min incubation at room temperature on a rotator (round 1 completed). Immediately after elution, the phage solution was neutralized by the addition of 500 ⁇ of 1 M Tris (HC1) pH 7.4. TGI cells were infected with round 1 output phages and incubated for 30 min at 37°C followed by an additional 30 min at 37°C with shaking. For output phage enrichment, the selected phages were used for reinfection of TG-1 cells.
  • the enriched output phages were used for another round of selection, 0.75 ml of the neutralized TEA-eluate were mixed with 5 ml of logarithmic TG-1 cells. For infection, the culture was incubated without shaking at 37°C for 30 min and then transferred to a shaking 37°C incubator at 100 RPM for 30 min. The infected cells were spread on 2xYT plates supplemented with 100 ⁇ g/ml ampicillin and 1% glucose and grown overnight at 37°C.
  • Single clone panned phages were validated for binding to antigens by standard enzyme-linked immunosorbent assays.
  • E. coli TG-1 cells were infected with fourth panning output phages and plated to yield individual colonies.
  • 96 individual colonies were picked into sterile 96-well plates containing 100 ⁇ of 2xYT with 100 ⁇ g/ml ampicillin and 1% glucose medium per well and incubated in a 30°C shaker at 150 RPM overnight. 10 ⁇ of inoculum was transferred to a second 96-well plate containing fresh 100 ⁇ of YTAG per well and grown with shaking for 2 hours at 37°C.
  • Helper phage was added, incubated for 30 minutes at 37°C without shaking and for an additional 30 min with shaking (150 rpm) at 37°C.
  • the infected cells were collected by centrifugation and resuspended in 100 ⁇ of 2xYT medium supplemented with 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin and grown overnight at 30°C while shaking (150 rpm).
  • the plates were centrifuged to pellet the cells. The supernatant containing the phages was used for analysis by ELISA.
  • the antibodies described herein are composed of heavy chains that are dimerized by the knobs into holes (KIH) approach (as disclosed in PCT Patent Application No. IL2012/050093).
  • bispecific antibodies were engineered by disulfide stabilization between the VH and VL domains and are mutated in the CHI and CL domains (as disclosed in PCT Patent Application No. IL2012/050093).
  • the monospecific heavy chains were not engineered in the VH, VL CHI or CL domains.
  • Vectors for the expression of monospecific antibodies anti HGF 2C3 and anti Ang2 1A2 were constructed as follows. After their isolation by antibody phage display, the coding sequences for the VH and VL domain were prepared as synthetic genes with optimization for expression in E. coli by GeneArt (Germany). The synthetic genes contained the Ndel and Nhel sites required for cloning into the pHAK-HC-knob vector or pHAK-HC-hole vector and the Ndel and Avrll sites required for cloning into the pHAK-IgLam vector (described herein below). The synthetic genes were subcloned into the pHAK expression vectors and the sequences were verified by DNA sequencing.
  • pHAK-IgLam plasmid A lambda light chain gene fragment was constructed by overlap extension PCR from a lambda VL that was isolated from the Ronit 1 library and a lambda constant domain that was PCR-amplified from human lymphoid cDNA (commercial cDNA from human spleen, lymph nodes and peripheral blood lymphocytes (Clontech, USA)).
  • the cloned fragment contained an Ndel site at the 5' end (CATATG (SEQ ID NO: 168) with the ATG serving as a translation initiation codon), an Avrll site (CCTAGG (SEQ ID NO: 169) with the first C being the last base of the penultimate codon of V-lambda, CTA being the last codon of V-lambda and GG being the first two bases of the first residue of C-lambda).
  • CAATG SEQ ID NO: 168
  • CCTAGG SEQ ID NO: 169
  • Plasmid pHAK-IgL (Kappa light chain) was linearized with Ndel and EcoRl restriction enzymes that removed the entire kappa light chain and the Lambda light chain fragment was cloned into it as Ndel-EcoRl fragment.
  • the resulting plasmid was named pHAK-IgLam and is used to express a wild-type lambda light chain.
  • Vectors for expression of bispecific IgGlantibodies in E. coli were constructed as follows.
  • VH and VL domains after their isolation by antibody phage display, were prepared as synthetic genes with optimization for expression in E. coli by GeneArt (Germany).
  • Anti HGF antibody 2C3 was synthesized with mutations G44C in the VH domain and G100C in the VL domain to facilitate disulfide stabilization of the association between the two engineered chains.
  • the variable domains of anti-Ang2 were not mutated since that side of the BIC104 molecule contains the native disulfide bond between CHI and CL.
  • the synthetic genes contained the Ndel and Nhel sites required for cloning the heavy variable regions of desired antibody were cloned into pHAK-HC-knob (carrying mutations T366W, S354C) vector or pHAK-HC-hole (carrying mutations T366S, L368A, Y407V, Y349C) (PCT Patent Application No. IL2012/050093).
  • the synthetic genes contained the Ndel and Avrll sites required for cloning the light variable regions of desired antibody were cloned into the pHAK-IgLam or into the pFUS 12 (described below) vectors. The sequences were verified by DNA sequencing.
  • pHAK-IgLam vector To express engineered lambda light chains for association with engineered heavy chain using disulfide stabilization, pHAK-IgLam vector was modified; the constant lambda domain was amplified with primer CLam- Avrll-FOR (for introduction of C214A mutation) and primer CLam-C214A-EcoRI- REV (Table 7, herein below), digested with Avrll-EcoRI restriction enzymes and cloned to pHAK-IgLam previously digested with the same enzymes. The resulted vector named pFUS 12.
  • variable domains from pHAK-HC-hole, pHAK-HC-Cys-knob, pHAK-IgLam and pFUS 12 which encode 2C3 and 1A2 were synthesized after codon optimization for expression in mammalian cells. Subsequently, synthetic DNA was cloned into GPEx® (Catalent Pharma Solution, MO, USA) retroviral IgGl vectors.
  • IgG production in E. coli Heavy and light chains constructs were expressed in separate E. coli BL21 (DE3) pUBS500 bacterial cultures as inclusion bodies. The inclusion bodies were purified, denatured, mixed and refolded according to the Hakim et al., MAbs. 2009 May-Jun;l(3):281-7.
  • Protein A purification Following the refolding process, refolded IgGs were concentrated (Vivaflow200, Sartorius) followed by diafiltration against 50mM Tris, 50g/L Arginine, pH7.0. Subsequently the IgG was purified by protein A chromatography. The proteins were eluted with 0.1 M citric acid pH3.0, neutralized with 1M Tris-HCl, pH8.5 followed by buffer exchange against 20 mM phosphate buffer solution pH6.5 (PD-10 column, GE Healthcare). The protein final concentration was determined by absorbance at 280 nm.
  • ELISA analysis 96-well ELISA plate was coated with 2-5 ⁇ g/ml of pure antigen in PBS overnight at 4°C. All subsequent steps were carried out at room temperature (25 °C). The plates were blocked with 3 % skim milk (in PBS) for 1 hour at room temperature. Protein A purified antibodies, serially diluted in PBST, were added to wells and incubated for 1 hour and washed three times with PBST. Following a 50 min incubation with HRP conjugated secondary antibodies, the plates were washed in PBST and developed using chromogenic HRP substrate TMB and colour development was terminated with 1M H 2 S0 4 . The plated were read at 450 nm.
  • Bispecific binding ELISA 96 wells plate was coated with 2.5 ⁇ g/ml human HGF (Sino Biologicals Inc.). Samples containing 1 ⁇ g/ml of biotinylated Ang2 (R&D Systems) and serial dilutions of antibodies were added for 1 hour incubation at room temperature. Following incubation with streptavidin-HRP (1:5000; Jackson), wells were washed with PBST. Reaction was developed using chromogenic HRP substrate TMB and colour development was terminated with 1M H 2 S0 4 . The plated were read at 450 nm.
  • SPR Surface Plasmon Resonance analysis: Experiments were performed on a ProteOn XPR36 instrument (Bio-Rad) using PBS, 0.05% polysorbate 20 as running and dilution buffer. Anti-human IgG polyclonal antibodies (Jackson ImmunoResearch Europe Ltd.) were immobilized to the surface of a CM5 sensorchip using standard amine-coupling chemistry to a surface density of ⁇ 200 response units (RU). After capturing the bispecific antibodies to the surface human HGF was injected at increasing concentrations at a flow rate of 80 ⁇ / ⁇ . The contact time (association phase) was 5 min. The dissociation time (washing with running buffer) was 15 min. The derived curves were fit to a 1: 1 Langmuir binding model using the ProteOn evaluation software.
  • Xenograft animal model Colo205 human colorectal cancer cells were originally obtained from ATCC. lxlO 6 Colo205 tumor cells in 50% Matrigel (0.2 ml per mouse) were injected subcutaneously in the flank to 8-12 weeks CR female NCr nu/nu mice. Pair match of the tumor size was done when tumors reached an average size of 100-150 mm , at which time antibody treatment was initiated. Antibodies were injected to a group of 9 animals (10 mg/kg for monospecific antibodies and 20 mg/kg for bispecific antibodies), once a week for 6 weeks. Non-relevant antibody was injected to a control group. Caliper measurements of tumors was performed twice a week. Tumor size analysis and survival analysis were used as an efficacy endpoint.
  • Survival data contain duration times until occurrence of a specific event, (i.e. the death of an animal). Survival data was analyzed with specialized methods, because they have specialized non-normal distributions, line the exponential or Weibyll. Furthermore, the censored observations cannot be ignored without biasing the analysis. Kaplan-Meier curves give an estimation of the survival functions for one or more group of right-censored data.
  • Tumor xenografts were initiated with SKOV3 human ovarian carcinomas maintained by serial subcutaneous transplantation in severe combined immunodeficient mice. On the day of tumor implant, each athymic nude test mouse received a 1-mm SKOV3 fragment implanted subcutaneously in the right flank, and tumor growth was monitored until 80 to 120 mm . Twenty-one days after tumor implantation, designated as Day 1 of dosing, the animals were sorted into groups; treatment with antibodies was once a week.
  • Anti-ANG2 antibody (1A2 - CDRs SEQ ID NOs: 7-

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Abstract

L'invention concerne un anticorps comprenant une région de reconnaissance d'angiopoïétine-2 humaine (ANG2) ou du facteur de croissance d'hépatocytes humain (HGF). L'invention concerne également des anticorps bispécifiques qui comprennent un premier site de liaison à un antigène qui se lie spécifiquement à HGF et un second site de liaison à un antigène qui se lie spécifiquement à ANG2. L'invention concerne également des utilisations de ceux-ci et des compositions les comprenant.
PCT/IB2014/058159 2013-01-09 2014-01-09 Anticorps anti-hgf et anti-ang2 monospécifiques, et anticorps anti-hgf/anti-ang2 bispécifiques WO2014108854A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108948195A (zh) * 2017-05-23 2018-12-07 胡毅 一种抗egfr/pd-l1双靶向抗体、其制备方法及用途

Citations (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3791932A (en) 1971-02-10 1974-02-12 Akzona Inc Process for the demonstration and determination of reaction components having specific binding affinity for each other
US3839153A (en) 1970-12-28 1974-10-01 Akzona Inc Process for the detection and determination of specific binding proteins and their corresponding bindable substances
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3850578A (en) 1973-03-12 1974-11-26 H Mcconnell Process for assaying for biologically active molecules
US3853987A (en) 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3867517A (en) 1971-12-21 1975-02-18 Abbott Lab Direct radioimmunoassay for antigens and their antibodies
US3879262A (en) 1972-05-11 1975-04-22 Akzona Inc Detection and determination of haptens
US3901654A (en) 1971-06-21 1975-08-26 Biological Developments Receptor assays of biologically active compounds employing biologically specific receptors
US3935074A (en) 1973-12-17 1976-01-27 Syva Company Antibody steric hindrance immunoassay with two antibodies
US3984533A (en) 1975-11-13 1976-10-05 General Electric Company Electrophoretic method of detecting antigen-antibody reaction
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4034074A (en) 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US4098876A (en) 1976-10-26 1978-07-04 Corning Glass Works Reverse sandwich immunoassay
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4879219A (en) 1980-09-19 1989-11-07 General Hospital Corporation Immunoassay utilizing monoclonal high affinity IgM antibodies
US5011771A (en) 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5281521A (en) 1992-07-20 1994-01-25 The Trustees Of The University Of Pennsylvania Modified avidin-biotin technique
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5650490A (en) 1994-10-07 1997-07-22 Regeneron Pharmaceuticals, Inc. Tie-2 ligand 2
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5747654A (en) 1993-06-14 1998-05-05 The United States Of America As Represented By The Department Of Health And Human Services Recombinant disulfide-stabilized polypeptide fragments having binding specificity
US5814464A (en) 1994-10-07 1998-09-29 Regeneron Pharma Nucleic acids encoding TIE-2 ligand-2
WO2003030833A2 (fr) 2001-10-11 2003-04-17 Amgen Inc. Agents de liaison spécifiques de l'angiopoïétine-2
WO2003057134A2 (fr) 2001-10-11 2003-07-17 Amgen, Inc. Agents de liaison specifiques de l'angiopoietine-2 humaine
US20050118643A1 (en) 2003-07-18 2005-06-02 Burgess Teresa L. Specific binding agents to hepatocyte growth factor
WO2006068953A2 (fr) 2004-12-21 2006-06-29 Astrazeneca Ab Anticorps dirigés contre l'angiopoïétine 2 et leurs utilisations
US20070036789A1 (en) 2003-11-11 2007-02-15 National Cancer Center Neutralizable Epitope of HGF and Neutralizing Antibody Binding to the Same
US7183076B2 (en) 1997-05-02 2007-02-27 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
WO2007068895A1 (fr) * 2005-12-15 2007-06-21 Astrazeneca Ab Combinaison d'un antagoniste de l'angiopoietine 2 et d'un antagoniste du vegf-a et/ou du kdr et/ou du fltl pour le traitement du cancer
US20090155284A1 (en) 1999-11-09 2009-06-18 Boliang Cao Hgf-sf monoclonal antibody combinations
WO2009107129A1 (fr) 2008-02-29 2009-09-03 Ramot At Tel Aviv University Ltd. Compositions d’immunoglobuline et procédés de production de celles-ci
US20100115589A1 (en) 2008-10-30 2010-05-06 Sharp Kabushiki Kaisha Apparatus for determining facsimile transmission authorizer and facsimile machine having the apparatus
WO2010069532A1 (fr) * 2008-12-16 2010-06-24 F. Hoffmann-La Roche Ag Anticorps dirigés contre l'angiopoïétine 2 humaine
US20100256340A1 (en) 2009-04-07 2010-10-07 Ulrich Brinkmann Trivalent, bispecific antibodies
US20110311546A1 (en) 2001-10-11 2011-12-22 Amgen, Inc. Angiopoietin-2 specific binding agents
US20120065380A1 (en) 2009-05-20 2012-03-15 Pharmabcine Inc. Dual targeting antibody of novel form, and use thereof
US20120100166A1 (en) 2010-07-15 2012-04-26 Zyngenia, Inc. Ang-2 Binding Complexes and Uses Thereof
US20120123949A1 (en) 2010-11-17 2012-05-17 Shreyank Gupta Method and system to automatically modify task status in a project management system via keywords in source code version control commit logs
US9601598B2 (en) 2010-07-15 2017-03-21 Taiwan Semiconductor Manufacturing Company, Ltd. Method of manufacturing a fin-like field effect transistor (FinFET) device

Patent Citations (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3839153A (en) 1970-12-28 1974-10-01 Akzona Inc Process for the detection and determination of specific binding proteins and their corresponding bindable substances
US3791932A (en) 1971-02-10 1974-02-12 Akzona Inc Process for the demonstration and determination of reaction components having specific binding affinity for each other
US3901654A (en) 1971-06-21 1975-08-26 Biological Developments Receptor assays of biologically active compounds employing biologically specific receptors
US3853987A (en) 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3867517A (en) 1971-12-21 1975-02-18 Abbott Lab Direct radioimmunoassay for antigens and their antibodies
US3879262A (en) 1972-05-11 1975-04-22 Akzona Inc Detection and determination of haptens
US3850578A (en) 1973-03-12 1974-11-26 H Mcconnell Process for assaying for biologically active molecules
US3935074A (en) 1973-12-17 1976-01-27 Syva Company Antibody steric hindrance immunoassay with two antibodies
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4034074A (en) 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US3984533A (en) 1975-11-13 1976-10-05 General Electric Company Electrophoretic method of detecting antigen-antibody reaction
US4098876A (en) 1976-10-26 1978-07-04 Corning Glass Works Reverse sandwich immunoassay
US4879219A (en) 1980-09-19 1989-11-07 General Hospital Corporation Immunoassay utilizing monoclonal high affinity IgM antibodies
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5011771A (en) 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5281521A (en) 1992-07-20 1994-01-25 The Trustees Of The University Of Pennsylvania Modified avidin-biotin technique
US5747654A (en) 1993-06-14 1998-05-05 The United States Of America As Represented By The Department Of Health And Human Services Recombinant disulfide-stabilized polypeptide fragments having binding specificity
US6147203A (en) 1993-06-14 2000-11-14 The United States Of America As Represented By The Department Of Health And Human Services Recombinant disulfide-stabilized polypeptide fragments having binding specificity
US6558672B1 (en) 1993-06-14 2003-05-06 The United States Of America As Represented By The Department Of Health And Human Services Methods of making recombinant disulfide-stabilized polypeptide fragments having binding specificity
US5650490A (en) 1994-10-07 1997-07-22 Regeneron Pharmaceuticals, Inc. Tie-2 ligand 2
US5814464A (en) 1994-10-07 1998-09-29 Regeneron Pharma Nucleic acids encoding TIE-2 ligand-2
US6166185A (en) 1994-10-07 2000-12-26 Regeneron Pharmaceuticals, Inc. Antibodies to human TIE-2 ligands
US7183076B2 (en) 1997-05-02 2007-02-27 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
US20090155284A1 (en) 1999-11-09 2009-06-18 Boliang Cao Hgf-sf monoclonal antibody combinations
WO2003030833A2 (fr) 2001-10-11 2003-04-17 Amgen Inc. Agents de liaison spécifiques de l'angiopoïétine-2
US20060122370A1 (en) 2001-10-11 2006-06-08 Amgen Inc. Specific binding agents of human angiopoietin-2
WO2003057134A2 (fr) 2001-10-11 2003-07-17 Amgen, Inc. Agents de liaison specifiques de l'angiopoietine-2 humaine
US20030124129A1 (en) 2001-10-11 2003-07-03 Oliner Jonathan Daniel Angiopoietin-2 specific binding agents
US8129331B2 (en) 2001-10-11 2012-03-06 Amgen Inc. Specific binding agents of human angiopoietin-2
US20110311546A1 (en) 2001-10-11 2011-12-22 Amgen, Inc. Angiopoietin-2 specific binding agents
US20050118643A1 (en) 2003-07-18 2005-06-02 Burgess Teresa L. Specific binding agents to hepatocyte growth factor
US20070036789A1 (en) 2003-11-11 2007-02-15 National Cancer Center Neutralizable Epitope of HGF and Neutralizing Antibody Binding to the Same
US20090023894A1 (en) 2003-11-11 2009-01-22 National Cancer Center Neutralizing Antibody Against HGF
WO2006068953A2 (fr) 2004-12-21 2006-06-29 Astrazeneca Ab Anticorps dirigés contre l'angiopoïétine 2 et leurs utilisations
US20120052073A1 (en) 2004-12-21 2012-03-01 Medlmmune Limited Antibodies Directed to Angiopoietin-2 And Uses Thereof
WO2007068895A1 (fr) * 2005-12-15 2007-06-21 Astrazeneca Ab Combinaison d'un antagoniste de l'angiopoietine 2 et d'un antagoniste du vegf-a et/ou du kdr et/ou du fltl pour le traitement du cancer
WO2009107129A1 (fr) 2008-02-29 2009-09-03 Ramot At Tel Aviv University Ltd. Compositions d’immunoglobuline et procédés de production de celles-ci
US20100115589A1 (en) 2008-10-30 2010-05-06 Sharp Kabushiki Kaisha Apparatus for determining facsimile transmission authorizer and facsimile machine having the apparatus
WO2010069532A1 (fr) * 2008-12-16 2010-06-24 F. Hoffmann-La Roche Ag Anticorps dirigés contre l'angiopoïétine 2 humaine
US20100256340A1 (en) 2009-04-07 2010-10-07 Ulrich Brinkmann Trivalent, bispecific antibodies
US20120065380A1 (en) 2009-05-20 2012-03-15 Pharmabcine Inc. Dual targeting antibody of novel form, and use thereof
US20120100166A1 (en) 2010-07-15 2012-04-26 Zyngenia, Inc. Ang-2 Binding Complexes and Uses Thereof
US9601598B2 (en) 2010-07-15 2017-03-21 Taiwan Semiconductor Manufacturing Company, Ltd. Method of manufacturing a fin-like field effect transistor (FinFET) device
US20120123949A1 (en) 2010-11-17 2012-05-17 Shreyank Gupta Method and system to automatically modify task status in a project management system via keywords in source code version control commit logs

Non-Patent Citations (88)

* Cited by examiner, † Cited by third party
Title
"A Practical Guide to Molecular Cloning", 1984
"Animal Cell Culture", 1986
"Antibody Purification Handbook", AMERSHAM BIOSCIENCES
"Basic and Clinical Immunology", 1994, APPLETON & LANGE
"Cell Biology: A Laboratory Handbook", vol. I-III, 1994
"Current Protocols in Immunology", vol. I-III, 1994
"Current Protocols in Molecular Biology", 1987, GREENE PUBLISHING AND WILEY INTERSCIENCE
"Current Protocols in Molecular Biology", vol. I-III, 1994
"Genome Analysis: A Laboratory Manual Series", vol. 1-4, 1998, COLD SPRING HARBOR LABORATORY PRESS
"Immobilized Cells and Enzymes", 1986, IRL PRESS
"Methods in Enzymology", vol. 1-317, ACADEMIC PRESS
"Nucleic Acid Hybridization", 1985
"Oligonucleotide Synthesis", 1984
"PCR Protocols: A Guide To Methods And Applications", 1990, ACADEMIC PRESS
"Selected Methods in Cellular Immunology", 1980, W. H. FREEMAN AND CO.
"Transcription and Translation", 1984
ADELMAN ET AL., DNA, vol. 2, 1983, pages 183
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1989, JOHN WILEY AND SONS
AZRIEL-ROSENFELD, J MOL BIOL., vol. 335, no. 1, 2 January 2004 (2004-01-02), pages 177 - 92
BOERNER ET AL., J. IMMUNOL., vol. 147, no. 1, 1991, pages 86 - 95
BRUCCOLERI ET AL., NATURE, vol. 335, 1988, pages 564 - 568
CAO Y; SURESH MR: "Bispecific antibodies as novel bioconjugates", BIOCONJUG CHEM., vol. 9, no. 6, November 1998 (1998-11-01), pages 635 - 44, XP000858673, DOI: doi:10.1021/bc980044l
CHEUNG, A. H. ET AL., GENOMICS, vol. 48, 1998, pages 389 - 91
CHOTHIA ET AL., SCIENCE, vol. 233, 1986, pages 755 - 758
COLE ET AL.: "Monoclonal Antibodies and Cancer Therapy", 1985, ALAN R. LISS, pages: 77
DAVIS, S. ET AL., CELL, vol. 87, 1996, pages 1161 - 69
DE BERNARDEZ-CLARK; GEORGIOU: "Protein Refolding", 1991, article "Inclusion bodies and recovery of proteins from the aggregated state", pages: 1 - 20
E. HARLOW; D. LANE: "Antibodies: A Laboratory Manual.", 1988, COLD SPRING HARBOR LABORATORY PRESS
E. KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NIH PUBLICATION NO. 91-3242
ERLICH, SCIENCE, vol. 252, 1991, pages 1643 - 1650
FERRIN ET AL., J. MOL. GRAPHICS, vol. 6, 1988, pages 13 - 27
FISHWILD ET AL., NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 845 - 51
FRESHNEY: "Culture of Animal Cells - A Manual of Basic Technique", 1994, WILEY-LISS
GILLMAN; SMITH, GENE, vol. 8, 1979, pages 81 - 97
GODING: "Monoclonal Antibodies: Principles and Practice", 1986, ACADEMIC PRESS, pages: 59 - 103
GUNASEKARAN K; PENTONY M; SHEN M; GARRETT L; FORTE C; WOODWARD A; NG SB; BORN T; RETTER M; MANCHULENKO K: "Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG", J BIOL CHEM., vol. 285, no. 25, 16 April 2010 (2010-04-16), pages 19637 - 46, XP055303838, DOI: doi:10.1074/jbc.M110.117382
HAKIM ET AL., MABS, vol. 1, no. 3, May 2009 (2009-05-01), pages 281 - 7
HAMID LAKHIARIA; DANIEL MULLERB, JOURNAL OF CHROMATOGRAPHY B, vol. 818, no. 1, 15 April 2005 (2005-04-15), pages 53 - 59
HARLOW; LANE: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
HELFRICH, I. ET AL.: "Angiopoietin-2 levels are associated with disease progression in metastatic malignant melanoma", CLIN. CANCER RES., vol. 15, 2009, pages 1384 - 1392, XP055189487, DOI: doi:10.1158/1078-0432.CCR-08-1615
HOOGENBOOM; WINTER, J. MOL. BIOL., vol. 227, 1991, pages 381
HU, B. ET AL.: "Angiopoietin-2 induces human glioma invasion through the activation of matrix metalloprotease-2", PROC. NATL ACAD. SCI. USA, vol. 100, 2003, pages 8904 - 8909
HUANG, Y. ET AL.: "Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis", CANCER RES., vol. 69, 2009, pages 7529 - 7537
HUGHES, R.A. ET AL., METHODS IN ENZYMOLOGY, vol. 498, 2011, pages 277 - 309
HUTCHINS BM; KAZANE SA; STAFLIN K; FORSYTH JS; FELDING-HABERMANN B; SCHULTZ PG; SMIDER VV: "Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids", J MOL BIOL., vol. 406, no. 4, 13 January 2011 (2011-01-13), pages 595 - 603, XP028363709, DOI: doi:10.1016/j.jmb.2011.01.011
IMANISHI, Y. ET AL.: "Angiopoietin-2 stimulates breast cancer metastasis through the a5pl integrin-mediated pathway", CANCER RES., vol. 67, 2007, pages 4254 - 4263
ISABELLE MEYNIAL-SALLES; DIDIER COMBES: "In vitro glycosylation of proteins: An enzymatic approach", JOURNAL OF BIOTECHNOLOGY, vol. 46, no. 1, 18 April 1996 (1996-04-18), pages 1 - 14, XP004036807, DOI: doi:10.1016/0168-1656(95)00174-3
JACKMAN J, J BIOL CHEM., vol. 285, no. 27, 5 May 2010 (2010-05-05), pages 20850 - 9
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
KABAT: "Sequences of proteins of immunological Interest", 1991, NIH PUBLICATION NO. 91-3242
KABAT; WU; LOEW ET AL., INT. J. QUANT. CHEM., QUANT. BIOL. SYMP., vol. 15, 1988, pages 55 - 66
KIM, I. ET AL., FEBS LET, vol. 443, 1999, pages 353 - 56
KIM, I. ET AL., J BIOL CHEM, vol. 274, 1999, pages 26523 - 28
KUNKEL, PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 488 - 492
LEOW CHING CHING ET AL: "MEDI3617, a human anti-angiopoietin 2 monoclonal antibody, inhibits angiogenesis and tumor growth in human tumor xenograft models.", INTERNATIONAL JOURNAL OF ONCOLOGY MAY 2012, vol. 40, no. 5, May 2012 (2012-05-01), pages 1321 - 1330, XP002721374, ISSN: 1791-2423 *
LIU CC; SCHULTZ PG: "Adding new chemistries to the genetic code", ANNU REV BIOCHEM., vol. 79, 2010, pages 413 - 44, XP055026250, DOI: doi:10.1146/annurev.biochem.052308.105824
LONBERG ET AL., NATURE, vol. 368, 1994, pages 856 - 859
LONBERG; HUSZAR, INTERN. REV. IMMUNOL., vol. 13, 1995, pages 65 - 93
MAISONPIERRE, P. C. ET AL., SCIENCE, vol. 277, 1997, pages 55 - 60
MARKS ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 779 - 783
MARKS ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581
MARSHAK ET AL.: "Strategies for Protein Purification and Characterization - A Laboratory Course Manual", 1996, CSHL PRESS
MAULIK ET AL., CYTOKINE & GROWTH FACTOR REVIEWS, vol. 13, no. 1, 2002, pages 1 - 59
MORRISON, NATURE, vol. 368, 1994, pages 812 - 13
NAKAMURA ET AL., NATURE, vol. 342, 1989, pages 440 - 443
NEUBERGER, NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 826
PARK, J. H. ET AL.: "Serum angiopoietin-2 as a clinical marker for lung cancer", CHEST, vol. 132, 2007, pages 200 - 206
PERBAL: "A Practical Guide to Molecular Cloning", 1988, JOHN WILEY & SONS
PONDERS; RICHARDS, J. MOL. BIOL., vol. 193, 1987, pages 775 - 791
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 327
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
ROBERTS, S. ET AL., NATURE, vol. 328, 1987, pages 731 - 734
SAMBROOK ET AL.: "Molecular Cloning: A laboratory Manual", 1989
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR
SARAGOVI ET AL., SCIENCE, vol. 253, 1991, pages 792
THOMAS M ET AL: "489 LC06, a novel angiopoietin-2 selective human antibody with potent anti-tumoral and anti-angiogenic efficacy in different xenograft models", EUROPEAN JOURNAL OF CANCER. SUPPLEMENT, PERGAMON, OXFORD, GB, vol. 8, no. 7, 1 November 2010 (2010-11-01), pages 156 - 157, XP027498179, ISSN: 1359-6349, [retrieved on 20101101], DOI: 10.1016/S1359-6349(10)72196-0 *
TSUTSUI, S. ET AL.: "Angiopoietin 2 expression in invasive ductal carcinoma of the breast: its relationship to the VeGF expression and microvessel density", BREAST CANCER RES. TREAT, vol. 98, 2006, pages 261 - 266, XP019392312, DOI: doi:10.1007/s10549-005-9157-9
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534 - 1536
VIJAYALAKSHMI, M.A., APPL. BIOCHEM. BIOTECH., vol. 75, 1998, pages 93 - 102
WANG MING-HAI ET AL: "Potential therapeutics specific to c-MET/RON receptor tyrosine kinases for molecular targeting in cancer therapy", ACTA PHARMACOLOGICA SINICA,, vol. 31, no. 9, 1 September 2010 (2010-09-01), pages 1181 - 1188, XP009146790 *
WARD ET AL., NATURE, vol. 341, 1989, pages 544
WATSON ET AL.: "Recombinant DNA", SCIENTIFIC AMERICAN BOOKS
WELLS ET AL., GENE, vol. 34, 1985, pages 315
WIELENGA ET AL., AMERICAN JOURNAL OF PATHOLOGY, vol. 157, no. 5, 2000, pages 1563 - 1573
WU, X.; SCHULTZ, P.G.: "Synthesis at the Interface of Chemistry and Biology", J. AM. CHEM. SOC., vol. 131, no. 35, 2009, pages 12497 - 515
YANCOPOULOS, G. D. ET AL., NATURE, vol. 407, 2000, pages 242 - 48
YAP, MOLECULAR CANCER THERAPEUTICS, vol. 9, 2012, pages 1077 - 1079

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108948195A (zh) * 2017-05-23 2018-12-07 胡毅 一种抗egfr/pd-l1双靶向抗体、其制备方法及用途
CN108948195B (zh) * 2017-05-23 2022-05-31 胡毅 一种抗egfr/pd-l1双靶向抗体、其制备方法及用途

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