WO2014089193A1 - Anti-fgfr2 antibodies - Google Patents
Anti-fgfr2 antibodies Download PDFInfo
- Publication number
- WO2014089193A1 WO2014089193A1 PCT/US2013/073090 US2013073090W WO2014089193A1 WO 2014089193 A1 WO2014089193 A1 WO 2014089193A1 US 2013073090 W US2013073090 W US 2013073090W WO 2014089193 A1 WO2014089193 A1 WO 2014089193A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- amino acid
- acid sequence
- variable region
- chain variable
- Prior art date
Links
- 101000827688 Homo sapiens Fibroblast growth factor receptor 2 Proteins 0.000 claims abstract description 38
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 31
- 102000055736 human FGFR2 Human genes 0.000 claims abstract description 29
- 201000011510 cancer Diseases 0.000 claims abstract description 22
- 241000282414 Homo sapiens Species 0.000 claims description 114
- 210000004027 cell Anatomy 0.000 claims description 79
- 230000027455 binding Effects 0.000 claims description 70
- 101000998953 Homo sapiens Immunoglobulin heavy variable 1-2 Proteins 0.000 claims description 44
- 102100036887 Immunoglobulin heavy variable 1-2 Human genes 0.000 claims description 44
- 101001008255 Homo sapiens Immunoglobulin kappa variable 1D-8 Proteins 0.000 claims description 43
- 101001047628 Homo sapiens Immunoglobulin kappa variable 2-29 Proteins 0.000 claims description 43
- 101001008321 Homo sapiens Immunoglobulin kappa variable 2D-26 Proteins 0.000 claims description 43
- 101001047619 Homo sapiens Immunoglobulin kappa variable 3-20 Proteins 0.000 claims description 43
- 101001008263 Homo sapiens Immunoglobulin kappa variable 3D-15 Proteins 0.000 claims description 43
- 102100022949 Immunoglobulin kappa variable 2-29 Human genes 0.000 claims description 43
- 150000007523 nucleic acids Chemical class 0.000 claims description 40
- 239000013604 expression vector Substances 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 32
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 claims description 29
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 claims description 29
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 claims description 28
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 claims description 28
- 102000039446 nucleic acids Human genes 0.000 claims description 26
- 108020004707 nucleic acids Proteins 0.000 claims description 26
- 229920001184 polypeptide Polymers 0.000 claims description 26
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 26
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 26
- 239000000427 antigen Substances 0.000 claims description 25
- 102000036639 antigens Human genes 0.000 claims description 25
- 108091007433 antigens Proteins 0.000 claims description 25
- 230000035755 proliferation Effects 0.000 claims description 22
- 239000012634 fragment Substances 0.000 claims description 18
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 241000124008 Mammalia Species 0.000 claims description 9
- 206010006187 Breast cancer Diseases 0.000 claims description 6
- 208000026310 Breast neoplasm Diseases 0.000 claims description 6
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 6
- 206010017758 gastric cancer Diseases 0.000 claims description 6
- 201000011549 stomach cancer Diseases 0.000 claims description 6
- 230000004614 tumor growth Effects 0.000 claims description 6
- 238000012575 bio-layer interferometry Methods 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 claims description 4
- 210000004881 tumor cell Anatomy 0.000 claims description 4
- 206010014733 Endometrial cancer Diseases 0.000 claims description 3
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 3
- 206010033128 Ovarian cancer Diseases 0.000 claims description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 3
- 201000005202 lung cancer Diseases 0.000 claims description 3
- 208000020816 lung neoplasm Diseases 0.000 claims description 3
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 2
- 201000002528 pancreatic cancer Diseases 0.000 claims description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 54
- 102100023600 Fibroblast growth factor receptor 2 Human genes 0.000 abstract description 52
- 101710182389 Fibroblast growth factor receptor 2 Proteins 0.000 abstract description 43
- 230000004913 activation Effects 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract 1
- 208000037765 diseases and disorders Diseases 0.000 abstract 1
- 230000002018 overexpression Effects 0.000 abstract 1
- 230000002062 proliferating effect Effects 0.000 abstract 1
- 150000001413 amino acids Chemical group 0.000 description 72
- 108090000623 proteins and genes Proteins 0.000 description 59
- 102000004169 proteins and genes Human genes 0.000 description 50
- 108091028043 Nucleic acid sequence Proteins 0.000 description 21
- 230000005764 inhibitory process Effects 0.000 description 19
- 102220525770 Sodium channel subunit beta-3_Q89L_mutation Human genes 0.000 description 16
- 102200123127 rs1555621454 Human genes 0.000 description 16
- 102220075003 rs796053016 Human genes 0.000 description 15
- 102220490832 Mannosyl-oligosaccharide glucosidase_Y32F_mutation Human genes 0.000 description 14
- 230000035772 mutation Effects 0.000 description 14
- 238000011282 treatment Methods 0.000 description 14
- 108091008794 FGF receptors Proteins 0.000 description 13
- 238000003556 assay Methods 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 12
- 102200131576 rs121912452 Human genes 0.000 description 12
- 102220065900 rs200290535 Human genes 0.000 description 12
- 102220005551 rs33960522 Human genes 0.000 description 12
- 102220257229 rs754231971 Human genes 0.000 description 12
- 238000002823 phage display Methods 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 10
- 102000052178 fibroblast growth factor receptor activity proteins Human genes 0.000 description 10
- 102200105384 rs387906979 Human genes 0.000 description 10
- 238000010494 dissociation reaction Methods 0.000 description 9
- 230000005593 dissociations Effects 0.000 description 9
- 101150081880 FGF1 gene Proteins 0.000 description 8
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 8
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 8
- 229940126864 fibroblast growth factor Drugs 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 6
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 6
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 6
- 241000282567 Macaca fascicularis Species 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 238000001990 intravenous administration Methods 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 102100024952 Protein CBFA2T1 Human genes 0.000 description 5
- 102000001708 Protein Isoforms Human genes 0.000 description 5
- 108010029485 Protein Isoforms Proteins 0.000 description 5
- 238000001516 cell proliferation assay Methods 0.000 description 5
- 239000012636 effector Substances 0.000 description 5
- 101150088071 fgfr2 gene Proteins 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000002708 random mutagenesis Methods 0.000 description 5
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 102000005720 Glutathione transferase Human genes 0.000 description 4
- 108010070675 Glutathione transferase Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 101000917148 Homo sapiens Fibroblast growth factor receptor 3 Proteins 0.000 description 4
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 4
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 238000002869 basic local alignment search tool Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000055709 human FGFR3 Human genes 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000012146 running buffer Substances 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000002864 sequence alignment Methods 0.000 description 4
- 229940124597 therapeutic agent Drugs 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 101150021185 FGF gene Proteins 0.000 description 3
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 108091000080 Phosphotransferase Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000023445 activated T cell autonomous cell death Effects 0.000 description 3
- 230000009824 affinity maturation Effects 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 239000012131 assay buffer Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 102000020233 phosphotransferase Human genes 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- ZEOWTGPWHLSLOG-UHFFFAOYSA-N Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F Chemical compound Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F ZEOWTGPWHLSLOG-UHFFFAOYSA-N 0.000 description 2
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 2
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 2
- 102100023593 Fibroblast growth factor receptor 1 Human genes 0.000 description 2
- 101710182386 Fibroblast growth factor receptor 1 Proteins 0.000 description 2
- 102100027842 Fibroblast growth factor receptor 3 Human genes 0.000 description 2
- 101710182396 Fibroblast growth factor receptor 3 Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 102000003839 Human Proteins Human genes 0.000 description 2
- 108090000144 Human Proteins Proteins 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 108091054455 MAP kinase family Proteins 0.000 description 2
- 102000043136 MAP kinase family Human genes 0.000 description 2
- 238000000134 MTT assay Methods 0.000 description 2
- 231100000002 MTT assay Toxicity 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 102000038030 PI3Ks Human genes 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 2
- 241000276498 Pollachius virens Species 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000012082 adaptor molecule Substances 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 230000035578 autophosphorylation Effects 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 238000004422 calculation algorithm Methods 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000007598 dipping method Methods 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 208000023965 endometrium neoplasm Diseases 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- SXTAYKAGBXMACB-UHFFFAOYSA-N methionine sulfoximine Chemical compound CS(=N)(=O)CCC(N)C(O)=O SXTAYKAGBXMACB-UHFFFAOYSA-N 0.000 description 2
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102100036329 Cyclin-dependent kinase 3 Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 101150082429 FGFR4 gene Proteins 0.000 description 1
- 102100027844 Fibroblast growth factor receptor 4 Human genes 0.000 description 1
- 102100021066 Fibroblast growth factor receptor substrate 2 Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 101000715946 Homo sapiens Cyclin-dependent kinase 3 Proteins 0.000 description 1
- 101001107795 Homo sapiens Cytosolic Fe-S cluster assembly factor NUBP2 Proteins 0.000 description 1
- 101000917134 Homo sapiens Fibroblast growth factor receptor 4 Proteins 0.000 description 1
- 101000818410 Homo sapiens Fibroblast growth factor receptor substrate 2 Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000012745 Immunoglobulin Subunits Human genes 0.000 description 1
- 108010079585 Immunoglobulin Subunits Proteins 0.000 description 1
- 102000016844 Immunoglobulin-like domains Human genes 0.000 description 1
- 108050006430 Immunoglobulin-like domains Proteins 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 101000935846 Magnetospirillum magnetotacticum Bacterioferritin subunit 1 Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000011579 SCID mouse model Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 238000010162 Tukey test Methods 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001028 anti-proliverative effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036765 blood level Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 150000001860 citric acid derivatives Chemical class 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- -1 coatings Substances 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000017066 negative regulation of growth Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 239000000346 nonvolatile oil Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000011330 nucleic acid test Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000008389 polyethoxylated castor oil Substances 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 238000002702 ribosome display Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000005747 tumor angiogenesis Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 229940099073 xolair Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- 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
- C07K2317/50—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
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- 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
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
Monoclonal antibodies that bind and inhibit biological activities of human FGFR2 are disclosed. The antibodies can be used to treat cell proliferative diseases and disorders, including certain forms of cancer, associated with activation or over-expression of FGFR2.
Description
ANTI-FGFR2 ANTIBODIES
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 61/733,285, filed December 4, 2012, the contents of which are incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The field of the invention is molecular biology, immunology and oncology. More particularly, the field is therapeutic antibodies.
BACKGROUND
[0003] Fibroblast Growth Factor Receptor 2 (FGFR2), also known as BEK, BFR-1, CD332, CEK3, CFD1, ECT1, JWS, KGFR (also known as FGFR2IIIb), K-SAM, TK14, and TK25, is one of four highly conserved receptor tyrosine kinases (FGFR1, FGFR2, FGFR3 and FGFR4) that mediate fibroblast growth factor (FGF) signaling by binding FGFs. The FGF receptors are characterized by two or three extracellular immunoglobulin-like domains (IgDl, IgD2 and IgD3), a single-pass transmembrane domain, and a cytoplasmic tyrosine kinase domain. FGF ligand binding induces FGF receptor dimerization and tyrosine
autophosphorylation, resulting in cell proliferation, differentiation and migration (Turner et al. (2010) NATURE REVIEWS CANCER 10: 116-129; Beenken et al. (2009) NATURE REVIEWS DRUG DISCOVERY 8:235-254; Gomez-Roman et al. (2005) CLIN. CANCER RES. 1 1 :459-65; Chang et al. (2005) BLOOD 106:353-6; Eswarakumar et al. (2005) CYTOKINE GROWTH FACTOR REV. 16: 139-49).
[0004] Alternative splicing in the IgD3 domain yields either the Illb or IIIc isoform of FGFR1, FGFR2 and FGFR3. The FGFR4 gene is expressed only as the IIIc isoform. The different isoforms of FGF receptors exhibit tissue-specific expression, and they respond to a different spectrum of 18 mammalian FGFs (Beenken et al., supra). Binding of FGFs to FGFRs in the presence of heparan sulfate proteoglycans induces autophosphorylation of FGFRs at specific intracellular tyrosine residues. This causes phosphorylation of adaptor molecules, such
as FGFR substrate 2a (FRS2a), which recruits other proteins to activate various signaling cascades, including the mitogen-activated protein kinase (MAPK) pathway and the
phosphoinositide 3-kinase (PI3K)/Akt pathway (Beenken et ah, supra; Eswarakumar et ah, supra; Turner et ah, supra).
[0005] It has been suggested that the dysregulated FGF signaling can directly drive the proliferation of cancer cells, promote the survival of cancer stem cells, and support tumor angiogenesis (Turner et ah, supra). FGFR2 signaling appears to play a role in cancer.
Missense mutations in the FGFR2 gene occur in various cancers, including endometrial cancer (Pollock et ah, 2007, ONCOGENE 26:7158-7162; Dutt et ah, 2008, PROC. NATL. ACAD. SCI. USA 105:8713-8717), ovarian cancer, breast cancer, lung cancer (Greenman et ah, 2007,
Nature 446: 153-158; Ding et ah, 2008, NATURE 455: 1069-1075; Davies et ah, 2005, CANCER RES. 65:7591-7595) and gastric cancer (Jang et ah, 2001, CANCER RES. 61 :3541-3543). Some of these activating mutations also have been reported in patients with skeletal disorders (Dutt et ah, supra). Two independent genome-wide association studies have linked specific single nucleotide polymorphisms (SNPs) in the FGFR2 gene to increased susceptibility to breast cancer (Easton et ah, 2007, NATURE 447: 1087-1093; Hunter et ah, 2007, NAT. GENET. 39:870- 874). These cancer-associated SNPs appear to elevate FGFR2 gene expression (Meyer et ah, 2008, PLOS BIOL. 6:el08). The FGFR2 gene, located at human chromosome 10q26, is amplified in a subset of breast cancers (Adnane et ah, 1991, ONCOGENE 6:659-663; Turner et ah, 2010, ONCOGENE 29:2013-2023) and gastric cancer (Hara et ah, 1998, LAB. INVEST.
78: 1 143-1 153; Mor e? ah, 1993, CANCER GENET. CYTOGENET. 65: 11 1-1 14).
[0006] Naturally occurring antibodies are multimeric proteins that contain four polypeptide chains (FIG. 1). Two of the polypeptide chains are called heavy chains (H chains), and two of the polypeptide chains are called light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region VL in FIG. 1) and one constant region (CL in FIG. 1). The heavy chain consists of one variable region (VH in FIG. 1) and at least three constant regions (CHi, CI¾ and CH3 in FIG. 1). The variable regions determine the specificity of the antibody. Each variable region comprises three hypervariable regions also known as complementarity determining regions (CDRs) flanked by four relatively conserved framework regions (FRs). The three CDRs, referred to as CDRi, CDR2, and CDR3, contribute to the antibody binding specificity. Naturally occurring
antibodies can be obtained from hybridoma technology, synthetic antibody technology, and human donor-derived technology (e.g., phage, yeast, or ribosome display technology).
Naturally occurring antibodies have been used as starting material for engineered and/or optimized antibodies to improve antibody characteristics, e.g., improved biochemical properties, biophysical properties and/or immunogenicity.
[0007] Although antibodies that bind FGFR2 are known in the art, there is still a need for improved FGFR2 antibodies that can be used as therapeutic agents.
SUMMARY
[0008] The invention is based, in part, upon the discovery of a family of antibodies that specifically bind human FGFR2. The antibodies contain FGFR2 binding sites based on the CDRs of the antibodies. The antibodies can be used as therapeutic agents. When used as therapeutic agents, the antibodies can be optimized, e.g., affinity-matured, to improve biochemical properties (e.g., affinity and/or specificity), to improve biophysical properties (e.g., aggregation, stability, precipitation, and/or non-specific interactions), and/or to reduce or eliminate immunogenicity, when administered to a human patient.
[0009] The antibodies prevent or inhibit the activation of (i.e., neutralize) human FGFR2. The antibodies of the invention can be used to inhibit the proliferation of tumor cells in vitro or in vivo. When administered to a human cancer patient or an animal model, the antibodies inhibit or reduce tumor growth in the human patient or animal model.
[0010] These and other aspects and advantages of the invention will become apparent upon consideration of the following figures, detailed description, and claims. As used herein, "including" means without limitation, and examples cited are non-limiting.
DESCRIPTION OF THE DRAWINGS
[0011] The invention can be more completely understood with reference to the following drawings.
[0012] FIG. 1 (prior art) is a schematic representation of a typical naturally-occurring antibody.
[0013] FIG. 2 is a sequence alignment showing the amino acid sequence of the complete immunoglobulin heavy chain variable regions 15492Hv (antibodies 15492 and 15492.6),
15492Hv optCH02 (antibody 15492.50), 15492Hv Y32F optCH02 (antibodies 15492.61 and 15492.77) and 15492Hv Y32N E35Q G52S V56Y Q58V optCH02 (antibody 15492.65). The amino acid sequences for each heavy chain variable region are aligned against one another, and CDRi, CDR2, and CDR3 are identified in boxes. The Kabat numbering scheme is used to identify the amino acid numbers at given positions. The unboxed sequences represent framework (FR) sequences.
[0014] FIG. 3 is a sequence alignment showing the CDRi, CDR2, and CDR3 sequences for each of the immunoglobulin heavy chain variable region sequences in FIG. 2.
[0015] FIG. 4 is a sequence alignment showing the amino acid sequence of the complete immunoglobulin light chain variable regions 15492Kv (antibody 15492), 15492Kv Q3V A40P L85V R103K (antibody 15492.6), 15492.6Kv Q89L Y92H optCH02 (antibodies 15492.61 and 15492.65) and 31288Kv Q3V I58V D81E optCH02 (antibodies 15492.50 and 15492.77). In the amino acid sequence 31288Kv Q3V I58V D81E optCH02, the point mutations were identified relative to the original sequence of antibody 31288, which is not shown. The amino acid sequences for each light chain variable region are aligned against one another, and CDRi, CDR2, and CDR3 are identified in boxes. The Kabat numbering scheme is used to identify the amino acid numbers at given positions. The unboxed sequences represent framework (FR) sequences.
[0016] FIG. 5 is a sequence alignment showing the CDRi, CDR2, and CDR3 sequences for each of the immunoglobulin light chain variable region sequences in FIG. 4.
[0017] FIG. 6 is a graph summarizing results from an experiment to measure inhibition of FGFl-induced proliferation of FDCP-1 cells expressing wild-type FGFR2 alllb (·) and wild- type FGFR2 alllc (■), by treatment with antibody 15492.
[0018] FIG. 7 is a graph summarizing results from an experiment to measure inhibition of FGFl-induced proliferation of FDCP-1 cells expressing wild-type FGFR2 alllb (·), truncated FGFR2 alllb (A ), FGFR2 βΙΙ¾-Ν550Κ (■), and FGFR2 IIIb-S252W (T), by treatment with antibody 15492.
[0019] FIG. 8 is a graph summarizing results from an experiment to measure inhibition of FGFl-induced proliferation of FDCP-1 cells expressing wild-type FGFR2 alllb, by treatment
with affinity matured 15492 variants: 15492.6 (■), 15492.50 (A), 15492.61 (T ), 15492.65 (♦), and 15492.77 (·).
[0020] FIG. 9 is a graph summarizing results from an experiment to measure inhibition of FGF 1 -induced proliferation of FDCP-1 cells expressing wild-type FGFR2 alllc, by treatment with affinity matured 15492 variants: 15492.6 (■), 15492.50 (A), 15492.61 (T ), 15492.65 (♦), and 15492.77 (·).
[0021] FIG. 10 is a graph summarizing results from an experiment to measure inhibition of FGF 1 -induced proliferation of FDCP-1 cells expressing FGFR2 IIIb-S252W, by treatment with affinity matured 15492 variants: 15492.6 (■), 15492.50 (A), 15492.61 (T ), 15492.65 (♦), and 15492.77 (·).
[0022] FIG. 11 is a graph summarizing results from an experiment to measure inhibition of FGF 1 -induced proliferation of FDCP-1 cells expressing FGFR2 IIIc-S252W, by treatment with affinity matured 15492 variants: 15492.6 (■), 15492.50 (A), 15492.61 (T ), 15492.65 (♦), and 15492.77 (·).
[0023] FIG. 12 is a graph summarizing results from an experiment to measure inhibition of FGF 1 -induced proliferation of FDCP-1 cells expressing FGFR2 βΙΙ¾-Ν550Κ, by treatment with affinity matured 15492 variants: 15492.6 (■), 15492.50 (A ), 15492.61 (T), 15492.65 (♦), and 15492.77 (·).
[0024] FIG. 13 is a graph summarizing results from an experiment to measure inhibition of FGF 1 -induced proliferation of FDCP-1 cells expressing truncated FGFR2 alllb, by treatment with affinity matured 15492 variants: 15492.6 (■), 15492.50 (A ), 15492.61 (T), 15492.65 (♦), and 15492.77 (·).
[0025] FIG. 14 is a graph summarizing results from an experiment to measure inhibition of growth of SNU-16 xenograft tumors by treatment with affinity-matured antibody variants of 15492 at 2 mg/kg (also referred to herein as "mpk"): 15492.6 (O), 15492.50 (Δ), 15492.61 (♦), 15492.65 (A ), 15492.77 (·), with PBS (■) and hlgG (□) serving as a negative control.
DETAILED DESCRIPTION
[0026] The anti-FGFR2 antibodies disclosed herein are based on the antigen binding sites of certain monoclonal antibodies that have been selected on the basis of binding and
neutralizing the activity of human FGFR2. The antibodies contain immunoglobulin variable region CDR sequences that define a binding site for human FGFR2.
[0027] In view of the neutralizing activity of these antibodies, they are useful for modulating the growth and/or proliferation of certain types of cancer cells. When used as a therapeutic agent, the antibodies can be optimized, e.g., affinity-matured, to improve biochemical properties and/or biophysical properties, and/or to reduce or eliminate
immunogenicity when administered to a human patient. Various features and aspects of the invention are discussed in more detail below.
[0028] As used herein, unless otherwise indicated, the term "antibody" means an intact antibody (e.g., an intact monoclonal antibody) or antigen-binding fragment of an antibody, including an intact antibody or antigen-binding fragment of an antibody (e.g., a phage display antibody including a fully human antibody, a semisynthetic antibody or a fully synthetic antibody) that has been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized are affinity-matured antibodies. Examples of antibodies that have been engineered are Fc optimized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, single chain antibodies (e.g., scFv), minibodies and diabodies. An antibody conjugated to a toxin moiety is an example of a chemically conjugated antibody.
I. Antibodies That Bind FGFR2
[0029] The antibodies disclosed herein comprise: (a) an immunoglobulin heavy chain variable region comprising the structure CDRHi-CDRH2-CDRH3 and (b) an immunoglobulin light chain variable region comprising the structure CDRLI-CDRL2-CDRL3, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR2 protein.
[0030] In some embodiments, the antibody comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDRHi-CDRH2-CDRH3 and (b) an immunoglobulin light chain variable region, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR2. A CDRHI comprises an amino acid sequence selected from the group consisting of SEQ ID NO: l (15492, 15492.6, 15492.50), SEQ ID NO:2 (15492.61, 15492.77), and SEQ ID NO:3 (15492.65); a CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4 (15492,
15492.6, 15492.50, 15492.61, 15492.77) and SEQ ID NO:5 (15492.65); and a CDRH3 comprises the amino acid sequence of SEQ ID NO:6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77). Throughout the specification a particular SEQ ID NO. is followed in parentheses by the antibody that was the origin of that sequence. For example, "SEQ ID NO:5 (15492.65)" means that SEQ ID NO:5 comes from antibody 15492.65.
[0031] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRm comprising the amino acid sequence of SEQ ID NO: l (15492, 15492.6, 15492.50); a CDRH2 comprising the amino acid sequence of SEQ ID NO:4 (15492, 15492.6, 15492.50, 15492.61, 15492.77), and a CDRH3 comprising the amino acid sequence of SEQ ID NO:6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77).
[0032] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRHI comprising the amino acid sequence of SEQ ID NO:2 (15492.61, 15492.77); a CDRH2 comprising the amino acid sequence of SEQ ID NO:4 (15492, 15492.6, 15492.50, 15492.61, 15492.77), and a CDRH3 comprising the amino acid sequence of SEQ ID NO:6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77).
[0033] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRHI comprising the amino acid sequence of SEQ ID NO:3 (15492.65); a CDRH2 comprising the amino acid sequence of SEQ ID NO:5 (15492.65), and a CDRH3 comprising the amino acid sequence of SEQ ID NO:6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77).
[0034] The CDRHi, CDRH2, and CDRH3 sequences are interposed between immunoglobulin framework regions (FRs).
[0035] In some embodiments, the antibody comprises (a) an immunoglobulin light chain variable region comprising the structure CDRLi-CDRL2-CDRL3, and (b) an immunoglobulin heavy chain variable region, wherein the IgG light chain variable region and the IgG heavy chain variable region together define a single binding site for binding human FGFR2. A CDRLI comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7 (15492, 15492.6, 15492.61, 15492.65) and SEQ ID NO:8 (15492.50, 15492.77); a CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9 (15492, 15492.6, 15492.61, 15492.65) and SEQ ID NO: 10 (15492.50, 15492.77); and a CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 1
(15492, 15492.6), SEQ ID NO: 12 (15492.61, 15492.65), and SEQ ID O: 13 (15492.50, 15492.77).
[0036] In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising a CDRLI comprising the amino acid sequence of SEQ ID NO:7 (15492, 15492.6, 15492.61, 15492.65), a CDRL2 comprising the amino acid sequence of SEQ ID NO: 9 (15492, 15492.6, 15492.61, 15492.65), and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 1 1 (15492, 15492.6).
[0037] In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising a CDRLI comprising the amino acid sequence of SEQ ID NO:7 (15492, 15492.6, 15492.61, 15492.65), a CDRL2 comprising the amino acid sequence of SEQ ID NO: 9 (15492, 15492.6, 15492.61, 15492.65), and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12 (15492.61, 15492.65).
[0038] In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising a CDRLI comprising the amino acid sequence of SEQ ID NO:8 (15492.50, 15492.77), a CDRL2 comprising the amino acid sequence of SEQ ID NO: 10
(15492.50, 15492.77), and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 13 (15492.50, 15492.77).
[0039] The CDRLi, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FRs.
[0040] In some embodiments, the antibody comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDRHI-CDRH2-CDRH3 and (b) an immunoglobulin light chain variable region comprising the structure CDRLI -CDRL2-CDRL3, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR2. The CDRHI is an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (15492, 15492.6, 15492.50), SEQ ID NO:2 (15492.61, 15492.77), and SEQ ID NO:3 (15492.65); the CDRH2 is an amino acid sequence selected from the group consisting SEQ ID NO:4 (15492, 15492.6, 15492.50, 15492.61, 15492.77) and SEQ ID NO:5 (15492.65); and the CDRH3 is the amino acid sequence of SEQ ID NO:6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77). The CDRLI is an amino acid sequence selected from the group consisting of SEQ ID NO:7 (15492, 15492.6, 15492.61, 15492.65) and SEQ ID NO: 8 (15492.50, 15492.77); the CDRL2 is an amino acid sequence selected from the group
consisting of SEQ ID NO:9 (15492, 15492.6, 15492.61, 15492.65) and SEQ ID NO: 10
(15492.50, 15492.77); and the CDRL3 is an amino acid sequence selected from the group consisting of SEQ ID NO: 11 (15492, 15492.6), SEQ ID NO: 12 (15492.61, 15492.65), and SEQ ID NO: 13 (15492.50, 15492.77).
[0041] In other embodiments, the antibodies disclosed herein comprise an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO: 14 (15492, 15492.6, 15492.50), SEQ ID NO: 16 (15492.61, 15492.77), and SEQ ID NO: 18 (15492.65), and an immunoglobulin light chain variable region.
[0042] In other embodiments, the antibody comprises an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO:20 (15492), SEQ ID NO:22 (15492.6), SEQ ID NO:24 (15492.61, 15492.65), and SEQ ID NO:26 (15492.50, 15492.77), and an immunoglobulin heavy chain variable region.
[0043] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO: 14 (15492, 15492.6, 15492.50), SEQ ID NO: 16 (15492.61, 15492.77), and SEQ ID NO: 18 (15492.65), and an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO:20 (15492), SEQ ID NO:22 (15492.6), SEQ ID NO:24 (15492.61, 15492.65), and SEQ ID NO:26 (15492.50, 15492.77).
[0044] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (15492, 15492.6, 15492.50), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:20 (15492).
[0045] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (15492, 15492.6, 15492.50), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:22 (15492.6).
[0046] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (15492, 15492.6,
15492.50), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:26 (15492.50, 15492.77).
[0047] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 (15492.61, 15492.77), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:24 (15492.61, 15492.65).
[0048] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 (15492.65), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:24 (15492.61, 15492.65).
[0049] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 (15492.61, 15492.77), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:26 (15492.50, 15492.77).
[0050] In certain embodiments, the antibodies disclosed herein comprise an
immunoglobulin heavy chain and an immunoglobulin light chain. In some embodiments, the antibody comprises an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO:32 (15492, 15492.6, 15492.50), SEQ ID NO:34 (15492.61, 15492.77), and SEQ ID NO:36 (15492.65), and an immunoglobulin light chain.
[0051] In other embodiments, the antibody comprises an immunoglobulin light chain selected from the group consisting of SEQ ID NO:38 (15492), SEQ ID NO:40 (15492.6), SEQ ID NO:42 (15492.61, 15492.65), and SEQ ID NO:44 (15492.50, 15492.77), and an
immunoglobulin heavy chain.
[0052] In some embodiments, the antibody comprises (i) an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO:32 (15492, 15492.6, 15492.50), SEQ ID
NO:34 (15492.61, 15492.77), and SEQ ID NO:36 (15492.65), and (ii) an immunoglobulin light chain selected from the group consisting of SEQ ID NO:38 (15492), SEQ ID NO:40 (15492.6), SEQ ID NO:42 (15492.61, 15492.65), and SEQ ID NO:44 (15492.50, 15492.77).
[0053] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:32 (15492, 15492.6, 15492.50), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:38 (15492).
[0054] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:32 (15492, 15492.6, 15492.50), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:40 (15492.6).
[0055] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:32 (15492, 15492.6, 15492.50), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:44 (15492.50, 15492.77).
[0056] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:34 (15492.61, 15492.77), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:42 (15492.61, 15492.65).
[0057] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:36 (15492.65), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:42 (15492.61, 15492.65).
[0058] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:34 (15492.61, 15492.77), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:44 (15492.50, 15492.77).
[0059] In certain embodiments, an isolated antibody that binds human FGFR2 comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the entire variable region or the framework region sequence of SEQ ID NO: 14 (15492, 15492.6, 15492.50), SEQ ID NO: 16 (15492.61, 15492.77), or SEQ ID NO: 18 (15492.65).
[0060] In certain embodiments, an isolated antibody that binds human FGFR2 comprises an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the entire variable region or the
framework region sequence of SEQ ID NO:20 (15492), SEQ ID NO:22 (15492.6), SEQ ID NO:24 (15492.61, 15492.65), or SEQ ID NO:26 (15492.50, 15492.77).
[0061] Sequence identity may be determined in various ways that are within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al, (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290- 300; Altschul et al, (1997) NUCLEIC ACIDS RES. 25:3389-3402, incorporated by reference herein) are tailored for sequence similarity searching. For a discussion of basic issues in searching sequence databases see Altschul et al, (1994) NATURE GENETICS 6: 1 19-129, which is fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the
BLOSUM62 matrix (Henikoff et al, (1992) PROC. NATL. ACAD. SCI. USA 89: 10915-10919, incorporated by reference herein). Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=l (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings may be Q=9; R=2; wink=l; and gapw=32. Searches may also be conducted using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameter (e.g.: -G, Cost to open gap [Integer]: default = 5 for nucleotides/ 1 1 for proteins; -E, Cost to extend gap [Integer]: default = 2 for nucleotides/ 1 for proteins; -q, Penalty for nucleotide mismatch [Integer]: default = -3; -r, reward for nucleotide match [Integer]: default = 1; -e, expect value [Real]: default = 10; -W, wordsize [Integer]: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins; -y, Dropoff (X) for blast extensions in bits: default = 20 for blastn/ 7 for others; -X, X dropoff value for gapped alignment (in bits): default = 15 for all programs, not applicable to blastn; and -Z, final X dropoff value for gapped alignment (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, e.g., Blosum62 matrix and Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1). A Bestfit comparison
between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.
[0062] In each of the foregoing embodiments, it is contemplated herein that
immunoglobulin heavy chain variable region sequences and/or light chain variable region sequences that together bind human FGFR2 may contain amino acid alterations (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the heavy and/or light chain variable regions.
[0063] In certain embodiments, the antibody binds human FGFR2 with a ¾ of 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or lower. Unless otherwise specified, KD values are determined by surface plasmon resonance or bio-layer interferometry methods under the conditions described in Example 2.
[0064] In some embodiments, monoclonal antibodies bind to the same epitope on human FGFR2 as any of the 15492 antibodies disclosed herein (e.g., antibodies 15492, 15492.6, 15492.50, 15492.61, 15492.65 or 15492.77). In some embodiments, monoclonal antibodies compete for binding to human FGFR2 with any of the 15492 antibodies disclosed herein.
[0065] Competition assays for determining whether an antibody binds to the same epitope as, or competes for binding with, a 15492 antibody disclosed herein are known in the art. Exemplary competition assays include immunoassays (e.g., ELISA assays, RIA assays), BIAcore analysis, bio-layer interferometry and flow cytometry.
[0066] Typically, a competition assay involves the use of an antigen (e.g., a FGFR2 protein or fragment thereof) bound to a solid surface or expressed on a cell surface, a test FGFR2- binding antibody and a reference antibody (e.g., antibodies 15492, 15492.6, 15492.50, 15492.61, 15492.65 or 15492.77). The reference antibody is labeled and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cells in the presence of the test antibody. Usually the test antibody is present in excess (e.g., lx, 5x, lOx, 20x or lOOx). Antibodies identified by competition assay (i.e., competing antibodies) include antibodies binding to the same epitope, or similar (e.g., overlapping) epitopes, as the reference antibody, and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.
[0067] In an exemplary competition assay, a reference FGFR2 antibody (e.g., antibody 15492, 15492.6, 15492.50, 15492.61, 15492.65 or 15492.77) is biotinylated using
commercially available reagents. The biotinylated reference antibody is mixed with serial dilutions of the test antibody or unlabeled reference antibody (self-competition control) resulting in a mixture of various molar ratios (e.g., lx, 5x, lOx, 20x or lOOx) of test antibody (or unlabeled reference antibody) to labeled reference antibody. The antibody mixture is added to a FGFR2 (e.g., extracellular domain of FGFR2) polypeptide coated-ELISA plate. The plate is then washed and horseradish peroxidase (HRP)-streptavidin is added to the plate as the detection reagent. The amount of labeled reference antibody bound to the target antigen is detected following addition of a chromogenic substrate (e.g., TMB (3, 3', 5,5'- tetramethylbenzidine) or ABTS (2,2"-azino-di-(3-ethylbenzthiazoline-6-sulfonate)), which are well-known in the art. Optical density readings (OD units) are measured using a SpectraMax M2 spectrometer (Molecular Devices). OD units corresponding to zero percent inhibition are determined from wells without any competing antibody. OD units corresponding to 100% inhibition, i.e., the assay background are determined from wells without any labeled reference antibody or test antibody. Percent inhibition of labeled reference antibody to FGFR2 by the test antibody (or the unlabeled reference antibody) at each concentration is calculated as follows: % inhibition = (l-(OD units - 100% inhibition)/(0% inhibition - 100% inhibition ))* 100. Persons skilled in the art will appreciate that the competition assay can be performed using various detection systems well known in the art.
[0068] A competition assay may be conducted in both directions to ensure that the presence of the label does not interfere or otherwise inhibit binding. For example, in the first direction the reference antibody is labeled and the test antibody is unlabeled, and in the second direction, the test antibody is labeled and the reference antibody is unlabeled.
[0069] A test antibody competes with the reference antibody for specific binding to the antigen if an excess of one antibody (e.g., lx, 5x, lOx, 20x or lOOx) inhibits binding of the other antibody, e.g., by at least 50%, 75%, 90%, 95% or 99% as measured in a competitive binding assay.
[0070] Two antibodies may be determined to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies may be determined to bind to overlapping
epitopes if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
II. Production of Antibodies
[0071] Methods for producing antibodies, such as those disclosed herein, are known in the art. For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized using the sequence information provided herein. Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. Production of defined gene constructs is within routine skill in the art.
[0072] Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E.coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions.
[0073] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed secreted protein accumulates in refractile or inclusion bodies, and can be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the proteins refolded and cleaved by methods known in the art.
[0074] If the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon, and, optionally, may contain enhancers, and various introns. This expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be introduced into eukaryotic host cells using conventional techniques. The
host cells express VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a moiety having another function (e.g., cytotoxicity). In some embodiments, a host cell is transfected with a single vector expressing a polypeptide expressing an entire, or part of, a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region). In other embodiments, a host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In still other embodiments, a host cell is co-transfected with more than one expression vector (e.g., one expression vector expressing a polypeptide comprising an entire, or part of, a heavy chain or heavy chain variable region, and another expression vector expressing a polypeptide comprising an entire, or part of a light chain or light chain variable region).
[0075] A polypeptide comprising an immunoglobulin heavy chain variable region or light chain variable region can be produced by growing (culturing) a host cell transfected with an expression vector encoding such variable region, under conditions that permit expression of the polypeptide. Following expression, the polypeptide can be harvested and purified or isolated using techniques well known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) and histidine tags.
[0076] A monoclonal antibody that binds human FGFR2, or an antigen-binding fragment of the antibody, can be produced by growing (culturing) a host cell transfected with: (a) an expression vector that encodes a complete or partial immunoglobulin heavy chain, and a separate expression vector that encodes a complete or partial immunoglobulin light chain; or (b) a single expression vector that encodes both chains (e.g., complete or partial heavy and light chains), under conditions that permit expression of both chains. The intact antibody (or antigen-binding fragment) can be harvested and purified or isolated using techniques well known in the art, e.g., Protein A, Protein G, affinity tags such as glutathione-S-transferase (GST) and histidine tags. It is within ordinary skill in the art to express the heavy chain and the light chain from a single expression vector or from two separate expression vectors.
III. Antibody Modifications
[0077] Human monoclonal antibodies can be isolated or selected from phage display libraries including immune, na'fve and synthetic libraries. Antibody phage display libraries are known in the art, see, e.g., Hoet et al, NATURE BIOTECH. 23 :344-348, 2005; Soderlind et al, NATURE BIOTECH. 18:852-856, 2000; Rothe ei a/., J. MOL. BIOL. 376: 1 182-1200, 2008;
Knappik et al, J. MOL. BIOL. 296:57-86, 2000; and Krebs et al, J. IMMUNOL. METH. 254:67-84, 2001. When used as a therapeutic, human antibodies isolated by phage display may be optimized (e.g., affinity-matured) to improve biochemical characteristics including affinity and/or specificity, improve biophysical properties including aggregation, stability, precipitation and/or non-specific interactions, and/or to reduce immunogenicity. Affinity-maturation procedures are within ordinary skill in the art. For example, diversity can be introduced into an immunoglobulin heavy chain and/or an immunoglobulin light chain by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis and/or site-specific mutagenesis.
[0078] In some embodiments, isolated human antibodies contain one or more somatic mutations in a framework region. In these cases, framework regions can be modified to a human germline sequence to optimize the antibody (i.e., a process referred to as germlining).
[0079] Generally, an optimized antibody has at least the same, or substantially the same, affinity for the antigen as the non-optimized (or parental) antibody from which it was derived. Preferably, an optimized antibody has a higher affinity for the antigen when compared to the parental antibody.
[0080] Human antibody fragments (e.g., parental and optimized variants) can be engineered to contain certain constant (i.e., Fc) regions with a specified effector function (e.g., antibody- dependent cellular cytotoxicity (ADCC)). Human constant regions are known in the art.
[0081] If the antibody is for use as a therapeutic, it can be conjugated to an effector agent such as a small molecule toxin or a radionuclide using standard in vitro conjugation
chemistries. If the effector agent is a polypeptide, the antibody can be chemically conjugated to the effector or joined to the effector as a fusion protein. Construction of fusion proteins is within ordinary skill in the art.
IV. Use of Antibodies
[0082] The antibodies disclosed herein can be used to treat various forms of cancer, e.g., gastric cancer, breast cancer, lung cancer, pancreatic cancer, endometrial cancer, and ovarian cancer. The cancer cells are exposed to a therapeutically effective amount of the antibody so as to inhibit or reduce proliferation of the cancer cell. In some embodiments, the antibodies inhibit cancer cell proliferation by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%.
[0083] In some embodiments, the antibody 15492, 15492.6, 15492.50, 15492.61, 15492.65, or 15492.77 is used in therapy. For example, the antibody 15492, 15492.6, 15492.50,
15492.61, 15492.65, or 15492.77 can be used to inhibit tumor growth in a mammal (e.g., a human patient). In some embodiments, use of the antibody to inhibit tumor growth in a mammal comprises administering to the mammal a therapeutically effective amount of the antibody. In other embodiments, antibody 15492, 15492.6, 15492.50, 15492.61, 15492.65, or 15492.77 can be used for inhibiting or reducing proliferation of a tumor cell.
[0084] As used herein, "treat," "treating," and "treatment" mean the treatment of a disease in a mammal, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state.
[0085] Generally, a therapeutically effective amount of active component is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 10 mg/kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level. Alternatively, the initial dosage can be smaller than the optimum, and the dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the antibody, and the disease being treated. Exemplary dosing frequencies are once per day, once per week, and once every two weeks. In some embodiments, dosing is once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. Formulation of
monoclonal antibody-based drugs is within ordinary skill in the art. In some embodiments, the antibody is lyophilized, and then reconstituted in buffered saline, at the time of administration.
[0086] For therapeutic use, an antibody preferably is combined with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier(s) should be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
[0087] Pharmaceutical compositions containing antibodies, such as those disclosed herein, can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration. A preferred route of administration for monoclonal antibodies is IV infusion. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's
Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
[0088] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
[0089] Pharmaceutical formulations preferably are sterile. Sterilization can be
accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
EXAMPLES
[0090] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.
Example 1: Anti-FGFR2 Antibodies
[0091] This Example describes the production of anti-FGFR2 monoclonal antibodies.
A. Isolation of Anti-FGFR2-Antibodies by Phage Display
[0092] Anti-FGFR2 antibodies were isolated from a phage display library constructed from human heavy chain and light chain variable region genes. More specifically, the anti-FGFR2 antibodies were isolated using a phagemid antibody library constructed from human B-cell donor light chain variable and constant genes paired with a single human heavy chain framework consisting of human B-cell donor heavy chain variable region CDR3 genes and a diversity of synthetic heavy chain variable region CDRi and CDR2 sequences (Dyax
Corporation, Burlington, MA). The anti-FGFR2 antibody 15492 was isolated from four rounds of phage display selection, with the target alternating between purified recombinant human proteins (100 pmol of biotinylated-recombinant human FGFR2 -IIIb (rhFGFR2 -IIIb) and 100 pmol biotinylated-rhFGFR2 -IIIc (round one antigens) and 100 pmol biotinylated-rhFGFR2 - IIIc (round two antigen)) and engineered Ba/F3 cell lines exogenously expressing human FGFR2 -IIIb or FGFR2 -IIIc (lxlO8 Ba/F3 expressing human FGFR2 -IIIb (round three antigen) and lxlO8 Ba/F3 expressing human FGFR2 -IIIc (round four antigen)).
[0093] Each round of selection was followed by infection of TGI E. coli (Agilent
Technologies, Santa Clara, CA) rescue with M13K07 helper phage (Life Technologies, Grand Island, NY), and phage amplification to enrich the input for each subsequent round. Colonies from round four were screened by ELISA for binding to both rhFGFR2 -IIIb and rhFGFR2 - IIIc. All positive clones were sequenced. Clones with unique sequences were expressed as to
Fabs, Protein A purified, and screened for potency as measured by inhibition in a human FGF 1 - dependent FGFR2-IIIb or FGFR2-IIIc cell-based proliferation assay. FDCP-1 cells expressing FGFR2-IIIb or FGFR2-IIIc were seeded in a 96-well plate in the absence of IL3. Purified Fabs were added to the FDC-P lcells expressing human FGFR2-IIIb or FGFR2-IIIc in the presence of FGF 1 and heparin, and MTT assays were carried out after three days.
B. Affinity-Maturation and Optimization of Anti-FGFR2 Antibodies
[0094] Several optimization methods, including affinity maturation, were used to derive variants of 15492 (i.e., antibodies 15492.6, 15492.50, 15492.61, 15492.65 and 15492.77). In one approach, light chain variable region or heavy chain CDRi and CDR2 variable region shuffling and/or random mutagenesis of the light chain variable region or heavy chain CDRi and CDR2 variable region was used to construct sub-libraries cloned into the phage display vectors. These particular chain shuffling and random mutagenesis methods replaced the light chain variable or heavy chain CDRi and CDR2 variable regions with either the original phage library repertoire or a diverse repertoire of sequences containing approximately one to six random mutations per sequence, respectively, while maintaining the 15492 sequence in the non-shuffled or non-mutated regions. Mutations were introduced using GeneMorph® II Random Mutagenesis kit (Agilent Technologies). The antibody chains corresponding to the 15492 derivatives were isolated from these sub-libraries by three rounds of phage selection with decreasing concentrations (50 pmol to 0.1 fmol) of recombinant human proteins, biotinylated-rhFGFR2 -IIIb and biotinylated-rhFGFR2 -IIIc for each round, while competing for target binding with excess amounts of non-biotinylated rhFGFR2 -IIIb and rhFGFR2 -IIIc at 37°C in later rounds. Derivatives of 15492 isolated from the phage display sub-libraries were ranked by IC50 using a competition ELISA. Phage supernatant from unique colonies was incubated with a 10 point dose curve of biotinylated-rhFGFR2 -IIIb (0.05 nM to 1000 nM) and then allowed to bind to a plate coated with biotinylated-rhFGFR2 -IIIb (1.0 μg/ml).
[0095] In another approach (non-phage display), random mutagenesis generated favorable mutations in the light chain CDR3 region of 15492. Additionally for lead variants, non-human framework light chain residues were changed to human germline sequences. Another approach incorporated heavy chain CDRi or CDR2 consensus mutations, which were favorable in shuffle derivatives from heavy chain sub-libraries.
[0096] Top derivatives of all strategies were ranked by equilibrium dissociation constant (KD) and dissociation rate constant (ka) as measured by Biacore T100 instrument (GE
Healthcare, Piscataway, NJ), as described below.
C. Sequences of Anti-FGFR2 Antibodies
[0097] The human heavy chains were subcloned into pEE6.4 (Lonza, Basel, Switzerland) via Hindlll and EcoRI sites using In-Fusion™ PCR cloning (Clontech, Mountain View, CA). The human kappa light chains were subcloned into pEE14.4 (Lonza) via Hindlll and EcoRI sites using In-Fusion™ PCR cloning.
[0098] Vectors encoding human antibody chains were transiently transfected into 293T cells to produce antibody. Antibody was either purified or used in cell culture media supernatant for subsequent in vitro analysis. Binding of the human antibodies to human FGFR2 was measured as described below. The results are summarized in Tables 8, 9, 10, and 1 1.
[0099] Some human antibody heavy and light chain combinations were stably expressed in CHOK1SV cells using the GS System™ (Lonza) in order to produce large quantities of purified human antibody. A single expression vector was constructed by combining pEE6.4 and pEE14.4 based vectors. First, pEE6.4 containing full length human heavy chain cDNA was digested with NotI and Sail to isolate the hCMV-MIE promoter + full length human heavy chain cDNA + SV40 poly A fragment. This fragment was inserted into the pEE14.4 vector already containing full length human light chain cDNA via Notl/Sall sites, thus creating an expression vector that simultaneously expresses heavy and light chains. The combined heavy and light chain vector was linearized and transfected into CHOK1 SV cells. Stable clones were selected in the presence of methionine sulfoximine.
[00100] The nucleic acid sequences encoding and the protein sequences defining variable regions of the human 15492, 15492.6, 15492.50, 15492.61, 15492.65, and 15492.77 antibodies are summarized below (amino terminal signal peptide sequences are not shown). CDR sequences (Kabat definition) are shown in bold and are underlined in the amino acid sequences.
[00101] Nucleic Acid Sequence Encoding the 15492Hv Heavy Chain Variable Region (SEQ ID NO: 15)
1 gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 61 tcttgcgctg cttccggatt cactttctct tggtactgga tggagtgggt tcgccaagct
121 cctggtaaag gtttggagtg ggtttcttat atcggtcctt ctggtggcgt tactcagtat
181 gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
241 ttgcagatga acagcttaag ggctgaggac acggccgtgt attactgtgc gagagatgga
301 gcagcagcct ttgactactg gggccaggga accctggtca ccgtctcaag c
[00102] Protein Sequence Defining the 15492Hv Heavy Chain Variable Region (SEQ ID NO: 14)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYWMEWVRQA PGKGLEWVSY IGPSGGVTQY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG AAAFDYWGQG TLVTVSS
[00103] Nucleic Acid Sequence Encoding the 15492Hv optCHQ2 Heavy Chain Variable Region (SEQ ID NO: 17)
1 gaggtacagc tccttgagtc aggcggaggt ttggtccaac cgggaggttc gctgcggctt
61 agctgtgcgg cgtcagggtt caccttctcc tggtactgga tggaatgggt acgacaggct
121 cccggaaaag gacttgaatg ggtgtcatac atcgggccat ccgggggagt cacgcaatac
181 gccgactccg tgaaggggag attcacgatc tcaagggaca actcgaagaa taccctctat 241 cttcagatga actcccttcg agcggaagat acagcggtgt attactgtgc ccgcgatggt
301 gctgccgcgt ttgactattg gggacagggg acgcttgtaa cagtgtcatc a
[00104] Protein Sequence Defining the 15492Hv optCHQ2 Heavy Chain Variable Region (SEQ ID NO: 14)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYWMEWVRQA PGKGLEWVSY IGPSGGVTQY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG AAAFDYWGQG TLVTVSS
[00105] Nucleic Acid Sequence Encoding the 15492Hv Y32F optCHQ2 Heavy Chain Variable Region (SEQ ID NO : 19)
1 gaggtacagc tccttgagtc aggcggaggt ttggtccaac cgggaggttc gctgcggctt 61 agctgtgcgg cgtcagggtt caccttctcc tggttctgga tggaatgggt acgacaggct
121 cccggaaaag gacttgaatg ggtgtcatac atcgggccat ccgggggagt cacgcaatac
181 gccgactccg tgaaggggag attcacgatc tcaagggaca actcgaagaa taccctctat
241 cttcagatga actcccttcg agcggaagat acagcggtgt attactgtgc ccgcgatggt
301 gctgccgcgt ttgactattg gggacagggg acgcttgtaa cagtgtcatc a
[00106] Protein Sequence Defining the 15492Hv Y32F optCHQ2 Heavy Chain Variable Region (SEQ ID NO: 16)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WFWMEWVRQA PGKGLEWVSY IGPSGGVTQY 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG AAAFDYWGQG TLVTVSS
[00107] Nucleic Acid Sequence Encoding the 15492Hv Y32N E35Q G52S V56Y 058V optCHQ2 Heavy Chain Variable Region (SEQ ID NO:21)
1 gaggtacagc tccttgagtc aggcggaggt ttggtccaac cgggaggttc gctgcggctt
61 agctgtgcgg cgtcagggtt caccttctcc tggaactgga tgcagtgggt acgacaggct
121 cccggaaaag gacttgaatg ggtgtcatac atctcgccat ccgggggata cacggtgtac
181 gccgactccg tgaaggggag attcacgatc tcaagggaca actcgaagaa taccctctat 241 cttcagatga actcccttcg agcggaagat acagcggtgt attactgtgc ccgcgatggt
301 gctgccgcgt ttgactattg gggacagggg acgcttgtaa cagtgtcatc a
[00108] Protein Sequence Defining the 15492Hv Y32N E35Q G52S V56Y 058V optCHQ2 Heavy Chain Variable Region (SEQ ID NO: 18)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WNWMQWVRQA PGKGLEWVSY ISPSGGYTVY 61 ADSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCARDG AAAFDYWGQG TLVTVSS
[00109] Nucleic Acid Sequence Encoding the 15492Kv Kappa Chain Variable Region (SEQ ID NO:23)
1 gacatccaga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc
61 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttaact 121 tggtaccagc agaaagcagg acagcctcct aagttgctca tttcgtgggc atctattcgg
181 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc
241 atcagcagcc tgcaggctga agatgtggca ctttattact gtcagcaata ttatgctatt
301 ccgtacactt ttggccaggg gaccaggctg gagatcaaa
[00110] Protein Sequence Defining the 15492Kv Kappa Chain Variable Region (SEQ ID NO:20)
1 DIQMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLT WYQQKAGQPP KLLISWASIR 61 ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA LYYCQQYYAI PYTFGQGTRL EIK
[00111] Nucleic Acid Sequence Encoding the 15492Kv Q3V A40P L85V R103K Kappa Chain Variable Region (SEQ ID NO:25)
1 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc
61 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttaact
121 tggtaccagc agaaacccgg acagcctcct aagttgctca tttcgtgggc atctattcgg
181 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc
241 atcagcagcc tgcaggctga agatgtggca gtctattact gtcagcaata ttatgctatt 301 ccgtacactt ttggccaggg gaccaagctg gagatcaaa
[00112] Protein Sequence Defining the 15492Kv Q3V A40P L85V R103K Kappa Chain Variable Region (SEQ ID NO:22)
1 DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLT WYQQKPGQPP KLLISWASIR 61 ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYAI PYTFGQGTKL EIK
[00113] Nucleic Acid Sequence Encoding the 15492.6Kv Q89L Y92H optCHQ2 Kappa Chain Variable Region (SEQ ID NO:27)
1 gacattgtga tgacgcagtc accggattca ctggcagtgt cgctgggaga aagagcgacc
61 atcaattgca agagcagcca atcagtgttg tactcatcga acaataagaa ctatctcaca
121 tggtatcaac agaaacccgg tcagcctccg aaattgctta tttcgtgggc gtccatccgc 181 gagtcaggcg tcccagacag gttttcgggg tcgggttcgg ggacagattt cactttgaca
241 atcagctcac tgcaggcgga ggacgtggcg gtctactact gcttgcaata tcacgcaatt
301 ccctatactt ttgggcaggg aactaagttg gaaatcaag
[00114] Protein Sequence Defining the 15492.6Kv Q89L Y92H optCHQ2 Kappa Chain
Variable Region (SEQ ID NO:24)
1 DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLT WYQQKPGQPP KLLISWASIR 61 ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCLQYHAI PYTFGQGTKL EIK
[00115] Nucleic Acid Sequence Encoding the 31288Kv Q3V I58V D81E optCHQ2 Kappa Chain Variable Region (SEQ ID NO:29)
1 gacattgtga tgacgcagtc accggattca ctggcagtgt cgctgggaga aagagcgacc
61 atcaattgca agagcagcca atcagtgttg tactcatcga acaataagaa cttcctcgcg
121 tggtatcaac agaaacccgg tcagcctccg aaattgctta tttcgtgggc gtccacgcgc 181 gagtcaggcg tcccagacag gttttcgggg tcgggttcgg ggacagattt cactttgaca
241 atcagctcac tgcaggcgga ggacgtggcg gtctactact gccagcaatt cttttccacg
301 cccttcactt ttgggccggg aactaaggtg gatatcaag
Protein Sequence Defining the 31288Kv Q3V I58V D81E optCHQ2 Kappa Chain Variable Region (SEQ ID NO:26)
1 DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNFLA WYQQKPGQPP KLLISWASTR
61 ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQFFST PFTFGPGTKV DIK
[00116] The amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies produced in Example 1 are aligned in FIG. 2. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDRi, CDR2, and CDR3 (Kabat definition) are identified by boxes. FIG. 3 shows an alignment of the separate CDRi, CDR2, and CDR3 sequences for each of the variable region sequences shown in FIG. 2.
[00117] The amino acid sequences defining the immunoglobulin light chain variable regions for the antibodies in Example 1 are aligned in FIG. 4. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDRi, CDR2 and CDR3 are identified by boxes. FIG. 5 shows an alignment of the separate CDRi, CDR2, and CDR3 sequences for each of the variable region sequences shown in FIG. 4.
[00118] Table 1 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.
Table 1
S l .O. I I) NO. Nucleic Acid or IVolcin
15 15492 Hv Heavy Chain Variable Region— nucleic acid
14 15492 Hv Heavy Chain Variable Region— protein
1 15492 Hv Heavy Chain CDRi
4 15492 Hv Heavy Chain CDR2
6 15492 Hv Heavy Chain CDR3
17 15492 Hv optCH02 Heavy Chain Variable Region— nucleic acid
14 15492 Hv optCH02 Heavy Chain Variable Region— protein
1 15492 Hv optCH02 Heavy Chain CDRi
4 15492 Hv optCH02 Heavy Chain CDR2
6 15492 Hv optCH02 Heavy Chain CDR3
19 15492 Hv Y32F optCH02 Heavy Chain Variable Region— nuclei c acid
16 15492 Hv Y32F optCH02 Heavy Chain Variable Region— protei n
2 15492 Hv Y32F optCH02 Heavy Chain CDRi
4 15492 Hv Y32F optCH02 Heavy Chain CDR2
6 15492 Hv Y32F optCH02 Heavy Chain CDR3
21 15492 Hv Y32N E35Q G52S V56Y Q58V optCH02 Heavy
Chain Variable Region— nucleic acid
18 15492 Hv Y32N E35Q G52S V56Y Q58V optCH02 Heavy
Chain Variable Region— protein
3 15492 Hv Y32N E35Q G52S V56Y Q58V optCH02 Heavy
Chain CDRi
5 15492 Hv Y32N E35Q G52S V56Y Q58V optCH02 Heavy
Chain CDR2
6 15492 Hv Y32N E35Q G52S V56Y Q58V optCH02 Heavy
Chain CDR3
23 15492 Kv Light (kappa) Chain Variable Region— nucleic acid
20 15492 Kv Light (kappa) Chain Variable Region— protein
7 15492 Kv Light (kappa) Chain CDRi
9 15492 Kv Light (kappa) Chain CDR2
1 1 15492 Kv Light (kappa) Chain CDR3
25 15492 Kv Q3V A40P L85V R103K Light (kappa) Chain
Varia ?le Region— nucleic acid
22 15492 Kv Q3V A40P L85V R103K Light (kappa) Chain
Varia ?le Region— protein
7 15492 Kv Q3V A40P L85V R103K Light (kappa) Chain CDRi
9 15492 Kv Q3V A40P L85V R103K Light (kappa) Chain CDR2
1 1 15492 Kv Q3V A40P L85V R103K Light (kappa) Chain CDR3
27 15492 .6Kv Q89L Y92H optCH02 Light (kappa) Chain
Varia ?le Region— nucleic acid
Table 1 continued
S EQ. I I) NO. Nucleic Acid or IVolcin
24 15492. 6Kv Q89L Y92H optCH02 Light (kappa) Chain
Variab e Region— protein
7 15492. 6Kv Q89L Y92H optCH02 Light (kappa) Chain CDRi
9 15492. 6Kv Q89L Y92H optCH02 Light (kappa) Chain CDR2
12 15492. 6Kv Q89L Y92H optCH02 Light (kappa) Chain CDR3
29 312881 v Q3V 158V D81E optCH02 Light (kappa) Chain
Variab e Region— nucleic acid
26 312881 v Q3V 158V D81E optCH02 Light (kappa) Chain
Variab e Region— protein
8 312881 v Q3V 158V D81E optCH02 Light (kappa) Chain
CDRi
10 312881 v Q3V 158V D81E optCH02 Light (kappa) Chain
CDR2
13 312881 v Q3V 158V D81E optCH02 Light (kappa) Chain
CDR3
[00119] Human monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 2.
Table 2
[00120] Human monoclonal antibody Kappa light chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 3.
Table 3
Kabul ( ho lk m
CDIil CDR2 CDK3
15492Kv KSSQSVLYSSNNKNYLT WAS IRES QQYYAIPYT
(SEQ ID NO: 7) (SEQ ID NO:9) (SEQ ID NO: 11)
15492Kv Q3V
A40PL85V KSSQSVLYSSNNKNYLT WAS IRES QQYYAIPYT R103K (SEQIDNO:7) (SEQIDNO:9) (SEQ ID NO: 11)
15492.6Kv Q89L KSSQSVLYSSNNKNYLT WAS IRES LQYHAIPYT Y92H optCH02 (SEQ ID NO : 7) (SEQ ID NO:9) (SEQ ID NO: 12)
31288KvQ3V KSSQSVLYSSNNKNFLA WASTRES QQFFSTPFT I58VD81E (SEQIDNO:8) (SEQ ID NO: 10) (SEQ ID NO: 13) optCH02
15492Kv QS VLYS SNNKNY WAS QQYYAIPYT
(SEQ ID NO: 59) (SEQ ID NO: 11)
15492Kv Q3V WAS
A40PL85V QS VLYS SNNKNY QQYYAIPYT R103K (SEQIDNO:59) (SEQ ID NO: 11)
15492.6Kv Q89L QS VLYS SNNKNY WAS LQYHAIPYT Y92H optCH02 (SEQ ID NO : 59) (SEQ ID NO: 12)
31288KvQ3V QS VLYS SNNKNF WAS QQFFSTPFT I58VD81E (SEQIDNO:60) (SEQ ID NO: 13) optCH02
[00121] To create the complete human heavy or kappa chain antibody sequences, each variable sequence above is combined with its respective human constant region. For example, a complete heavy chain comprises a heavy variable sequence followed by a human IgGl heavy chain constant sequence. A complete kappa chain comprises a kappa variable sequence followed by the human kappa light chain constant sequence.
[00122] Nucleic Acid Sequence Encoding the Human IgGl Heavy Chain Constant Region (SEQ ID O:31)
1 gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 61 ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 121 tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 181 ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 241 tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 301 aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 361 cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 421 gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 481 tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 541 agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 601 gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 661 aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 721 atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 781 gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 841 ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 901 cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 961 cagaagtcac tgagcctgag cccagggaag
[00123] Protein Sequence Defining the Human IgGl Heavy Chain Constant Region (SEQ
ID NO:28)
1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS
61 GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG
121 PSVFLFPPKP KDTLMISRTP EVTCWVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN
181 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE
241 MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW
301 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
[00124] Nucleic Acid Sequence Encoding the Human Kappa Light Chain Constant Region (SEQ ID NO:33)
1 cgcacagtcg ccgctccctc cgtgttcatc tttccaccaa gtgatgagca actgaagtct
61 ggtactgctt cagtcgtgtg tctgctgaac aatttctacc ctcgagaagc caaagtccaa
121 tggaaggtag acaacgcact gcagtccggc aatagccaag aatcagttac cgaacaggat
181 tcaaaggaca gtacatattc cctgagcagc actctgaccc tgtcaaaggc cgattacgag
241 aaacacaagg tctatgcttg cgaagtgaca catcagggac tgtccagccc agtgacaaaa 301 tcttttaacc gtggggagtg t
[00125] Protein Sequence Defining the Human Kappa Light Chain Constant Region (SEP ID NO:30)
1 RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD 61 SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC
[00126] The following sequences represent the actual or contemplated full length heavy and light chain sequence (i.e., containing both the variable and constant regions sequences) for each antibody described in this Example. Signal sequences for proper secretion of the antibodies (e.g., signal sequences at the 5' end of the DNA sequences or the amino terminal end of the protein sequences) are not shown in the full length heavy and light chain sequences disclosed herein, and are not included in the final secreted protein. Also not shown are stop codons for termination of translation required at the 3 ' end of the DNA sequences. It is within ordinary skill in the art to select a signal sequence and a stop codon for expression of the disclosed full length IgG heavy chain and light chain sequences. The variable region sequences can be ligated to other constant region sequences to produce active full length IgG heavy and light chains.
[00127] Nucleic Acid Sequence Encoding the Full Length 15492Hv Heavy Chain (Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 35)
1 gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
61 tcttgcgctg cttccggatt cactttctct tggtactgga tggagtgggt tcgccaagct 121 cctggtaaag gtttggagtg ggtttcttat atcggtcctt ctggtggcgt tactcagtat 181 gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 241 ttgcagatga acagcttaag ggctgaggac acggccgtgt attactgtgc gagagatgga 301 gcagcagcct ttgactactg gggccaggga accctggtca ccgtctcaag cgcctcaaca 361 aaaggaccaa gtgtgttccc actcgcccct agcagcaaga gtacatccgg gggcactgca 421 gcactcggct gcctcgtcaa ggattatttt ccagagccag taaccgtgag ctggaacagt 481 ggagcactca cttctggtgt ccatactttt cctgctgtcc tgcaaagctc tggcctgtac 541 tcactcagct ccgtcgtgac cgtgccatct tcatctctgg gcactcagac ctacatctgt 601 aatgtaaacc acaagcctag caatactaag gtcgataagc gggtggaacc caagagctgc 661 gacaagactc acacttgtcc cccatgccct gcccctgaac ttctgggcgg tcccagcgtc 721 tttttgttcc caccaaagcc taaagatact ctgatgataa gtagaacacc cgaggtgaca 781 tgtgttgttg tagacgtttc ccacgaggac ccagaggtta agttcaactg gtacgttgat 841 ggagtcgaag tacataatgc taagaccaag cctagagagg agcagtataa tagtacatac 901 cgtgtagtca gtgttctcac agtgctgcac caagactggc tcaacggcaa agaatacaaa 961 tgcaaagtgt ccaacaaagc actcccagcc cctatcgaga agactattag taaggcaaag 1021 gggcagcctc gtgaaccaca ggtgtacact ctgccaccca gtagagagga aatgacaaag 1081 aaccaagtct cattgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgttgag 1141 tgggagagta acggtcagcc tgagaacaat tacaagacaa cccccccagt gctggatagt 1201 gacgggtctt tctttctgta cagtaagctg actgtggaca agtcccgctg gcagcagggt 1261 aacgtcttca gctgttccgt gatgcacgag gcattgcaca accactacac ccagaagtca 1321 ctgagcctga gcccagggaa g
[00128] Protein Sequence Defining the Full Length 15492Hv Heavy Chain (Heavy Chain
Variable Region and Human IgGl Constant Region) (SEQ ID NO: 32)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYWMEWVRQA PGKGLEWVSY IGPSGGVTQY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG AAAFDYWGQG TLVTVSSAST
121 KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY
181 SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV
241 FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
301 RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK
361 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
421 NVFSCSVMHE ALHNHYTQKS LSLSPGK
[00129] Nucleic Acid Sequence Encoding the Full Length 15492Hv optCHQ2 Heavy Chain
(Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 37)
1 gaggtacagc tccttgagtc aggcggaggt ttggtccaac cgggaggttc gctgcggctt 61 agctgtgcgg cgtcagggtt caccttctcc tggtactgga tggaatgggt acgacaggct 121 cccggaaaag gacttgaatg ggtgtcatac atcgggccat ccgggggagt cacgcaatac 181 gccgactccg tgaaggggag attcacgatc tcaagggaca actcgaagaa taccctctat 241 cttcagatga actcccttcg agcggaagat acagcggtgt attactgtgc ccgcgatggt 301 gctgccgcgt ttgactattg gggacagggg acgcttgtaa cagtgtcatc agcgtcaaca 361 aaaggaccgt cggtattccc cctggctcct tcctcgaaga gcaccagcgg tggcacggct 421 gcgctgggat gtttggtgaa agattacttc ccggaacccg tgacggtgtc atggaactcg 481 ggggcattga cgtcaggggt gcatacgttt ccggccgtgc tgcagtccag cggactctac 541 tcgctttcat ccgtggtaac tgtgccttcc tcctcattgg gaactcagac atacatctgc 601 aatgtcaacc acaagccctc caacacgaaa gtcgataaac gggtggagcc aaagagctgc 661 gataaaacac acacctgccc gccatgtccg gctcccgagt tgttgggcgg tccctccgtg 721 tttttgttcc ctcccaagcc caaagacacc cttatgatta gcaggacacc ggaggtgacg 781 tgcgtggtgg tagacgtcag ccatgaagat cccgaggtca agtttaactg gtatgtggac 841 ggagtcgaag tacataacgc caaaactaaa cccagagagg agcagtataa ctcgacctac 901 cgcgtggtat cagtcttgac agtgcttcac caggactggc tcaatgggaa ggagtacaag 961 tgcaaggtat ccaataaggc actccccgca ccgattgaaa agaccatctc gaaagcgaag 1021 ggtcagccca gggagccgca agtatatacg ctcccgccgt cccgcgagga aatgaccaag 1081 aatcaggtgt cgctcacatg cctcgtgaaa ggattctacc cctcggatat tgcagtggaa 1141 tgggagtcga acgggcagcc cgagaataac tataagacga caccgcctgt gctggactcc 1201 gacggttcgt ttttcctcta tagcaaattg acggtggata agtcaagatg gcaacaagga 1261 aatgtctttt cgtgctcggt catgcacgag gccctgcata atcactatac gcagaagtcc 1321 ctgagcttgt cgccgggaaa g
[00130] Protein Sequence Defining the Full Length 15492Hv optCHQ2 Heavy Chain (Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 32)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYWMEWVRQA PGKGLEWVSY IGPSGGVTQY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG AAAFDYWGQG TLVTVSSAST
121 KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY
181 SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV
241 FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
301 RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK
361 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
421 NVFSCSVMHE ALHNHYTQKS LSLSPGK
[00131] Nucleic Acid Sequence Encoding the Full Length 15492Hv Y32F optCH02 Heavy
Chain (Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 39)
1 gaggtacagc tccttgagtc aggcggaggt ttggtccaac cgggaggttc gctgcggctt
61 agctgtgcgg cgtcagggtt caccttctcc tggttctgga tggaatgggt acgacaggct
121 cccggaaaag gacttgaatg ggtgtcatac atcgggccat ccgggggagt cacgcaatac
181 gccgactccg tgaaggggag attcacgatc tcaagggaca actcgaagaa taccctctat
241 cttcagatga actcccttcg agcggaagat acagcggtgt attactgtgc ccgcgatggt
301 gctgccgcgt ttgactattg gggacagggg acgcttgtaa cagtgtcatc agcgtcaaca
361 aaaggaccgt cggtattccc cctggctcct tcctcgaaga gcaccagcgg tggcacggct
421 gcgctgggat gtttggtgaa agattacttc ccggaacccg tgacggtgtc atggaactcg
481 ggggcattga cgtcaggggt gcatacgttt ccggccgtgc tgcagtccag cggactctac
541 tcgctttcat ccgtggtaac tgtgccttcc tcctcattgg gaactcagac atacatctgc
601 aatgtcaacc acaagccctc caacacgaaa gtcgataaac gggtggagcc aaagagctgc
661 gataaaacac acacctgccc gccatgtccg gctcccgagt tgttgggcgg tccctccgtg
721 tttttgttcc ctcccaagcc caaagacacc cttatgatta gcaggacacc ggaggtgacg
781 tgcgtggtgg tagacgtcag ccatgaagat cccgaggtca agtttaactg gtatgtggac
841 ggagtcgaag tacataacgc caaaactaaa cccagagagg agcagtataa ctcgacctac
901 cgcgtggtat cagtcttgac agtgcttcac caggactggc tcaatgggaa ggagtacaag
961 tgcaaggtat ccaataaggc actccccgca ccgattgaaa agaccatctc gaaagcgaag
1021 ggtcagccca gggagccgca agtatatacg ctcccgccgt cccgcgagga aatgaccaag
1081 aatcaggtgt cgctcacatg cctcgtgaaa ggattctacc cctcggatat tgcagtggaa
1141 tgggagtcga acgggcagcc cgagaataac tataagacga caccgcctgt gctggactcc
1201 gacggttcgt ttttcctcta tagcaaattg acggtggata agtcaagatg gcaacaagga
1261 aatgtctttt cgtgctcggt catgcacgag gccctgcata atcactatac gcagaagtcc 1321 ctgagcttgt cgccgggaaa g
[00132] Protein Sequence Defining the Full Length 15492Hv Y32F optCHQ2 Heavy Chain (Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 34)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WFWMEWVRQA PGKGLEWVSY IGPSGGVTQY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG AAAFDYWGQG TLVTVSSAST
121 KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY
181 SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV
241 FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
301 RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK
361 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG 421 NVFSCSVMHE ALHNHYTQKS LSLSPGK
[00134] Nucleic Acid Sequence Encoding the Full Length 15492Hv Y32N E35Q G52S V56Y 058V optCHQ2 Heavy Chain (Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO:41)
1 gaggtacagc tccttgagtc aggcggaggt ttggtccaac cgggaggttc gctgcggctt
61 agctgtgcgg cgtcagggtt caccttctcc tggaactgga tgcagtgggt acgacaggct
121 cccggaaaag gacttgaatg ggtgtcatac atctcgccat ccgggggata cacggtgtac
181 gccgactccg tgaaggggag attcacgatc tcaagggaca actcgaagaa taccctctat
241 cttcagatga actcccttcg agcggaagat acagcggtgt attactgtgc ccgcgatggt
301 gctgccgcgt ttgactattg gggacagggg acgcttgtaa cagtgtcatc agcgtcaaca
361 aaaggaccgt cggtattccc cctggctcct tcctcgaaga gcaccagcgg tggcacggct
421 gcgctgggat gtttggtgaa agattacttc ccggaacccg tgacggtgtc atggaactcg
481 ggggcattga cgtcaggggt gcatacgttt ccggccgtgc tgcagtccag cggactctac
541 tcgctttcat ccgtggtaac tgtgccttcc tcctcattgg gaactcagac atacatctgc
601 aatgtcaacc acaagccctc caacacgaaa gtcgataaac gggtggagcc aaagagctgc
661 gataaaacac acacctgccc gccatgtccg gctcccgagt tgttgggcgg tccctccgtg 721 tttttgttcc ctcccaagcc caaagacacc cttatgatta gcaggacacc ggaggtgacg 781 tgcgtggtgg tagacgtcag ccatgaagat cccgaggtca agtttaactg gtatgtggac 841 ggagtcgaag tacataacgc caaaactaaa cccagagagg agcagtataa ctcgacctac 901 cgcgtggtat cagtcttgac agtgcttcac caggactggc tcaatgggaa ggagtacaag 961 tgcaaggtat ccaataaggc actccccgca ccgattgaaa agaccatctc gaaagcgaag 1021 ggtcagccca gggagccgca agtatatacg ctcccgccgt cccgcgagga aatgaccaag 1081 aatcaggtgt cgctcacatg cctcgtgaaa ggattctacc cctcggatat tgcagtggaa 1141 tgggagtcga acgggcagcc cgagaataac tataagacga caccgcctgt gctggactcc 1201 gacggttcgt ttttcctcta tagcaaattg acggtggata agtcaagatg gcaacaagga 1261 aatgtctttt cgtgctcggt catgcacgag gccctgcata atcactatac gcagaagtcc 1321 ctgagcttgt cgccgggaaa g
[00135] Protein Sequence Defining the Full Length 15492Hv Y32N E35Q G52S V56Y Q58V optCHQ2 Heavy Chain (Heavy Chain Variable Region and Human IgGl Constant Region) (SEP ID NO: 36)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WNWMQWVRQA PGKGLEWVSY ISPSGGYTVY
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG AAAFDYWGQG TLVTVSSAST
121 KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY
181 SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV
241 FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
301 RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK
361 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
421 NVFSCSVMHE ALHNHYTQKS LSLSPGK
[00136] Nucleic Acid Sequence Encoding the Full Length 15492Kv Light Chain (Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO:43)
1 gacatccaga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 61 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttaact 121 tggtaccagc agaaagcagg acagcctcct aagttgctca tttcgtgggc atctattcgg 181 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 241 atcagcagcc tgcaggctga agatgtggca ctttattact gtcagcaata ttatgctatt 301 ccgtacactt ttggccaggg gaccaggctg gagatcaaac gcacagtcgc cgctccctcc 361 gtgttcatct ttccaccaag tgatgagcaa ctgaagtctg gtactgcttc agtcgtgtgt 421 ctgctgaaca atttctaccc tcgagaagcc aaagtccaat ggaaggtaga caacgcactg 481 cagtccggca atagccaaga atcagttacc gaacaggatt caaaggacag tacatattcc 541 ctgagcagca ctctgaccct gtcaaaggcc gattacgaga aacacaaggt ctatgcttgc 601 gaagtgacac atcagggact gtccagccca gtgacaaaat cttttaaccg tggggagtgt
[00137] Protein Sequence Defining the Full Length 15492Kv Light Chain (Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO: 38)
1 DIQMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLT WYQQKAGQPP KLLISWASIR
61 ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA LYYCQQYYAI PYTFGQGTRL EIKRTVAAPS
121 VFIFPPSDEQ LKSGTASWC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS
181 LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC
[00138] Nucleic Acid Sequence Encoding the Full Length 15492Kv Q3V A40P L85V R103K Light Chain (Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO:45)
1 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc
61 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttaact
121 tggtaccagc agaaacccgg acagcctcct aagttgctca tttcgtgggc atctattcgg
181 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc
241 atcagcagcc tgcaggctga agatgtggca gtctattact gtcagcaata ttatgctatt
301 ccgtacactt ttggccaggg gaccaagctg gagatcaaac gcacagtcgc cgctccctcc
361 gtgttcatct ttccaccaag tgatgagcaa ctgaagtctg gtactgcttc agtcgtgtgt
421 ctgctgaaca atttctaccc tcgagaagcc aaagtccaat ggaaggtaga caacgcactg
481 cagtccggca atagccaaga atcagttacc gaacaggatt caaaggacag tacatattcc
541 ctgagcagca ctctgaccct gtcaaaggcc gattacgaga aacacaaggt ctatgcttgc
601 gaagtgacac atcagggact gtccagccca gtgacaaaat cttttaaccg tggggagtgt
[00139] Protein Sequence Defining the Full Length 15492Kv 03V A40P L85V R103K Light Chain (Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO:40)
1 DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLT WYQQKPGQPP KLLISWASIR
61 ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYAI PYTFGQGTKL EIKRTVAAPS
121 VFIFPPSDEQ LKSGTASWC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS
181 LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC
[00140] Nucleic Acid Sequence Encoding the Full Length 15492.6Kv Q89L Y92H optCHQ2 Light Chain (Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO:47)
1 gacattgtga tgacgcagtc accggattca ctggcagtgt cgctgggaga aagagcgacc 61 atcaattgca agagcagcca atcagtgttg tactcatcga acaataagaa ctatctcaca 121 tggtatcaac agaaacccgg tcagcctccg aaattgctta tttcgtgggc gtccatccgc 181 gagtcaggcg tcccagacag gttttcgggg tcgggttcgg ggacagattt cactttgaca 241 atcagctcac tgcaggcgga ggacgtggcg gtctactact gcttgcaata tcacgcaatt 301 ccctatactt ttgggcaggg aactaagttg gaaatcaagc ggacggtagc cgctccctcc 361 gtgttcatct ttccgccttc ggatgaacag ttgaaaagcg gaaccgcgtc ggtggtctgt 421 cttttgaata acttctaccc acgcgaggcc aaagtccagt ggaaagtaga taacgcgctt 481 cagtcaggga actcgcaaga gtcggtcact gaacaggaca gcaaagactc gacatactcc 541 ctctcctcca cgcttacgct tagcaaggcc gattatgaga agcacaaggt atacgcatgt 601 gaagtaaccc atcagggact ttcgtcccct gtgacgaaga gcttcaatcg gggagagtgc
[00141] Protein Sequence Defining the Full Length 15492.6Kv Q89L Y92H optCHQ2 Light Chain (Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO:42)
1 DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLT WYQQKPGQPP KLLISWASIR
61 ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCLQYHAI PYTFGQGTKL EIKRTVAAPS
121 VFIFPPSDEQ LKSGTASWC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS
181 LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC
[00142] Nucleic Acid Sequence Encoding the Full Length 31288Kv Q3V I58V D81E optCHQ2 Light Chain (Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO:49)
1 gacattgtga tgacgcagtc accggattca ctggcagtgt cgctgggaga aagagcgacc 61 atcaattgca agagcagcca atcagtgttg tactcatcga acaataagaa cttcctcgcg
121 tggtatcaac agaaacccgg tcagcctccg aaattgctta tttcgtgggc gtccacgcgc 181 gagtcaggcg tcccagacag gttttcgggg tcgggttcgg ggacagattt cactttgaca 241 atcagctcac tgcaggcgga ggacgtggcg gtctactact gccagcaatt cttttccacg 301 cccttcactt ttgggccggg aactaaggtg gatatcaagc ggacggtagc cgctccctcc 361 gtgttcatct ttccgccttc ggatgaacag ttgaaaagcg gaaccgcgtc ggtggtctgt 421 cttttgaata acttctaccc acgcgaggcc aaagtccagt ggaaagtaga taacgcgctt 481 cagtcaggga actcgcaaga gtcggtcact gaacaggaca gcaaagactc gacatactcc 541 ctctcctcca cgcttacgct tagcaaggcc gattatgaga agcacaaggt atacgcatgt 601 gaagtaaccc atcagggact ttcgtcccct gtgacgaaga gcttcaatcg gggagagtgc
[00143] Protein Sequence Defining the Full Length 31288Kv Q3V I58V D81E optCHQ2 Light Chain (Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO:44)
1 DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNFLA WYQQKPGQPP KLLISWASTR 61 ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQFFST PFTFGPGTKV DIKRTVAAPS 121 VFIFPPSDEQ LKSGTASWC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS 181 LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC
[00144] Table 4 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.
Table 4
[00145] Each of the possible combinations of the human immunoglobulin heavy chain and immunoglobulin light chain variable regions are set forth below in Table 5. Heavy chain variable regions denoted 15492Hv and 15492Hv optCH02 have identical amino acid sequences, but different nucleic acid sequences. The heavy chain variable region denoted 15492Hv optCH02 is a codon optimized variant of 15492Hv.
Table 5
[00146] Table 6 below shows 15492 antibody variants containing combinations of heavy chain variable regions and light chain variable regions described in this Example.
Table 6
15492 = Human 15492Hv Heavy Chain Variable Region (SEQ ID NO: 14, including CDRHI SEQ ID NO: l, CDRH2 SEQ ID NO:4, and CDRH3 SEQ ID NO:6) plus 15492Kv Light Chain Variable Region (SEQ ID NO:20, including CDRLi SEQ ID NO:7, CDRL2 SEQ ID NO:9, and CDRL3 SEQ ID NO: 11)
15492.6 = Human 15492Hv Heavy Chain Variable Region (SEQ ID NO: 14, including CDRHI SEQ ID NO: l, CDRH2 SEQ ID NO:4, and CDRH3 SEQ ID NO:6) plus 15492Kv Q3V A40P L85V R103K Light Chain Variable Region (SEQ ID NO:22, including CDRLi SEQ ID NO:7, CDRL2 SEQ ID NO:9, and CDRL3 SEQ ID NO: 11)
15492.50 = Human 15492Hv optCH02 Heavy Chain Variable Region (SEQ ID NO: 14, including CDRHI SEQ ID NO: l, CDRH2 SEQ ID NO:4, and CDRH3 SEQ ID NO:6) plus 31288Kv Q3V I58V D81E optCH02 Light Chain Variable Region (SEQ ID NO:26, including CDRLi SEQ ID NO:8, CDRL2 SEQ ID NO: 10, and CDRL3 SEQ ID NO: 13)
15492.61 = Human 15492Hv Y32F optCH02 Heavy Chain Variable Region (SEQ ID NO: 16, including CDRHI SEQ ID NO:2, CDRH2 SEQ ID NO:4, and CDRH3 SEQ ID NO:6) plus 15492.6Kv Q89L Y92H optCH02 Light Chain Variable Region (SEQ ID NO:24, including CDRLi SEQ ID NO:7, CDRL2 SEQ ID NO:9, and CDRL3 SEQ ID NO: 12)
15492.65 = Human 15492Hv Y32N E35Q G52S V56Y Q58V optCH02 Heavy Chain Variable Region (SEQ ID NO: 18, including CDRHI SEQ ID NO:3, CDRH2 SEQ ID NO:5, and CDRH3 SEQ ID NO:6) plus 15492.6Kv Q89L Y92H optCH02 Light Chain Variable Region (SEQ ID NO:24, including CDRLi SEQ ID NO:7, CDRL2 SEQ ID NO:9, and CDRL3 SEQ ID NO: 12)
15492.77 = Human 15492Hv Y32F optCH02 Heavy Chain Variable Region (SEQ ID NO: 16, including CDRHI SEQ ID NO:2, CDRH2 SEQ ID NO:4, and CDRH3 SEQ ID NO:6) plus 31288Kv Q3V I58V D81E optCH02 Light Chain Variable
Region (SEQ ID NO:26, including CDRLi SEQ ID NO:8, CDRL2 SEQ ID NO: 10, and CDRL3 SEQ ID NO: 13)
[00148] Table 7 below shows antibodies containing combinations of the full-length human immunoglobulin heavy and light chains described in this Example. Heavy chains denoted 15492Hv and 15492Hv optCH02 have identical amino acid sequences, but different nucleic acid sequences. The heavy chain denoted 15492Hv optCH02 is a codon optimized variant of 15492Hv.
Table 7
15492 = Human 15492Hv Heavy Chain Variable Region and Human IgGl Constant Region (SEQ ID NO:32) plus 15492Kv Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 38)
15492.6 = Human 15492Hv Heavy Chain Variable Region and Human IgGl Constant
Region (SEQ ID NO:32) plus 15492Kv Q3V A40P L85V R103K Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO:40)
15492.50 = Human 15492Hv optCH02 Heavy Chain Variable Region and Human IgGl Constant Region (SEQ ID NO:32) plus 3 1288Kv Q3V I58V D81E optCH02
Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO:44)
15492.61 = Human 15492Hv Y32F optCH02 Heavy Chain Variable Region and
Human IgGl Constant Region (SEQ ID NO:34) plus 15492.6Kv Q89L Y92H optCH02 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO:42)
15492.65 = Human 15492Hv Y32N E35Q G52S V56Y Q58V optCH02 Heavy Chain Variable Region and Human IgGl Constant Region (SEQ ID NO: 36) plus 15492.6Kv Q89L Y92H optCH02 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO:42)
15492.77 = Human 15492Hv Y32F optCH02 Heavy Chain Variable Region and
Human IgGl Constant Region (SEQ ID NO:34) plus 31288Kv Q3V I58V D81E optCH02 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO:44)
Example 2: Binding Affinities of Human Anti-FGFR2 Monoclonal Antibodies
[00150] In certain experiments described herein, the binding affinities and binding kinetics of monoclonal antibody 15492 were measured with respect to the following proteins containing FGFR extracellular domains available from R&D Systems, Inc. (Minneapolis, MN):
recombinant human FGFRlbeta-IIIb-Fc chimera (rhFGFRl -IIIb-Fc),
recombinant human FGFRlbeta-IIIc-Fc chimera (rhFGFRl -IIIc-Fc),
recombinant human FGFR2beta-IIIb-Fc chimera (rhFGFR2 -IIIb-Fc), or
recombinant human FGFR2beta-IIIc-Fc chimera (rhFGFR2 -IIIc-Fc).
Alternatively, binding affinities and binding kinetics of monoclonal antibody 15492 were measured with respect to the following proteins produced at AVEO Pharmaceuticals, Inc.: recombinant human FGFR3 Ig domains 2+3-IIIb-Fc (rhFGFR3-IIIb-Fc),
recombinant human FGFR3 Ig domains 2+3-IIIc-Fc (rhFGFR3-IIIc-Fc), or recombinant human FGFR4-IIIc-Fc (rhFGFR4-IIIc-Fc).
[00151] The binding affinities and binding kinetics of monoclonal antibody 15492, 15492.6, 15492.50, 15492.61, 15492.65 and 15492.77 were also measured with respect to a version of the following monomeric proteins:
recombinant human FGFRlbeta-IIIb (rhFGFRl p-IIIb),
recombinant human FGFRlbeta-IIIc (rhFGFRl β-IIIc),
recombinant human FGFR2beta-IIIb (rhFGFR2 -IIIb),
recombinant human FGFR2beta-IIIc (rhFGFR2 -IIIc),
recombinant human FGFR3 Ig domains 2+3-IIIb (rhFGFR3-IIIb),
recombinant human FGFR3 Ig domains 2+3-IIIc (rhFGFR3-IIIc), or
recombinant human FGFR4-IIIc (rhFGFR4-IIIc).
[00152] Binding affinities and binding kinetics were measured using biolayer interferometry (BLI) on an Octet QK instrument (ForteBio, Inc., Menlo Park, CA) for 15492 and 15492.6. The Octet analysis was performed at 30°C using IX Kinetics Buffer (ForteBio, Inc.) as assay buffer. Anti-human IgG Fc Capture (AHC) biosensors (ForteBio, Inc.) were used to capture human antibodies onto the sensors. Sensors were saturated in assay buffer for 300 seconds before the assay. Antibodies were loaded onto sensors by dipping the sensors into antibody supernatant solution for 200-300 seconds, which typically resulted in capture levels of 1-2 nm within a row of eight sensors. Baseline was established by dipping the sensors into lx assay buffer for 200-300 seconds. Next, association was monitored for 300 seconds in 50-800 nM FGFR proteins and dissociation was followed for 500-900 seconds in buffer alone. When Fc fused FGFR proteins were monitored for binding, an additional blocking step was included before baseline with 500 nM human IgG Fc Fragments (Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA) for 200-300 seconds.
[00153] Binding affinities and binding kinetics were also measured with surface plasmon resonance using a Biacore T100 instrument (GE Healthcare, Piscataway, NJ) for 15492.50, 15492.61, 15492.65, and 15492.77. Goat anti-human IgGs (Fc fragment specific, Jackson ImmunoResearch) were immobilized on carboxymethylated dextran CM4 sensor chips (GE Healthcare) by amine coupling, using a standard coupling protocol, according to the vendor's
instructions (GE Healthcare). The analyses were performed at 37°C, using PBS containing 0.05% surfactant P20 (GE Healthcare) as running buffer.
[00154] The antibodies were captured in individual flow cells at a flow rate of 10 μΐ/minute. Injection time was varied for each antibody to typically yield an Rmax between 30 and 60 RU. Buffer and FGFR proteins diluted in running buffer were injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 240 seconds at 60 μΐ/minute. The dissociation phase was monitored for up to 1500 seconds. The surface was then regenerated with two 30-second injections of 10 mM Glycine (pH 2.0), at a flow rate of 30 μΐ/minute. The FGFR protein concentrations tested ranged from 365 to 6.25 nM (two-fold dilutions).
[00155] Kinetic parameters for 15492 and 15492.6 were determined using the kinetic function of the Octet analysis software Origin 7.5 (ForteBio, Inc.). Kinetic parameters of the antibody, ka (association rate constant), ka (dissociation rate constant), and KD (equilibrium dissociation constant) were determined. Kinetic values of the monoclonal antibodies 15492 and 15492.6 to FGFR2 proteins at 30°C are summarized in Table 8.
Table 8
[00156] The results in Table 8 demonstrate that antibody 15492 binds rhFGFR2p-IIIb-Fc, rhFGFR2p-IIIc-Fc, monomeric rhFGFR2p-IIIb, and monomeric rhFGFR2p-IIIc with a ¾ of about 9 nM, 10 nM, 2 nM, and 6 nM, respectively. The germline-version, antibody 15492.6, binds with a KD of about 5 nM, 6 nM, 7 nM, and 10 nM, respectively.
[00157] The kinetic values for monoclonal antibody 15492 binding to other recombinant human FGFR proteins at 30°C are summarized in Table 9.
Table 9
[00158] The results in Table 9 demonstrate that antibody 15492 binds to rhFGFRl β-IIIb-Fc, rhFGFRl β-IIIc-Fc, rhFGFR3 -IIIb-Fc, rhFGFR3-IIIc-Fc, and rhFGFR4-IIIc-Fc with a KD of about 19 nM, 4 nM, 70 nM, 7 nM, and 2 nM, respectively. The 15492 antibody also binds more weakly to monomeric rhFGFR3-IIIb, monomeric rhFGFR3-IIIc, and monomeric rhFGFR4-IIIc with a KD of about 350 nM, 38 nM, 200 nM, respectively, and very weakly to monomeric rhFGFRl β-IIIb, and monomeric rhFGFRl β-IIIc.
[00159] Kinetic parameters for antibodies 15492.50, 15492.61, 15492.65, and 15492.77 were determined using the kinetic function of the BIAevaluation software (Biacore) with double reference subtraction. Kinetic parameters for each antibody, ka, kd, and KD were determined. The kinetic values of certain purified monoclonal antibodies (i.e., 15492.50, 15492.61 , 15492.65, and 15492.77) binding to monomeric rhFGFR2 -IIIb and monomeric rhFGFR2 -IIIc at 37°C are summarized in Table 10.
Table 10
mom jmori ; ΐ· K2|M I Ib mom muTic K2|!-I I I
Ani i xK kil l l M M kd ( l M K l ) 11 kii 1 1 MM kd i l s ) K D i M i 11
15492.50 6.5E+04 2.7E-04 4.5E-09 6 4.8E+04 2.9E-04 6.5E-09 6
15492.61 3.3E+04 1.1E-04 3.5E-09 4 2.7E+04 1.1E-04 4.2E-09 4
15492.65 8.1E+04 1.9E-04 2.4E-09 4 6.9E+04 1.8E-04 2.5E-09 4
15492.77 5.7E+04 3.3E-04 5.7E-09 4 4.5E+04 2.3E-04 6.7E-09 4
[00160] The results in Table 10 demonstrate the purified antibodies have KD values ranging from about 2 nM to about 7 nM, when tested at 37°C.
[00161] The kinetic values of certain purified monoclonal antibodies (i.e., 15492.50, 15492.61 , 15492.65, and 15492.77) binding to other monomeric recombinant human FGFR proteins at 37°C are summarized in Table 1 1.
Table 11
[00162] The results in Table 1 1 demonstrate that the purified 15492.50, 15492.61 , 15492.65 and 15492.77 antibodies bind to monomeric rhFGFR3-IIIc with a KD between about 10 and 39 nM and more weakly to the FGFRl proteins, about 59 nM to 1.6 μΜ, as also reflected by faster dissociation rates. Binding to monomeric rhFGFR4-IIIc was considered very weak after examination of the sensograph files showed only traces at the two highest concentrations of the five tested in the dilution series described above. However, quick dissociation rates did not allow for accurate calculation of values. None of these antibodies showed binding to rhFGFR3-IIIb.
[00163] To determine the binding of the antibodies 15492.65 and 15492.77 to FGFR2 from cynomolgus monkey and mouse, the binding affinities and binding kinetics of 15492.65 and 15492.77 Fab fragments were measured with respect to the following proteins (R&D Systems, Inc., Minneapolis, MN):
recombinant human FGFR2beta-IIIb-Fc chimera (rhFGFR2 -IIIb-Fc),
recombinant human FGFR2beta-IIIc-Fc chimera (rhFGFR2 -IIIc-Fc),
recombinant murine FGFR2beta-IIIb-Fc chimera (rmFGFR2 -IIIb-Fc), or
recombinant murine FGFR2beta-IIIc-Fc chimera (rmFGFR2 -IIIc-Fc).
[00164] The binding affinities and binding kinetics of 15492.65 and 15492.77 Fab fragments to FGFR2 from cynomolgus monkey and mouse were also measured with respect to the following proteins produced at AVEO Pharmaceuticals, Inc.:
recombinant cynomolgus monkey FGFR2beta-IIIb-Fc chimera (rcFGFR2 -IIIb-Fc), or recombinant cynomolgus monkey FGFR2beta-IIIc-Fc chimera (rcFGFR2 -IIIc-Fc).
[00165] Binding affinities and binding kinetics of 15492.65 and 15492.77 Fab fragments were measured with surface plasmon resonance using a Biacore T100 instrument. Fab fragments were used in order to avoid the contribution of avidity to the assay. Goat anti-human IgGs (Fc fragment specific) were immobilized on carboxymethylated dextran CM4 sensor chips by amine coupling, using a standard coupling protocol, according to the vendor's instruction. The analyses were performed at 37°C, using PBS containing 0.05% surfactant P20 (GE Healthcare) as running buffer.
[00166] The FGFR2-Fc fusion proteins were captured in individual flow cells at a flow rate of 10 μΐ/minute. Injection time was varied for each protein to typically yield an Rmax between 30 and 60 RU. 250 μg/mL human IgG Fc was injected at 30 μΐ/minute for 120 seconds to block non-specific binding of goat anti-human IgGs (Fc fragment specific) to any residual human Fc portion 15492.65 and 15492.77 Fab proteins. Buffer, 15492.65 Fab, and 15492.77 Fab diluted in running buffer were injected sequentially over a reference surface (no Fc protein captured) and the active surface (FGFR-Fc to be tested) for 240 seconds at 60 μΐ/minute. The dissociation phase was monitored for up to 1200 seconds. The surface was then regenerated with two 30-second injections of 10 mM Glycine (pH 2.25) at a flow rate of 30 μΐ/minute. The 15492.65 and 15492.77 Fab protein concentration range tested was 150 to 18.75nM (two-fold dilutions).
[00167] Kinetic parameters for 15492.65 and 15492.77 Fabs were determined using the kinetic function of the BIA evaluation software (Biacore) with double reference subtraction. Kinetic parameters for each antibody, ka, ka, and KD were determined. The kinetic values of 15492.65 and 15492.77 purified monoclonal antibody Fab fragments binding to human, cynomolgus monkey, and mouse FGFR2-Fc fusion proteins at 37°C are summarized in Table 12.
Table 12
[00168] The results in Table 12 demonstrate that the purified 15492.65 and 15492.77 antibody Fab fragments bind cynomolgus monkey FGFR2-IIIb and FGFR2-IIIc similar to human and mouse.
Example 3: Anti-Proliferative Activity
[00169] To establish cell-based assays to screen for functional FGFR2 antibodies, FDCP-1 cells were engineered to express wild type FGFR2 and cancer-associated mutant variants of FGFR2. FGFR2-driven FDCP- 1 cells were obtained by the following methods. FDCP- 1 cells were transfected by electroporation with plasmids encoding the Illb, IIIc isoform or C- terminally truncated variant of human FGFR2 as well as cancer-associated FGFR2-IIIb S252W, or FGFR2-IIIb N550K mutants. Following selection with G418 (600 μ^πιΐ), single clones were isolated and tested for their FGF l -dependent proliferation in the absence of IL3 by
the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay (Sigma- Aldrich, St. Louis, MO). MTT reagent (10 μΐ) was added to the cells and the reaction was stopped with 100 μΐ of 10% SDS with 2N HCL after four hours. The plates were analyzed the following day. The clones that exhibited robust FGF-1 -dependent proliferation in the absence of IL3 were used for subsequent studies.
[00170] The potency of FGFR2 antibodies was assessed in a cell-based proliferation assay. FDCP-1 cells expressing various human FGFR2 isoforms or mutant variants were seeded in a 96-well plate in IL-3 free medium containing 8 ng/ml of FGF 1 and 5 μg/ml of heparin. Serial dilutions of the antibodies were prepared and added to the plate. After 2 days of incubation, cell proliferation was examined by a MTT assay.
[00171] Antibody 15492 potently inhibited FGF1 -induced proliferation of FDCP-1 cells driven by wild-type FGFR2a-IIIb or wild-type FGFR2-aIIIc in a dose-dependent manner (FIG. 6).
[00172] C-terminally truncated FGFR2-aIIIb, which causes constitutive phosphorylation of FRS2 adaptor molecule and activation of downstream signaling, is found in gastric and breast cancer cell lines (Itoh et al, 1994, CANCER RES. 54:3237-3241 ; Moffa et al, 2004, MOL.
CANCER RES. 2:643-652). Antibody 15492 potently inhibited the FGF l-induced proliferation of FDCP-1 cells driven by the C-terminally truncated FGFR2-IIIb (FIG. 7).
[00173] FGFR2 mutations have been reported in approximately 12% of endometrial tumor samples (Pollock et al, supra; Dutt et al, supra). Somatic activating mutations in FGFR2 cluster within the linker region between IgD2 and IgD3, the extracellular juxtamembrane domain, or the kinase domain. Two of the most common mutations in endometrial tumors are the S252W mutation (which alters ligand specificity and increases affinity of ligand binding) and the N550K mutation in the kinase domain (which enhances kinase activity). Purified antibody 15492 potently inhibited FGFl-induced cell proliferation driven by the wild type FGFR2-aIIIb, FGFR2-aIIIb-truncated, FGFR2- IIIb S252W and FGFR2- IIIb N550K, with IC50 values of 5.1 nM, 1.8 nM, 3.2 nM and 14.9 nM, respectively (FIG. 7 and Table 13).
[00174] Affinity maturated 15492 variants were tested in FDCP proliferation assays driven by wild-type FGFR2-IIIb and wild-type FGFR2-IIIC. Antibodies 15492.6, 15492.50, 15492.61, 15492.65 and 15492.77 potently inhibited FGF l-induced proliferation of FDCP-1 cells driven
by wild-type FGFR2-aIIIb (FIG. 8) or wild-type FGFR2-aIIIc (FIG. 9) in a dose-dependent manner.
[00175] Affinity maturated 15492 variants were tested in FDCP proliferation assays driven by cancer-associated mutant variants of FGFR2. Antibodies 15492.6, 15492.50, 15492.61 , 15492.65 and 15492.77 potently inhibited FGF l-induced proliferation of FDCP- 1 cells driven by FGFR2- IIIb S252W (FIG. 10), FGFR2- IIIc S252W (FIG. 11), FGFR2- IIIb N550K (FIG. 12), or FGFR2-aIIIb-truncated (FIG. 13) in a dose-dependent manner.
[00176] Table 13 provides the IC50 (nM) of 15492 variants on wild type (FGFR2 alllb and FGFR2 alllc) and mutant FGFR2 (FGFR2-piIIb-S252W, FGFR2-piIIc-S252W, FGFR2-piIIb- N550K and FGFR2-aIIIb-truncated) in proliferation assays.
Table 13
Example 4: Inhibition of Tumor Xenograft Growth
[00177] To assess the activity of these antibodies in vivo, the effect of antibody 15492 variants (i.e., antibodies 15492.6, 15492.50, 15492.61, 15492.65 and 15492.77) were tested on the growth of SNU-16 xenograft, a human gastric cancer model with an FGFR2 gene amplification.
[00178] All mice were treated in accordance with the OLAW Public Health Service Policy on Human Care and Use of Laboratory Animals and the ILAR Guide for the Care and Use of Laboratory Animals. All in vivo studies were conducted following the protocols approved by the AVEO Institutional Animal Care and Use Committee. For the SNU- 16 in vivo studies, approximately 8 week old female C.B-17 SCID mice (Taconic, Germantown, NY) were inoculated subcutaneously into the right flank with 5xl06 cells in 1 : 1 RPMI 1640 (Invitrogen, Carlsbad, CA)/Matrigel (BD Biosciences, San Jose CA). Tumor measurements were taken
twice weekly, using vernier calipers. Tumor volume was calculated using the formula: V=0.5 x width x width x length. When tumors approached a volume of 150 mm3, mice were randomized into seven groups often animals each. The next day, mice were treated with PBS or 2 mg/kg hlgG (Xolair), or 2 mg/kg of antibodies 15492.6, 15492.50, 15492.61, 15492.65 or 15492.77 by intraperitoneal injection. Mice were dosed twice weekly for the duration of the study. Seventy-two hours after the final dose, tumor volumes were measured again for calculation of tumor growth inhibition. All statistical analysis was done using GraphPad PRISM® Version 4.00. Final tumor volumes were analyzed using with a one-way analysis of variance and Tukey multiple comparison test.
[00179] As shown in FIG. 14, antibodies 15492.6, 15492.50, 15492.61, 15492.65 and
15492.77 treatment groups showed significant tumor growth inhibition, as compared to hlgG treated controls (72%, 75%, 60%, 68%, and 78%, respectively) at day 33, which was the last day for the control group to remain in the study. All treatments were well-tolerated with no significant body weight loss.
INCORPORATION BY REFERENCE
[00180] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.
EQUIVALENTS
[00181] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and the range of equivalency of the claims are intended to be embraced therein.
Claims
1. An isolated antibody that binds human FGFR2 comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region selected from the group consisting of:
(a) (i) an immunoglobulin heavy chain variable region comprising a CDRHI comprising the amino acid sequence of SEQ ID NO:2 (15492.61, 15492.77), a CDRH2 comprising the amino acid sequence of SEQ ID NO:4 (15492, 15492.6, 15492.50, 15492.61, 15492.77), and a CDRH3 comprising the amino acid sequence of SEQ ID NO:6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77); and
(ii) an immunoglobulin light chain variable region comprising a CDRLI comprising the amino acid sequence of SEQ ID NO: 8 (15492.50, 15492.77), a CDRL2 comprising the amino acid sequence of SEQ ID NO: 10 (15492.50, 15492.77), and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 13 (15492.50, 15492.77);
(b) (i) an immunoglobulin heavy chain variable region comprising a CDRHI comprising the amino acid sequence of SEQ ID NO: 1 (15492, 15492.6, 15492.50), a CDRH2 comprising the amino acid sequence of SEQ ID NO:4 (15492, 15492.6, 15492.50, 15492.61, 15492.77), and a CDRH3 comprising the amino acid sequence of SEQ ID NO:6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77); and
(ii) an immunoglobulin light chain variable region comprising a CDRLI comprising the amino acid sequence of SEQ ID NO: 7 (15492, 15492.6, 15492.61, 15492.65), a CDRL2 comprising the amino acid sequence of SEQ ID NO:9 (15492, 15492.6, 15492.61, 15492.65), and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 1 1 (15492, 15492.6);
(c) (i) an immunoglobulin heavy chain variable region comprising a CDRHI comprising the amino acid sequence of SEQ ID NO: 1 (15492, 15492.6, 15492.50), a CDRH2 comprising the amino acid sequence of SEQ ID NO:4 (15492, 15492.6, 15492.50, 15492.61, 15492.77), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77); and
(ii) an immunoglobulin light chain variable region comprising a CDRLI comprising the amino acid sequence of SEQ ID NO: 8 (15492.50, 15492.77), a CDRL2 comprising the amino acid sequence of SEQ ID NO: 10 (15492.50, 15492.77), and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 13 (15492.50, 15492.77);
(d) (i) an immunoglobulin heavy chain variable region comprising a CDRHI comprising the amino acid sequence of SEQ ID NO:2 (15492.61, 15492.77), a CDRH2 comprising the amino acid sequence of SEQ ID NO:4 (15492, 15492.6, 15492.50, 15492.61, 15492.77), and a CDRH3 comprising the amino acid sequence of SEQ ID NO:6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77); and
(ii) an immunoglobulin light chain variable region comprising a CDRLI comprising the amino acid sequence of SEQ ID NO: 7 (15492, 15492.6, 15492.61, 15492.65), a CDRL2 comprising the amino acid sequence of SEQ ID NO:9 (15492, 15492.6, 15492.61, 15492.65), and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12 (15492.61, 15492.65); and (e) (i) an immunoglobulin heavy chain variable region comprising a CDRHI comprising the amino acid sequence of SEQ ID NO: 3 (15492.65), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 5 (15492.65), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 6 (15492, 15492.6, 15492.50, 15492.61, 15492.65, 15492.77); and
(ii) an immunoglobulin light chain variable region comprising a CDRLI comprising the amino acid sequence of SEQ ID NO: 7 (15492, 15492.6, 15492.61, 15492.65), a CDRL2 comprising the amino acid sequence of SEQ ID NO:9 (15492, 15492.6, 15492.61, 15492.65), and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12 (15492.61, 15492.65).
2. An isolated nucleic acid comprising a nucleotide sequence encoding an
immunoglobulin heavy chain variable region of claim 1.
3. An isolated nucleic acid comprising a nucleotide sequence encoding an
immunoglobulin light chain variable region of claim 1.
4. An expression vector comprising the nucleic acid of claim 2.
5. An expression vector comprising the nucleic acid of claim 3.
6. The expression vector of claim 5, further comprising the nucleic acid of claim 2.
7. A host cell comprising the expression vector of claim 4.
8 A host cell comprising the expression vector of claim 5.
9. A host cell comprising the expression vector of claim 6.
10. The host cell of claim 8, further comprising the expression vector of claim 4.
1 1. A method of producing a polypeptide comprising an immunoglobulin heavy chain variable region or an immunoglobulin light chain variable region, the method comprising:
(a) growing the host cell of claim 7 or 8 under conditions so that the host cell expresses the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region; and
(b) purifying the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region.
12. A method of producing an antibody that binds human FGFR2 or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 9 or 10 under conditions so that the host cell expresses a polypeptide comprising the immunoglobulin heavy chain variable region and/or the immunoglobulin light chain variable region, thereby producing the antibody or the antigen-binding fragment of the antibody; and
(b) purifying the antibody or the antigen-binding fragment of the antibody.
13. An isolated antibody that binds human FGFR2, comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region selected from the group consisting of:
(a) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 (15492.61, 15492.77), and an immunoglobulin light chain
variable region comprising the amino acid sequence of SEQ ID NO:26 (15492.50, 15492.77);
(b) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (15492, 15492.6, 15492.50), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 (15492); (c) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (15492, 15492.6, 15492.50), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:22 (15492.6); (d) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (15492, 15492.6, 15492.50), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:26 (15492.50, 15492.77);
(e) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 (15492.61, 15492.77), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:24 (15492.61, 15492.65);
(f) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 (15492.65), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:24 (15492.61, 15492.65).
14. An isolated nucleic acid comprising a nucleotide sequence encoding an
immunoglobulin heavy chain variable region of claim 13.
15. An isolated nucleic acid comprising a nucleotide sequence encoding an
immunoglobulin light chain variable region of claim 13.
16. An expression vector comprising the nucleic acid of claim 14.
17. An expression vector comprising the nucleic acid of claim 15.
18. The expression vector of claim 17, further comprising the nucleic acid of claim 14.
19. A host cell comprising the expression vector of claim 16.
20. A host cell comprising the expression vector of claim 17.
21. A host cell comprising the expression vector of claim 18.
22. The host cell of claim 20, further comprising the expression vector of claim 16.
23. A method of producing a polypeptide comprising an immunoglobulin heavy chain variable region or an immunoglobulin light chain variable region, the method comprising:
(a) growing the host cell of claim 19 or 20 under conditions so that the host cell expresses the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region; and
(b) purifying the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region.
24. A method of producing an antibody that binds human FGFR2 or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 21 or 22 under conditions so that the host cell expresses a polypeptide comprising the immunoglobulin heavy chain variable region and/or the immunoglobulin light chain variable region, thereby producing the antibody or the antigen-binding fragment of the antibody; and
(b) purifying the antibody or the antigen-binding fragment of the antibody.
25. An isolated antibody that binds human FGFR2 comprising an immunoglobulin heavy chain and an immunoglobulin light chain selected from the group consisting of:
(a) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:34 (15492.61, 15492.77), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:44 (15492.50, 15492.77);
(b) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 32 (15492, 15492.6, 15492.50), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:38 (15492);
(c) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 32 (15492, 15492.6, 15492.50), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:40 (15492.6);
(d) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 32 (15492, 15492.6, 15492.50), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:44 (15492.50, 125492.77);
(e) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:34 (15492.61, 15492.77), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:42 (15492.61, 15492.65);
(f) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO:36 (15492.65), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO:42 (15492.61, 15492.65).
26. An isolated nucleic acid comprising a nucleotide sequence encoding an
immunoglobulin heavy chain of claim 25.
27. An isolated nucleic acid comprising a nucleotide sequence encoding an
immunoglobulin light chain of claim 25.
28. An expression vector comprising the nucleic acid of claim 26.
29. An expression vector comprising the nucleic acid of claim 27.
30. The expression vector of claim 29, further comprising the nucleic acid of claim 26.
31. A host cell comprising the expression vector of claim 28.
32. A host cell comprising the expression vector of claim 29.
33. A host cell comprising the expression vector of claim 30.
34. The host cell of claim 32, further comprising the expression vector of claim 28.
35. A method of producing a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain, the method comprising:
(a) growing the host cell of claim 31 or 32 under conditions so that the host cell expresses the polypeptide comprising the immunoglobulin heavy chain or the immunoglobulin light chain; and
(b) purifying the polypeptide comprising the immunoglobulin heavy chain or the immunoglobulin light chain.
36. A method of producing an antibody that binds human FGFR2 or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 33 or 34 under conditions so that the host cell expresses a polypeptide comprising the immunoglobulin heavy chain and/or the immunoglobulin light chain, thereby producing the antibody or the antigen-binding fragment of the antibody; and
(b) purifying the antibody or the antigen-binding fragment of the antibody.
37. The antibody of any one of claims 1, 13, or 25, wherein the antibody has a ¾ of 10 nM or lower, as measured by surface plasmon resonance or bio-layer interferometry.
38. A method of inhibiting or reducing proliferation of a tumor cell comprising exposing the cell to an effective amount of the antibody of any one of claims 1, 13, or 25 to inhibit or reduce proliferation of the tumor cell.
39. A method of inhibiting or reducing tumor growth in a mammal, the method comprising exposing the mammal to an effective amount of the antibody of any one of claims 1, 13, or 25 to inhibit or reduce proliferation of the tumor.
40. A method of treating cancer in a mammal, the method comprising administering an effective amount of the antibody of any one of claims 1, 13, or 25 to a mammal in need thereof.
41. The method of claim 40, wherein the cancer is selected from the group consisting of gastric cancer, breast cancer, lung cancer, pancreatic cancer, endometrial cancer, and ovarian cancer.
42. The method of claim 40, wherein the mammal is a human.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201261733285P | 2012-12-04 | 2012-12-04 | |
| US61/733,285 | 2012-12-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2014089193A1 true WO2014089193A1 (en) | 2014-06-12 |
Family
ID=49877023
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2013/073090 WO2014089193A1 (en) | 2012-12-04 | 2013-12-04 | Anti-fgfr2 antibodies |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2014089193A1 (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014160160A3 (en) * | 2013-03-13 | 2015-01-08 | Novartis Ag | Antibody drug conjugates and corresponding antibodies |
| US9415118B2 (en) | 2013-03-13 | 2016-08-16 | Novartis Ag | Antibody drug conjugates |
| US10172937B2 (en) | 2013-08-01 | 2019-01-08 | Five Prime Therapeutics, Inc. | Method of treatment of malignant solid tumors with afucosylated anti-FGFR2IIIb antibodies |
| EP3287522A4 (en) * | 2015-04-20 | 2019-03-27 | Daiichi Sankyo Company, Limited | Detection of fgfr2 |
| US11091555B2 (en) | 2017-05-16 | 2021-08-17 | Five Prime Therapeutics, Inc. | Method of treating gastric cancer with anti-FGFR2-IIIb antibodies and modified FOLFOX6 chemotherapy |
| US20220162323A1 (en) * | 2020-10-22 | 2022-05-26 | Regeneron Pharmaceuticals, Inc. | Anti-fgfr2 antibodies and methods of use thereof |
| US11447553B2 (en) | 2015-11-23 | 2022-09-20 | Five Prime Therapeutics, Inc. | FGFR2 inhibitors alone or in combination with immune stimulating agents in cancer treatment |
| WO2023185778A1 (en) * | 2022-03-28 | 2023-10-05 | Genor Biopharma Co., Ltd. | Novel anti-fgfr2 antibodies |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010054265A2 (en) * | 2008-11-07 | 2010-05-14 | Galaxy Biotech, Llc. | Monoclonal antibodies to fibroblast growth factor receptor 2 |
| WO2011143318A2 (en) * | 2010-05-11 | 2011-11-17 | Aveo Pharmaceuticals, Inc. | Anti-fgfr2 antibodies |
| WO2012021841A2 (en) * | 2010-08-12 | 2012-02-16 | Attogen Inc. | Antibody molecules to oncogenic isoforms of fibroblast growth factor receptor-2 and uses thereof |
-
2013
- 2013-12-04 WO PCT/US2013/073090 patent/WO2014089193A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010054265A2 (en) * | 2008-11-07 | 2010-05-14 | Galaxy Biotech, Llc. | Monoclonal antibodies to fibroblast growth factor receptor 2 |
| WO2011143318A2 (en) * | 2010-05-11 | 2011-11-17 | Aveo Pharmaceuticals, Inc. | Anti-fgfr2 antibodies |
| WO2012021841A2 (en) * | 2010-08-12 | 2012-02-16 | Attogen Inc. | Antibody molecules to oncogenic isoforms of fibroblast growth factor receptor-2 and uses thereof |
Non-Patent Citations (39)
| Title |
|---|
| "Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING COMPANY |
| A. BAI ET AL: "GP369, an FGFR2-IIIb-Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling", CANCER RESEARCH, vol. 70, no. 19, 13 August 2010 (2010-08-13), pages 7630 - 7639, XP055079445, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-10-1489 * |
| ADNANE ET AL., ONCOGENE, vol. 6, 1991, pages 659 - 663 |
| AHMAD I ET AL: "Mechanisms of FGFR-mediated carcinogenesis", BIOCHIMICA ET BIOPHYSICA ACTA - MOLECULAR CELL RESEARCH 2012 ELSEVIER NLD, vol. 1823, no. 4, April 2012 (2012-04-01), pages 850 - 860, XP002719573, ISSN: 0167-4889 * |
| ALTSCHUL ET AL., NATURE GENETICS, vol. 6, 1994, pages 119 - 129 |
| ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402 |
| ALTSCHUL, J. MOL. EVOL., vol. 36, 1993, pages 290 - 300 |
| ASAOKA YOSHINARI ET AL: "New targeted therapies for gastric cancer.", EXPERT OPINION ON INVESTIGATIONAL DRUGS MAY 2011, vol. 20, no. 5, May 2011 (2011-05-01), pages 595 - 604, XP008167057, ISSN: 1744-7658 * |
| BEENKEN ET AL., NATURE REVIEWS DRUG DISCOVERY, vol. 8, 2009, pages 235 - 254 |
| CHANG ET AL., BLOOD, vol. 106, 2005, pages 353 - 6 |
| CHEN G ET AL: "FGFR2: A key molecule in the progression of gastric cancer", WORLD CHINESE JOURNAL OF DIGESTOLOGY 2011 WCJ PRESS CHN, vol. 19, no. 4, 2011, pages 384 - 388, XP008167077, ISSN: 1009-3079 * |
| DANIELE G ET AL: "FGF Receptor inhibitors: Role in cancer therapy", CURRENT ONCOLOGY REPORTS 2012 CURRENT MEDICINE GROUP LLC GBR, vol. 14, no. 2, April 2012 (2012-04-01), pages 111 - 119, XP002719572, ISSN: 1523-3790 * |
| DAVIES ET AL., CANCER RES., vol. 65, 2005, pages 7591 - 7595 |
| DING ET AL., NATURE, vol. 455, 2008, pages 1069 - 1075 |
| DUTT ET AL., PROC. NATL. ACAD. SCI. USA, vol. 105, 2008, pages 8713 - 8717 |
| EASTON ET AL., NATURE, vol. 447, 2007, pages 1087 - 1093 |
| ESWARAKUMAR ET AL., CYTOKINE GROWTH FACTOR REV., vol. 16, 2005, pages 139 - 49 |
| GOMEZ-ROMAN ET AL., CLIN. CANCER RES., vol. 11, 2005, pages 459 - 65 |
| GREENMAN ET AL., NATURE, vol. 446, 2007, pages 153 - 158 |
| HARA ET AL., LAB. INVEST., vol. 78, 1998, pages 1143 - 1153 |
| HENIKOFF ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 10915 - 10919 |
| HOET ET AL., NATURE BIOTECH., vol. 23, 2005, pages 344 - 348 |
| HUNTER ET AL., NAT. GENET., vol. 39, 2007, pages 870 - 874 |
| ISHIWATA TOSHIYUKI ET AL: "Enhanced Expression of Fibroblast Growth Factor Receptor 2 IIIc Promotes Human Pancreatic Cancer Cell Proliferation", AMERICAN JOURNAL OF PATHOLOGY, vol. 180, no. 5, May 2012 (2012-05-01), pages 1928 - 1941, XP002719575, ISSN: 0002-9440 * |
| ITOH ET AL., CANCER RES., vol. 54, 1994, pages 3237 - 3241 |
| JANG ET AL., CANCER RES., vol. 61, 2001, pages 3541 - 3543 |
| KNAPPIK ET AL., J. MOL. BIOL., vol. 296, 2000, pages 57 - 86 |
| KREBS ET AL., J. IMMUNOL. METH., vol. 254, 2001, pages 67 - 84 |
| MATSUDA YOKO ET AL: "Fibroblast Growth Factor Receptor 2 IIIc as a Therapeutic Target for Colorectal Cancer Cells", MOLECULAR CANCER THERAPEUTICS, vol. 11, no. 9, September 2012 (2012-09-01), pages 2010 - 2020, XP002719574, ISSN: 1535-7163 * |
| MEYER ET AL., PLOS BIOL., vol. 6, 2008, pages E108 |
| MOFFA ET AL., MOL. CANCER RES., vol. 2, 2004, pages 643 - 652 |
| MOR ET AL., CANCER GENET. CYTOGENET., vol. 65, 1993, pages 111 - 114 |
| POLLOCK ET AL., ONCOGENE, vol. 26, 2007, pages 7158 - 7162 |
| PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 2264 - 2268 |
| ROTHE ET AL., J. MOL. BIOL., vol. 376, 2008, pages 1182 - 1200 |
| SODERLIND ET AL., NATURE BIOTECH., vol. 18, 2000, pages 852 - 856 |
| TURNER ET AL., NATURE REVIEWS CANCER, vol. 10, 2010, pages 116 - 129 |
| TURNER ET AL., ONCOGENE, vol. 29, 2010, pages 2013 - 2023 |
| WEI-MENG ZHAO ET AL: "Monoclonal Antibodies to Fibroblast Growth Factor Receptor 2 Effectively Inhibit Growth of Gastric Tumor Xenografts", CLINICAL CANCER RESEARCH, THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 16, no. 23, 1 December 2010 (2010-12-01), pages 5750 - 5758, XP002673994, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-10-0531 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014160160A3 (en) * | 2013-03-13 | 2015-01-08 | Novartis Ag | Antibody drug conjugates and corresponding antibodies |
| US9415118B2 (en) | 2013-03-13 | 2016-08-16 | Novartis Ag | Antibody drug conjugates |
| US9498532B2 (en) | 2013-03-13 | 2016-11-22 | Novartis Ag | Antibody drug conjugates |
| AU2017202150B2 (en) * | 2013-03-13 | 2018-12-20 | Novartis Ag | Antibody drug conjugates and corresponding antibodies |
| US10172937B2 (en) | 2013-08-01 | 2019-01-08 | Five Prime Therapeutics, Inc. | Method of treatment of malignant solid tumors with afucosylated anti-FGFR2IIIb antibodies |
| US11235059B2 (en) | 2013-08-01 | 2022-02-01 | Five Prime Therapeutics, Inc. | Afucosylated anti-FGFR2IIIB antibodies |
| US10881734B2 (en) | 2015-04-20 | 2021-01-05 | Daiichi Sankyo Company, Limited | Monoclonal antibodies to human fibroblast growth factor receptor 2 (HFGFR2) and methods of use thereof |
| EP3287522A4 (en) * | 2015-04-20 | 2019-03-27 | Daiichi Sankyo Company, Limited | Detection of fgfr2 |
| US11447553B2 (en) | 2015-11-23 | 2022-09-20 | Five Prime Therapeutics, Inc. | FGFR2 inhibitors alone or in combination with immune stimulating agents in cancer treatment |
| US11091555B2 (en) | 2017-05-16 | 2021-08-17 | Five Prime Therapeutics, Inc. | Method of treating gastric cancer with anti-FGFR2-IIIb antibodies and modified FOLFOX6 chemotherapy |
| US20220162323A1 (en) * | 2020-10-22 | 2022-05-26 | Regeneron Pharmaceuticals, Inc. | Anti-fgfr2 antibodies and methods of use thereof |
| US11866502B2 (en) * | 2020-10-22 | 2024-01-09 | Regeneron Pharmaceuticals, Inc. | Anti-FGFR2 antibodies and methods of use thereof |
| WO2023185778A1 (en) * | 2022-03-28 | 2023-10-05 | Genor Biopharma Co., Ltd. | Novel anti-fgfr2 antibodies |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110606891B (en) | Antibody molecule aiming at human CLDN18.2, antigen binding fragment and medical application thereof | |
| AU2016306090B2 (en) | 5-bromo-2,6-di-(1H-pyrazol-l-yl)pyrimidin-4-amine for use in the treatment of cancer | |
| US8481688B2 (en) | Anti-FGFR2 antibodies | |
| CA2896076C (en) | Anti-gdf15 antibodies | |
| WO2014089193A1 (en) | Anti-fgfr2 antibodies | |
| WO2011140249A2 (en) | Antibodies that bind csf1r | |
| US20210171627A1 (en) | Anti-notch3 antibodies | |
| WO2012003472A1 (en) | Anti-notch1 antibodies | |
| WO2022104697A1 (en) | Modified egfr antibody with reduced affinity | |
| CA2966551A1 (en) | Assay and method for determining cdc eliciting antibodies | |
| US20220235127A1 (en) | Anti-notch3 antibody | |
| US20230357398A1 (en) | Novel human antibodies binding to human cd3 epsilon | |
| KR20230041810A (en) | Anti-PVRIG Protein Antibodies or Antibody Fragments and Uses Thereof | |
| WO2014172653A2 (en) | Anti-notch1 antibodies | |
| IL294921A (en) | Anti-e-selectin antibodies, compositions and methods of use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13811704 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 13811704 Country of ref document: EP Kind code of ref document: A1 |