NZ618635B2 - High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as pichia pastoris - Google Patents
High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as pichia pastoris Download PDFInfo
- Publication number
- NZ618635B2 NZ618635B2 NZ618635A NZ61863512A NZ618635B2 NZ 618635 B2 NZ618635 B2 NZ 618635B2 NZ 618635 A NZ618635 A NZ 618635A NZ 61863512 A NZ61863512 A NZ 61863512A NZ 618635 B2 NZ618635 B2 NZ 618635B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- seq
- antibody
- ngf
- cdr2
- chain cdr1
- Prior art date
Links
- 102000004965 antibodies Human genes 0.000 title claims abstract description 808
- 108090001123 antibodies Proteins 0.000 title claims abstract description 808
- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 66
- 102000004169 proteins and genes Human genes 0.000 title claims description 77
- 108090000623 proteins and genes Proteins 0.000 title claims description 77
- 238000004519 manufacturing process Methods 0.000 title claims description 39
- 244000052616 bacterial pathogens Species 0.000 title description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 332
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 154
- 230000014509 gene expression Effects 0.000 claims abstract description 94
- 241000235648 Pichia Species 0.000 claims abstract description 75
- 238000007792 addition Methods 0.000 claims abstract description 56
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 24
- 230000012010 growth Effects 0.000 claims abstract description 13
- 230000003698 anagen phase Effects 0.000 claims abstract description 9
- 229920001184 polypeptide Polymers 0.000 claims description 270
- 210000004027 cells Anatomy 0.000 claims description 199
- 108091006028 chimera Proteins 0.000 claims description 122
- 235000018102 proteins Nutrition 0.000 claims description 74
- 239000000203 mixture Substances 0.000 claims description 70
- 239000000047 product Substances 0.000 claims description 44
- 239000002609 media Substances 0.000 claims description 38
- 239000003795 chemical substances by application Substances 0.000 claims description 30
- 239000001963 growth media Substances 0.000 claims description 27
- 229910052760 oxygen Inorganic materials 0.000 claims description 27
- 239000001301 oxygen Substances 0.000 claims description 27
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims description 27
- 230000028327 secretion Effects 0.000 claims description 26
- 241000238631 Hexapoda Species 0.000 claims description 24
- 230000001939 inductive effect Effects 0.000 claims description 24
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 22
- 241000282414 Homo sapiens Species 0.000 claims description 21
- 238000001542 size-exclusion chromatography Methods 0.000 claims description 20
- 230000002829 reduced Effects 0.000 claims description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 15
- 230000001594 aberrant Effects 0.000 claims description 15
- 239000008103 glucose Substances 0.000 claims description 15
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-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 claims description 15
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 14
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 13
- 101710044706 SFA1 Proteins 0.000 claims description 13
- 235000001014 amino acid Nutrition 0.000 claims description 12
- 230000015572 biosynthetic process Effects 0.000 claims description 12
- 239000007789 gas Substances 0.000 claims description 12
- 150000001413 amino acids Chemical class 0.000 claims description 11
- 230000003899 glycosylation Effects 0.000 claims description 11
- 238000006206 glycosylation reaction Methods 0.000 claims description 11
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 101700080270 ADH2 Proteins 0.000 claims description 8
- 101700013560 CUP1 Proteins 0.000 claims description 8
- BJHIKXHVCXFQLS-UYFOZJQFSA-N Fructose Natural products OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 claims description 8
- GUBGYTABKSRVRQ-YOLKTULGSA-N Maltose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)O[C@H]1CO)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 GUBGYTABKSRVRQ-YOLKTULGSA-N 0.000 claims description 8
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 8
- 101700005118 SEI1 Proteins 0.000 claims description 8
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 8
- SRBFZHDQGSBBOR-SQOUGZDYSA-N Xylose Natural products O[C@@H]1CO[C@@H](O)[C@@H](O)[C@@H]1O SRBFZHDQGSBBOR-SQOUGZDYSA-N 0.000 claims description 8
- 230000002708 enhancing Effects 0.000 claims description 8
- 238000005755 formation reaction Methods 0.000 claims description 8
- 235000019157 thiamine Nutrition 0.000 claims description 8
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims description 8
- 229960003495 thiamine Drugs 0.000 claims description 8
- 239000011721 thiamine Substances 0.000 claims description 8
- 229960002180 Tetracycline Drugs 0.000 claims description 7
- OFVLGDICTFRJMM-WESIUVDSSA-N Tetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O OFVLGDICTFRJMM-WESIUVDSSA-N 0.000 claims description 7
- 239000004098 Tetracycline Substances 0.000 claims description 7
- 238000001502 gel electrophoresis Methods 0.000 claims description 7
- 235000019364 tetracycline Nutrition 0.000 claims description 7
- 101700045663 ACP5 Proteins 0.000 claims description 6
- 229940041514 Candida albicans extract Drugs 0.000 claims description 6
- 102000006602 Glyceraldehyde-3-Phosphate Dehydrogenases Human genes 0.000 claims description 6
- 108020004445 Glyceraldehyde-3-Phosphate Dehydrogenases Proteins 0.000 claims description 6
- 101700049607 HIS4 Proteins 0.000 claims description 6
- 235000006708 antioxidants Nutrition 0.000 claims description 6
- 230000003612 virological Effects 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- LJQLQCAXBUHEAZ-UHFFFAOYSA-N 1,3-Bisphosphoglyceric acid Chemical compound OP(=O)(O)OCC(O)C(=O)OP(O)(O)=O LJQLQCAXBUHEAZ-UHFFFAOYSA-N 0.000 claims description 5
- 241000271566 Aves Species 0.000 claims description 5
- 210000004507 Chromosomes, Artificial Anatomy 0.000 claims description 5
- GZCGUPFRVQAUEE-KCDKBNATSA-N D-(+)-Galactose Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 claims description 5
- 229940066779 Peptones Drugs 0.000 claims description 5
- 101700018328 ccdB Proteins 0.000 claims description 5
- WQZGKKKJIJFFOK-PHYPRBDBSA-N α-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 5
- KRKNYBCHXYNGOX-UHFFFAOYSA-K 2qpq Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 4
- 102100000356 ADH1A Human genes 0.000 claims description 4
- 101700007462 ADH1A Proteins 0.000 claims description 4
- 101700085948 ADH4 Proteins 0.000 claims description 4
- 241000269328 Amphibia Species 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 4
- 101710039162 CDC19 Proteins 0.000 claims description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 4
- FBPFZTCFMRRESA-KAZBKCHUSA-N D-Mannitol Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KAZBKCHUSA-N 0.000 claims description 4
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 4
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 4
- 239000005715 Fructose Substances 0.000 claims description 4
- 101700058973 GAL4 Proteins 0.000 claims description 4
- 101710015850 LGALS4 Proteins 0.000 claims description 4
- 102100011539 LGALS4 Human genes 0.000 claims description 4
- GUBGYTABKSRVRQ-UUNJERMWSA-N Lactose Natural products O([C@@H]1[C@H](O)[C@H](O)[C@H](O)O[C@@H]1CO)[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 GUBGYTABKSRVRQ-UUNJERMWSA-N 0.000 claims description 4
- 241000270322 Lepidosauria Species 0.000 claims description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 4
- 230000036091 Metabolic activity Effects 0.000 claims description 4
- 241000283898 Ovis Species 0.000 claims description 4
- 101710016939 PHO3 Proteins 0.000 claims description 4
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N Raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims description 4
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims description 4
- PNNNRSAQSRJVSB-BXKVDMCESA-N Rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N Trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 4
- 229960003487 Xylose Drugs 0.000 claims description 4
- 101710026144 acu-3 Proteins 0.000 claims description 4
- 101710025952 adhT Proteins 0.000 claims description 4
- 150000001479 arabinose derivatives Chemical class 0.000 claims description 4
- 235000018417 cysteine Nutrition 0.000 claims description 4
- 101710004278 icl Proteins 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 4
- 239000000594 mannitol Substances 0.000 claims description 4
- 235000010355 mannitol Nutrition 0.000 claims description 4
- 230000002503 metabolic Effects 0.000 claims description 4
- 230000000051 modifying Effects 0.000 claims description 4
- 239000000600 sorbitol Substances 0.000 claims description 4
- DLRVVLDZNNYCBX-ZZFZYMBESA-N β-melibiose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1 DLRVVLDZNNYCBX-ZZFZYMBESA-N 0.000 claims description 4
- 241000283707 Capra Species 0.000 claims description 3
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 3
- 241000700159 Rattus Species 0.000 claims description 3
- 230000003834 intracellular Effects 0.000 claims description 3
- 101700029703 och1 Proteins 0.000 claims description 3
- 239000002831 pharmacologic agent Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 210000000805 Cytoplasm Anatomy 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 238000011105 stabilization Methods 0.000 claims description 2
- 108060000428 AOX Proteins 0.000 claims 3
- 102100010989 AOX1 Human genes 0.000 claims 3
- 101700068416 H1L1 Proteins 0.000 claims 1
- 150000001945 cysteines Chemical class 0.000 claims 1
- 208000002193 Pain Diseases 0.000 description 541
- 102100009139 NGF Human genes 0.000 description 306
- 230000027455 binding Effects 0.000 description 267
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 243
- 230000002265 prevention Effects 0.000 description 243
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 150
- 230000035693 Fab Effects 0.000 description 82
- 102000015434 Humanized Monoclonal Antibodies Human genes 0.000 description 50
- 108010064750 Humanized Monoclonal Antibodies Proteins 0.000 description 50
- 101700073818 CDR1 Proteins 0.000 description 42
- 102100002977 CDR1 Human genes 0.000 description 40
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 40
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 40
- 230000000694 effects Effects 0.000 description 37
- 239000000427 antigen Substances 0.000 description 33
- 108091007172 antigens Proteins 0.000 description 32
- 102000038129 antigens Human genes 0.000 description 32
- 230000023298 conjugation with cellular fusion Effects 0.000 description 30
- 230000013011 mating Effects 0.000 description 30
- 230000021037 unidirectional conjugation Effects 0.000 description 30
- 201000010099 disease Diseases 0.000 description 28
- 238000000855 fermentation Methods 0.000 description 28
- 230000004151 fermentation Effects 0.000 description 28
- 230000001965 increased Effects 0.000 description 27
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 27
- 239000000014 opioid analgesic Substances 0.000 description 27
- 241000196324 Embryophyta Species 0.000 description 26
- 230000003247 decreasing Effects 0.000 description 26
- 241000894007 species Species 0.000 description 26
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 23
- 239000004365 Protease Substances 0.000 description 22
- 230000002255 enzymatic Effects 0.000 description 22
- 210000004962 mammalian cells Anatomy 0.000 description 22
- 239000003550 marker Substances 0.000 description 22
- 229920001405 Coding region Polymers 0.000 description 21
- 101700043017 NTRK1 Proteins 0.000 description 21
- 230000029087 digestion Effects 0.000 description 21
- 230000000813 microbial Effects 0.000 description 21
- 229940055729 Papain Drugs 0.000 description 20
- 108090000526 Papain Proteins 0.000 description 20
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 20
- 230000002538 fungal Effects 0.000 description 20
- 235000019834 papain Nutrition 0.000 description 20
- 230000001976 improved Effects 0.000 description 18
- 238000000034 method Methods 0.000 description 15
- 239000000523 sample Substances 0.000 description 15
- 239000006227 byproduct Substances 0.000 description 14
- 229960001031 Glucose Drugs 0.000 description 12
- 102000018358 Immunoglobulins Human genes 0.000 description 12
- 108060003951 Immunoglobulins Proteins 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 238000005215 recombination Methods 0.000 description 12
- 230000035897 transcription Effects 0.000 description 12
- 229920000023 polynucleotide Polymers 0.000 description 11
- 239000002157 polynucleotide Substances 0.000 description 11
- 238000011067 equilibration Methods 0.000 description 10
- 239000000499 gel Substances 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 10
- 230000001105 regulatory Effects 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 230000001131 transforming Effects 0.000 description 10
- 230000035899 viability Effects 0.000 description 10
- 102000008100 Human Serum Albumin Human genes 0.000 description 9
- 108091006822 Human Serum Albumin Proteins 0.000 description 9
- 230000002068 genetic Effects 0.000 description 9
- 229940021015 I.V. solution additive Amino Acids Drugs 0.000 description 8
- 108020004999 Messenger RNA Proteins 0.000 description 8
- 108010014251 Muramidase Proteins 0.000 description 8
- 102000016943 Muramidase Human genes 0.000 description 8
- 241000320412 Ogataea angusta Species 0.000 description 8
- 230000002759 chromosomal Effects 0.000 description 8
- 230000000295 complement Effects 0.000 description 8
- 229960000274 lysozyme Drugs 0.000 description 8
- 239000004325 lysozyme Substances 0.000 description 8
- 235000010335 lysozyme Nutrition 0.000 description 8
- 229920002106 messenger RNA Polymers 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 239000003814 drug Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- -1 AOX1 Proteins 0.000 description 6
- 229940072221 IMMUNOGLOBULINS Drugs 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 210000001840 diploid cell Anatomy 0.000 description 6
- 229940079593 drugs Drugs 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 235000003642 hunger Nutrition 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 238000005457 optimization Methods 0.000 description 6
- 230000037351 starvation Effects 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- 229920002676 Complementary DNA Polymers 0.000 description 5
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 5
- 229920001850 Nucleic acid sequence Polymers 0.000 description 5
- 241001452677 Ogataea methanolica Species 0.000 description 5
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 230000003115 biocidal Effects 0.000 description 5
- 230000001276 controlling effect Effects 0.000 description 5
- 210000004748 cultured cells Anatomy 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 238000004949 mass spectrometry Methods 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000002194 synthesizing Effects 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 101710015954 HVA1 Proteins 0.000 description 4
- 101700065814 LEA2 Proteins 0.000 description 4
- 101700021338 LEC Proteins 0.000 description 4
- 101700077545 LECC Proteins 0.000 description 4
- 101700028499 LECG Proteins 0.000 description 4
- 101700063913 LECT Proteins 0.000 description 4
- 101710034340 Os04g0173800 Proteins 0.000 description 4
- 108010001267 Protein Subunits Proteins 0.000 description 4
- 102000002067 Protein Subunits Human genes 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K Tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 230000000875 corresponding Effects 0.000 description 4
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000000977 initiatory Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 101700036391 lecA Proteins 0.000 description 4
- 239000002523 lectin Substances 0.000 description 4
- 230000000670 limiting Effects 0.000 description 4
- 101700001016 mbhA Proteins 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000000717 retained Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000002103 transcriptional Effects 0.000 description 4
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 3
- 210000000628 Antibody-Producing Cells Anatomy 0.000 description 3
- 241000251730 Chondrichthyes Species 0.000 description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 3
- 229940110715 ENZYMES FOR TREATMENT OF WOUNDS AND ULCERS Drugs 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 3
- 108010033725 Recombinant Proteins Proteins 0.000 description 3
- 102000007312 Recombinant Proteins Human genes 0.000 description 3
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M Sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 3
- 108010084455 Zeocin Proteins 0.000 description 3
- VBJHPXDIVMXHJU-UHFFFAOYSA-N Zeocin Chemical compound N=1C(C=2SC=C(N=2)C(=O)NCCCCN=C(N)N)CSC=1CCNC(=O)C(C(O)C)NC(=O)C(C)C(O)C(C)NC(=O)C(C(OC1C(C(O)C(O)C(CO)O1)OC1C(C(OC(N)=O)C(O)C(CO)O1)O)C=1[N]C=NC=1)NC(=O)C1=NC(C(CC(N)=O)NCC(N)C(N)=O)=NC(N)=C1C VBJHPXDIVMXHJU-UHFFFAOYSA-N 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 230000001580 bacterial Effects 0.000 description 3
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 3
- 230000001851 biosynthetic Effects 0.000 description 3
- 238000006664 bond formation reaction Methods 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
- 239000010949 copper Substances 0.000 description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 3
- 150000002019 disulfides Chemical class 0.000 description 3
- 210000003783 haploid cell Anatomy 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 229940020899 hematological Enzymes Drugs 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 239000001573 invertase Substances 0.000 description 3
- 235000011073 invertase Nutrition 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000000087 stabilizing Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000001502 supplementation Effects 0.000 description 3
- 101700044023 ACVR1 Proteins 0.000 description 2
- 102100003919 ACVR1 Human genes 0.000 description 2
- 101700039383 AOX1 Proteins 0.000 description 2
- 108010059616 Activins Proteins 0.000 description 2
- 102000005606 Activins Human genes 0.000 description 2
- 229940064005 Antibiotic throat preparations Drugs 0.000 description 2
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 description 2
- 229940042052 Antibiotics for systemic use Drugs 0.000 description 2
- 108010083359 Antigen Receptors Proteins 0.000 description 2
- 102000006306 Antigen Receptors Human genes 0.000 description 2
- 229940042786 Antitubercular Antibiotics Drugs 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 2
- 241000255789 Bombyx mori Species 0.000 description 2
- 101710004053 CWINV2 Proteins 0.000 description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 2
- 210000000349 Chromosomes Anatomy 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N DEOXYTHYMIDINE Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 101700070526 DNMT1 Proteins 0.000 description 2
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 2
- 229940093922 Gynecological Antibiotics Drugs 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 229920001204 Intergenic region Polymers 0.000 description 2
- 229920002459 Intron Polymers 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N Kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 102100008175 MGAM Human genes 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N PMSF Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 108091005771 Peptidases Proteins 0.000 description 2
- OTYBMLCTZGSZBG-UHFFFAOYSA-L Potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 2
- 102100009178 RUNX1T1 Human genes 0.000 description 2
- 101710034060 RUNX1T1 Proteins 0.000 description 2
- 102000004389 Ribonucleoproteins Human genes 0.000 description 2
- 108010081734 Ribonucleoproteins Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 101700016532 SUC2 Proteins 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- 241001123650 Schwanniomyces occidentalis Species 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 description 2
- 231100000765 Toxin Toxicity 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K Trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 101700051226 US11 Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000000488 activin Substances 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000002596 correlated Effects 0.000 description 2
- 238000009510 drug design Methods 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 108091008472 immunoglobulin binding proteins Proteins 0.000 description 2
- 102000029028 immunoglobulin binding proteins Human genes 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 229940079866 intestinal antibiotics Drugs 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 229940005935 ophthalmologic Antibiotics Drugs 0.000 description 2
- 230000036961 partial Effects 0.000 description 2
- 230000001323 posttranslational Effects 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 description 2
- 235000011151 potassium sulphates Nutrition 0.000 description 2
- 230000012846 protein folding Effects 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 230000002797 proteolythic Effects 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 230000000754 repressing Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 108091007521 restriction endonucleases Proteins 0.000 description 2
- 101700059496 rnb Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000003248 secreting Effects 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 210000004215 spores Anatomy 0.000 description 2
- 230000002459 sustained Effects 0.000 description 2
- 230000001225 therapeutic Effects 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 108020003112 toxins Proteins 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 235000019798 tripotassium phosphate Nutrition 0.000 description 2
- 239000011778 trisodium citrate Substances 0.000 description 2
- 235000019263 trisodium citrate Nutrition 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229910052720 vanadium Inorganic materials 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- BRZYSWJRSDMWLG-DJWUNRQOSA-N (2R,3R,4R,5R)-2-[(1S,2S,3R,4S,6R)-4,6-diamino-3-[(2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-[(1R)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-DJWUNRQOSA-N 0.000 description 1
- KWTQSFXGGICVPE-WCCKRBBISA-N (2S)-2-amino-5-(diaminomethylideneamino)pentanoic acid;hydron;chloride Chemical compound Cl.OC(=O)[C@@H](N)CCCN=C(N)N KWTQSFXGGICVPE-WCCKRBBISA-N 0.000 description 1
- OIRDTQYFTABQOQ-GAWUUDPSSA-N 9-β-D-XYLOFURANOSYL-ADENINE Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@H](O)[C@H]1O OIRDTQYFTABQOQ-GAWUUDPSSA-N 0.000 description 1
- 102100002312 ADH5 Human genes 0.000 description 1
- OIRDTQYFTABQOQ-SXVXDFOESA-N Adenosine Natural products Nc1ncnc2c1ncn2[C@@H]3O[C@@H](CO)[C@H](O)[C@@H]3O OIRDTQYFTABQOQ-SXVXDFOESA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N Ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N Ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 241001508785 Arxiozyma Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 210000003719 B-Lymphocytes Anatomy 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N Boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 241000178289 Botryozyma Species 0.000 description 1
- 241000722885 Brettanomyces Species 0.000 description 1
- 241000235172 Bullera Species 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 101700017623 CALCA Proteins 0.000 description 1
- 102000033175 CALCA Human genes 0.000 description 1
- 101700041770 CALCR Proteins 0.000 description 1
- 102100008428 CCL2 Human genes 0.000 description 1
- 101700006000 CCL2 Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241001508787 Citeromyces Species 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L Cobalt(II) chloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 241000710127 Cricket paralysis virus Species 0.000 description 1
- 102000001493 Cyclophilins Human genes 0.000 description 1
- 108010068682 Cyclophilins Proteins 0.000 description 1
- 101710035145 DHODH Proteins 0.000 description 1
- 101710027899 DOTSEDRAFT_139328 Proteins 0.000 description 1
- 241000235035 Debaryomyces Species 0.000 description 1
- 206010011953 Decreased activity Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 102100016350 EIF2AK4 Human genes 0.000 description 1
- 102000033147 ERVK-25 Human genes 0.000 description 1
- 229940088598 Enzyme Drugs 0.000 description 1
- 241001465321 Eremothecium Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 230000036809 Fabs Effects 0.000 description 1
- 108060006676 GCN2 Proteins 0.000 description 1
- 102100014292 GZMM Human genes 0.000 description 1
- 101710014261 GZMM Proteins 0.000 description 1
- 210000002816 Gills Anatomy 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N HEPES Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102100015710 HSPA5 Human genes 0.000 description 1
- 102000006479 Heterogeneous-Nuclear Ribonucleoproteins Human genes 0.000 description 1
- 108010019372 Heterogeneous-Nuclear Ribonucleoproteins Proteins 0.000 description 1
- 210000004408 Hybridomas Anatomy 0.000 description 1
- 102000012745 Immunoglobulin Subunits Human genes 0.000 description 1
- 108010079585 Immunoglobulin Subunits Proteins 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N Inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 Inositol Drugs 0.000 description 1
- 108020004391 Introns Proteins 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L Iron(II) sulfate Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 241000235644 Issatchenkia Species 0.000 description 1
- 241001233945 Kazachstania Species 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 241001302784 Kodamaea Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 101700065946 LYS4 Proteins 0.000 description 1
- 241000221479 Leucosporidium Species 0.000 description 1
- 241001508815 Lodderomyces Species 0.000 description 1
- 101710033910 MT-I Proteins 0.000 description 1
- 108010038049 Mating Factor Proteins 0.000 description 1
- 108020003593 Met Transfer RNA Proteins 0.000 description 1
- UASDAHIAHBRZQV-YUMQZZPRSA-N Met-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N UASDAHIAHBRZQV-YUMQZZPRSA-N 0.000 description 1
- 206010027599 Migraine Diseases 0.000 description 1
- 208000008085 Migraine Disorders Diseases 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 108091007229 NSP3 Papain-like protease domain Proteins 0.000 description 1
- 241000537797 Ogataea nitratoaversa Species 0.000 description 1
- 229920000272 Oligonucleotide Polymers 0.000 description 1
- 241000415294 Orectolobidae Species 0.000 description 1
- 210000003463 Organelles Anatomy 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 101710043203 P23p89 Proteins 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 102100013574 POU2F1 Human genes 0.000 description 1
- 101710006192 POU2F1 Proteins 0.000 description 1
- 241000235652 Pachysolen Species 0.000 description 1
- 102000035443 Peptidases Human genes 0.000 description 1
- 206010034960 Photophobia Diseases 0.000 description 1
- 241000223252 Rhodotorula Species 0.000 description 1
- 102000004167 Ribonuclease P Human genes 0.000 description 1
- 108090000621 Ribonuclease P Proteins 0.000 description 1
- 210000003705 Ribosomes Anatomy 0.000 description 1
- 101710023382 S100A12 Proteins 0.000 description 1
- 235000003534 Saccharomyces carlsbergensis Nutrition 0.000 description 1
- 229940081969 Saccharomyces cerevisiae Drugs 0.000 description 1
- 241000235344 Saccharomycetaceae Species 0.000 description 1
- 241001149673 Saturnispora Species 0.000 description 1
- 241000235346 Schizosaccharomyces Species 0.000 description 1
- 210000002966 Serum Anatomy 0.000 description 1
- 108010070144 Single-Chain Antibodies Proteins 0.000 description 1
- 102000005632 Single-Chain Antibodies Human genes 0.000 description 1
- 102000004598 Small Nuclear Ribonucleoproteins Human genes 0.000 description 1
- 108010003165 Small Nuclear Ribonucleoproteins Proteins 0.000 description 1
- 229940083599 Sodium Iodide Drugs 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N Sodium molybdate Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 241000228389 Sporidiobolus Species 0.000 description 1
- 229920000978 Start codon Polymers 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102000004591 Telomerase Human genes 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- 241000183049 Tetrapisispora Species 0.000 description 1
- 229940104230 Thymidine Drugs 0.000 description 1
- 241000235006 Torulaspora Species 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Tris Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108020003635 Untranslated Regions Proteins 0.000 description 1
- 229920000146 Untranslated region Polymers 0.000 description 1
- 229940029983 VITAMINS Drugs 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 229940021016 Vitamin IV solution additives Drugs 0.000 description 1
- 241000235152 Williopsis Species 0.000 description 1
- 241000235013 Yarrowia Species 0.000 description 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L Zinc chloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 1
- 241000235017 Zygosaccharomyces Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 108010025188 alcohol oxidase Proteins 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 238000002617 apheresis Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000011087 biopharmaceutical technology Methods 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 235000010338 boric acid Nutrition 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001876 chaperonelike Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 1
- 230000001809 detectable Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 201000008286 diarrhea Diseases 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000000534 elicitor Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 108010014507 erythroagglutinating phytohemagglutinin Proteins 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 102000035428 foldases Human genes 0.000 description 1
- 108091005721 foldases Proteins 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108010051015 glutathione-independent formaldehyde dehydrogenase Proteins 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 230000002045 lasting Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 101700072735 lys-1 Proteins 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated Effects 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- NQMRYBIKMRVZLB-UHFFFAOYSA-M methanamine;chloride Chemical compound [Cl-].NC NQMRYBIKMRVZLB-UHFFFAOYSA-M 0.000 description 1
- 239000006151 minimal media Substances 0.000 description 1
- 108010000066 molecular chaperone GRP78 Proteins 0.000 description 1
- 102000005614 monoclonal antibodies Human genes 0.000 description 1
- 108010045030 monoclonal antibodies Proteins 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000000869 mutational Effects 0.000 description 1
- 229920001894 non-coding RNA Polymers 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 101700050775 oct-1 Proteins 0.000 description 1
- 230000003204 osmotic Effects 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic Effects 0.000 description 1
- 244000052769 pathogens Species 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000865 phosphorylative Effects 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000036417 physical growth Effects 0.000 description 1
- 230000001402 polyadenylating Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002035 prolonged Effects 0.000 description 1
- 230000001737 promoting Effects 0.000 description 1
- 230000002285 radioactive Effects 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 230000001718 repressive Effects 0.000 description 1
- 238000009781 safety test method Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000001568 sexual Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000009518 sodium iodide Nutrition 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- 230000000392 somatic Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 101700075565 sumT Proteins 0.000 description 1
- 230000004083 survival Effects 0.000 description 1
- 210000001519 tissues Anatomy 0.000 description 1
- 230000002588 toxic Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 108090000464 transcription factors Proteins 0.000 description 1
- 102000003995 transcription factors Human genes 0.000 description 1
- 108091006090 transcriptional activators Proteins 0.000 description 1
- 238000004450 types of analysis Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 101710035147 ura3 Proteins 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamins Natural products 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
Abstract
Discloses a method of producing full length antibodies, comprising: (a) providing a growth culture comprising Pichia pastoris cells comprising genes that provide for the expression of said full length antibodies and growing said Pichia yeast in said growth culture for about 12-16 hours (“growth phase”); (b) adding ethanol to said culture after said growth phase in a single bolus addition (“bolus”) which bolus results in a concentration of ethanol in said culture of between 0.5% and 1.5% w/v, and (c) culturing said culture to produce said secreted full length antibodies , wherein said culturing comprises adding a feed comprising a fermentable carbon source to said culture. se”); (b) adding ethanol to said culture after said growth phase in a single bolus addition (“bolus”) which bolus results in a concentration of ethanol in said culture of between 0.5% and 1.5% w/v, and (c) culturing said culture to produce said secreted full length antibodies , wherein said culturing comprises adding a feed comprising a fermentable carbon source to said culture.
Description
HIGH-PURITY PRODUCTION OF MULTI-SUBUNIT PROTEINS SUCH AS
ANTIBODIES IN TRANSFORMED MICROBES SUCH AS PICHIA PASTORIS
RELATED APPLICATION DISCLOSURE
The reader’s attention is also directed to our related divisional New Zealand
patent application No 718187.
This application claims the benefit of U.S. Provisional Application Ser. No.
61/525,307 (Atty. Docket No. 67858.730200), filed August 19, 2011 (entitled “MULTI-COPY
STRATEGY FOR HIGH-TITER AND HIGH-PURITY PRODUCTION OF MULTI-SUBUNIT
PROTEINS SUCH AS ANTIBODIES IN TRANSFORMED MICROBES SUCH AS PICHIA
PASTORIS,” U.S. Provisional Application Ser. No. 61/488,660 (Atty. Docket No.
67858.730300), filed May 20, 2011, entitled “ANTI-CGRP COMPOSITIONS AND USE
THEREOF,” U.S. Provisional Application Ser. No. 61/496,860 (Atty. Docket No.
67858.760000), filed June 14, 2011, entitled “USE OF ANTI-CGRP ANTIBODIES AND
ANTIBODY FRAGMENTS TO PREVENT OR INHIBIT PHOTOPHOBIA IN SUBJECTS IN
NEED THEREOF, ESPECIALLY MIGRAINE SUFFERERS,” and U.S. Provisional
Application Ser. No. 61/496,873 (Atty. Docket No. 67858.770000), filed June 14, 2011, entitled
“USE OF ANTI-CGRP ANTIBODIES AND ANTIBODY FRAGMENTS TO TREAT
DIARRHEA IN SUBJECTS WITH DISEASES OR TREATMENTS THAT RESULT IN
ELEVATED CGRP LEVELS” each of which is incorporated by reference herein in its entirety.
This application includes a Sequence Listing which is being submitted in ASCII
format via EFS-Web, in a file named “67858o711002.txt” created May 8, 2012 and having a size
of 315,392 bytes, which is hereby incorporated by reference in its entirety.
FIELD
The present disclosure generally relates to methods for producing heterologous
proteins in transformed cells. In particular, the present disclosure provides improved methods of
producing multi-subunit proteins, including antibodies and other multi-subunit proteins, which
may or may not be secreted, with decreased production of undesired side-products and/or
increased yield. In exemplary embodiments, the transformed cells are a yeast, such as Pichia
pastoris or Saccharomyces cerevisiae.
BACKGROUND
Conventional antibodies are tetrameric proteins composed of two identical light
chains and two identical heavy chains. Pure human antibodies of a specific type can be difficult
or impossible to purify from natural sources in sufficient amounts for many purposes. As a
consequence, biotechnology and pharmaceutical companies have turned to recombinant DNA-
based methods to prepare antibodies on a large scale. The production of functional antibodies
generally involves not just the synthesis of the two polypeptides but also a number of post-
translational events, including proteolytic processing of the N-terminal secretion signal
sequence; proper folding and assembly of the polypeptides into tetramers; formation of disulfide
bonds; and typically includes a specific N-linked glycosylation. All of these events take place in
the eukaryotic cell secretory pathway, an organelle complex unique to eukaryotic cells.
Recombinant synthesis of such complex proteins has typically relied on cultures
of higher eukaryotic cells to produce biologically active material, with cultured mammalian cells
being very commonly used. However, mammalian tissue culture-based production systems incur
significant added expense and complication relative to microbial fermentation methods.
Additionally, products derived from mammalian cell culture may require additional safety testing
to ensure freedom from mammalian pathogens (including viruses) that might be present in the
cultured cells or animal-derived products used in culture, such as serum.
Prior work has help to establish the yeast Pichia pastoris as a cost-effective
platform for producing functional antibodies that are potentially suitable for research, diagnostic,
and therapeutic use. See co-owned U.S. Patents 7,935,340 and 7,927,863, each of which is
incorporated by reference herein in its entirety. Methods are also known in the literature for
design and optimization of P. pastoris fermentations for expression of recombinant proteins,
including optimization of the cell density, broth volume, substrate feed rate, and the length of
each phase of the reaction. See Zhang et al., “Rational Design and Optimization of Fed-Batch
and Continuous Fermentations” in Cregg, J. M., Ed., 2007, Pichia Protocols (2nd edition),
Methods in Molecular Biology, vol. 389, Humana Press, Totowa, N.J., pgs. 43-63.
Though recombinant multi-subunit proteins can be produced from cultured cells,
undesired side-products may also be produced. For example, the cultured cells may produce the
desired multi-subunit protein along with free monomers, complexes having incorrect
stoichiometry, or proteins having undesired or aberrant glycosylation. Purification of the desired
multi-subunit protein can increase production cost, and the steps involved in purification may
decrease total yield of active complexes. Moreover, even after purification, undesired side-
products may be present in amounts that cause concern. For example, glycosylated side-
products may be present in amounts that increase the risk of an immune reaction after
administration, while aberrant complexes or aggregates may decrease specific activity and may
also be potentially immunogenic.
[0007A] In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that such
documents, or such sources of information, in any jurisdiction, are prior art, or form part of the
common general knowledge in the art.
[0007B] In the description in this specification reference may be made to subject matter
that is not within the scope of the claims of the current application. That subject matter should be
readily identifiable by a person skilled in the art and may assist in putting into practice the
invention as defined in the claims of this application.
SUMMARY
[0007C] The present invention relates to a method of producing a full length antibody,
comprising:
(a) providing a growth culture comprising Pichia pastoris cells comprising genes that
provide for the expression of said full length antibodies and growing said Pichia yeast in
said growth culture for about 12-16 hours (“growth phase”);
(b) adding ethanol to said culture after said growth phase in a single bolus addition
(“bolus”) which bolus results in a concentration of ethanol in said culture of between
0.5% and 1.5% w/v, and
(c) culturing said culture to produce said secreted full length antibodies, wherein said
culturing comprises adding a feed comprising a fermentable carbon source to said culture;
and further wherein said antibody does not comprise any of:
(i) an anti-human NGF antibody comprising the variable light (V ) chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 5, 6, and 7 and the variable heavy
(V ) chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 8, 9, and 10;
(ii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 15, 16, and 17 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 18, 19, and 20;
(iii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 25, 26, and 27 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 28, 29, and 30;
(iv) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 35, 36, and 37 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 38, 39, and 40;
(v) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 45, 46, and 47 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 48, 49, and 50;
(vi) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 55, 56, and 57 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 58, 59, and 60;
(vii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 65, 66, and 67 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 68, 69, and 70;
(viii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 75, 76, and 77 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 78, 79, and 80;
(ix) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 85, 86, and 87 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 88, 89, and 90;
(x) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 95, 96, and 97 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 98, 99, and 100;
(xi) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 105, 106, and 107 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 108, 109, and 110;
(xii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 115, 116, and 117 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 118, 119, and 120;
(xiii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 125, 126, and 127 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 128, 129, and 130;
(xiv) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 135, 136, and 137 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 138, 139, and 140;
(xv) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 145, 146, and 147 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 148, 149, and 150;
(xvi) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 155, 156, and 157 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 158, 159, and 160;
(xvii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 165, 166, and 167 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 168, 169, and 170;
(xviii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 175, 176, and 177 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 178, 179, and 180;
(xix) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 185, 186, and 187 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 188, 189, and 190; and
(xx) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and
CDR3 polypeptides of SEQ ID NO: 195, 196, and 197 and the V chain CDR1,
CDR2, and CDR3 polypeptides of SEQ ID NO: 198, 199, and 200.
[0007D] The present invention also provides full-length antibodies when produced by a
method of the invention.
Most IgG1 antibody molecules are stabilized by a total of 16 intra-chain and inter-
chain disulfide bridges. The intra-chain disulfide bridges stabilize the folding of the IgG
domains in both heavy and light chains, while the inter-chain disulfide bridges stabilize the
association between heavy and light chains. As a result of these bonds, antibodies form a stable
complex containing two heavy chains and two light chains (H2L2). However, due to improper
disulfide bond formation, product-associated variants are sometimes found in recombinant
antibody preparations, including a complex having one light and one heavy chain (H1L1) and a
complex having two heavy chains and one light chain (H2L1). Further, higher order complexes
may also form in which additional inter-chain disulfide bonds form, resulting in a greater number
of covalently linked subunits.
As further described below, Applicants have now identified methods of
decreasing the production of these complexes containing aberrant disulfide bonds during
recombinant production of antibodies from yeast culture. Specifically, the method involves
addition of an ethanol bolus to the culture, and resulted in decreased production of the H1L1,
H2L1, and H4L4 products-associated variants, and increased purity of the desired H2L2 product.
The H1L1 and H2L1 complexes were detected by non-reduced, denaturing SDS-PAGE, and the
H4L4 complexes were detected by size exclusion chromatography. Using the subject methods,
proper disulfide bond formation was facilitated, resulting in increased antibody purity. This was
demonstrated for three different antibodies, all three of which exhibited improved purity when
produced with the addition of the ethanol bolus (FIGS. 1-6). These three antibodies are not only
are different in sequence but also recognize three different antigens. Moreover, when produced
in the absence of an ethanol bolus, two of the antibodies contained greater amounts of the H1L1
product (FIGS. 1-4), compared with the third antibody (. The two antibodies containing
greater amounts of the H1L1 product have a non-canonical or additional disulfide bridge,
whereas the third does not. The antibody exemplified in FIGS. 1, 2 and 3 has an additional intra-
chain disulfide bridge in the variable light chain domain, while the antibody exemplified in has an additional intra-chain disulfide bridge in its heavy chain. It has been reported in the
literature that the presence of disulfide-bridges in overexpressed proteins increases intracellular
stress in the host (see Gasser et al., Biotechnology and Bioengineering, Vol. 94, No. 2, pg. 353-
61, June 5, 2006; Inan et al., Biotechnology And Bioengineering, Vol. 93, No. 4, pg. 771-78,
March 5, 2006; Li et al., Biochem Biophys Res Commun. 2010 November 19; 402(3): 519–524).
This increased stress can also lead to lower viability, as is demonstrated in FIGS. 11, 12, and 13
where both antibodies with the extra intra-chain disulfide bridge have lower viability under the
“no-bolus” conditions. The addition of the ethanol bolus, therefore leads to increased viability
and increased purity. This may be of use in particular when difficult to express proteins with
multiple disulfide bridges are being expressed.
Described is a method of producing a multi-subunit complex, comprising: (a)
providing a culture comprising a eukaryotic cells comprising genes that provide for the
expression of the subunits of said multi-subunit complex; (b) adding a bolus of ethanol to said
culture; and (c) culturing said culture to produce said multi-subunit complex.
The ethanol bolus may enhance the formation of stable disulfide bonds relative to
the same method effected in the absence of the bolus of ethanol.
Said multi-subunit complex may contain one or more polypeptides comprising at
least one disulfide bond.
Said multi-subunit complex may comprise an antibody.
The method may decrease the relative abundance of one or more product-
associated variants relative to the same method effected in the absence of the bolus of ethanol.
The method may decrease the relative abundance of product-associated variants
having a higher or lower apparent molecular weight than said desired multi-subunit complex as
detected by size exclusion chromatography or gel electrophoresis relative to the same method
effected in the absence of the bolus of ethanol.
The method may decrease the relative abundance of complexes having aberrant
stoichiometry relative to the same method effected in the absence of the bolus of ethanol.
The method may decrease the relative abundance of complexes having aberrant
disulfide bonds relative to the same method effected in the absence of the bolus of ethanol.
The method may decrease the relative abundance of complexes having reduced
cysteines relative to the same method effected in the absence of the bolus of ethanol.
The method may decrease the relative abundance of complexes having aberrant
glycosylation relative to the same method effected in the absence of the bolus of ethanol.
The method may modulate the formation or stability of inter-heavy chain
disulfide bonds.
The method may modulate the formation or stability of disulfide bonds linking the
light and heavy chains.
The method may decrease the relative abundance of one or more product-
associated variants relative to the same method effected in the absence of the bolus of ethanol.
Said product-associated variants may comprise one or more of the H1L1, H2L1,
and H4L4 product-associate variants.
The method increase the purity of said antibody relative to said method effected
in the absence of said bolus of ethanol.
Step (b) may be effected prior to step (c).
Step (b) may be effected subsequent to step (c).
Step (b) may be effected concurrently with step (c).
Step (b) may result in a concentration of ethanol in said culture of between about
0.01% and about 4% (w/v).
Step (b) may result in a concentration of ethanol in said culture of between about
0.01% and about 4%, between about 0.02% and about 3.75%, between about 0.04% and about
3.5%, between about 0.08% and about 3.25%, between about 0.1% and about 3%, between about
0.2% and about 2.75%, between about 0.3% and about 2.5%, between about 0.4% and about
2.25%, between about 0.5% and about 1.5%, between about 0.5% and about 2%, between about
0.6% and about 1.75%, between about 0.7% and about 1.5%, or between about 0.8% and about
1.25%.
Step (b) may result in a concentration of ethanol in said culture that may be at
least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%,
0.3%, 0.4%, 0.6%, 0.6%, 0.7%, 0.8% or 0.9% (w/v).
Step (b) may result in a concentration of ethanol in said culture that may be at
most about 4%, 3.5%, 3%, 2.5%, 2%, 1.8%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%,
0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.35%, 0.3%, 0.25%, 0.2%, or 0.15% (w/v).
Step (b) may comprise adding ethanol to said culture, adding a carrier comprising
ethanol to said culture, adding said cells to a medium or carrier comprising ethanol, or replacing
part of the culture medium.
Said bolus of ethanol may be added to the culture medium over a period of time
between 1 and 20 minutes.
Step (c) may comprise providing oxygen to said cells.
Said providing oxygen may comprise agitating said culture.
Said providing oxygen may comprise contacting said culture with a gas mixture
comprising oxygen.
Step (c) may comprise adding a feed comprising a carbon source to said culture.
Said feed may comprise at least one fermentable carbon source.
Said feed may comprise one or more of glucose, ethanol, citrate, sorbitol, xylose,
trehalose, arabinose, galactose, fructose, melibiose, lactose, maltose, rhamnose, ribose, mannose,
mannitol, and raffinose.
The method may further comprise maintaining the concentration of ethanol
between an upper set point and a lower set point during step (c).
Said lower set point may be about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%,
0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.6%, 0.6%, 0.7%, 0.8% or 0.9% (w/v).
Said upper set point may be about 4%, 3.5%, 3%, 2.5%, 2%, 1.8%, 1.6%, 1.5%,
1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.35%, 0.3%, 0.25%,
0.2%, or 0.15% (w/v).
Said upper set point may be at most about 1.5%, 1.4%, 1.3, 1.2%, or 1.1% (w/v).
The method may further comprise maintaining the concentration of ethanol at a
set point during step (c).
Said set point may be about 0.1%, 0.2%, 0. 3%, 0. 4%, 0.5%, 0.6%, 0.7%, 0.8%,
0.9%, 01.%, 01.1%, 01.2%, 01.3%, 01.4%, or 01.5% (w/v).
Step (c) may comprise maintaining the concentration of ethanol in said culture
between about 0.01% and about 4%, between about 0.02% and about 3.75%, between about
0.04% and about 3.5%, between about 0.08% and about 3.25%, between about 0.1% and about
3%, between about 0.2% and about 2.75%, between about 0.3% and about 2.5%, between about
0.4% and about 2.25%, between about 0.5% and about 2%, between about 0.6% and about
1.75%, between about 0.7% and about 1.5%, or between about 0.8% and about 1.25%.
The concentration of ethanol in said culture may be maintained by controlling
production of ethanol by said cells or by addition of ethanol to said culture.
The step of controlling production of ethanol may comprise controlling one or
more of the concentration of glucose, availability of oxygen, intensity of agitation, gas pressure,
flow rate of supplied air or other gas mixture, viscosity of the culture, culture density,
concentration of oxygen in the supplied air or other gas mixture, and temperature.
The time between step (a) and step (b) may be less than about 72 hours, less than
about 48 hours, less than about 24 hours, less than about 12 hours, less than about 9 hours, less
than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less
than about 90 minutes, less than about 30 minutes, less than about 5 minutes, or less than about 1
minute.
The time between step (b) and step (c) may be less than about 10 hours, less than
about 9 hours, less than about 8 hours, less than about 7 hours, less than about 6 hours, less than
about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, less than
about 90 minutes, less than about 80 minutes, less than about 70 minutes, less than about 60
minutes, less than about 50 minutes, less than about 40 minutes, less than about 30 minutes, less
than about 20 minutes, less than about 10 minutes, less than about 5 minutes, or less than about 1
minute.
The culture of step (a) may be produced by adding a carbon source to said culture,
and culturing said culture until the carbon source may be depleted.
Said carbon source may comprise one or more of: glycerol, glucose, ethanol,
citrate, sorbitol, xylose, trehalose, arabinose, galactose, fructose, melibiose, lactose, maltose,
rhamnose, ribose, mannose, mannitol, and raffinose.
The depletion of the carbon source may be determined by detecting a decrease in
the metabolic activity of said eukaryotic cells.
Said decrease in the metabolic activity of said eukaryotic cells may be identified
by detecting a decrease in the consumption of oxygen by said eukaryotic cells, by detecting an
increase in pH in the culture, by detecting stabilization of the wet cell mass, or by detecting an
increase in the concentration of ammonia in the culture.
Said decrease in the consumption of oxygen by said eukaryotic cells may be
identified by detecting an increase in the concentration of dissolved oxygen in said culture.
Said eukaryotic cells may comprise yeast cells.
Said yeast cells may comprise methylotrophic yeast.
Said methylotrophic yeast may be of the genus Pichia.
Said methylotrophic yeast of the genus Pichia may be Pichia pastoris.
Said methylotrophic yeast of the genus Pichia may be selected from the group
consisting of: Pichia angusta, Pichia guillermordii, Pichia methanolica, and Pichia inositovera.
The genes that provide for expression of said multi-subunit complex may be
integrated into one or more genomic loci.
At least one of said genomic loci may be selected from the group consisting of the
pGAP locus, 3’ AOX TT locus; PpURA5; OCH1; AOX1; HIS4; GAP; pGAP; 3’ AOX TT;
ARG; and the HIS4 TT locus.
At least one of the genes encoding said subunits of the multi-subunit complex
may be expressed under control of an inducible or constitutive promoter.
Said inducible promoter may be selected from the group consisting of the AOX1,
CUP1, tetracycline inducible, thiamine inducible, and FLD1 promoters.
At least one of the genes encoding said subunits of the multi-subunit complex
may be expressed under control of a promoter selected from the group consisting of: the CUP1,
AOX1, ICL1, glyceraldehydephosphate dehydrogenase (GAP), FLD1, ADH1, alcohol
dehydrogenase II, GAL4, PHO3, PHO5, and Pyk promoters, tetracycline inducible promoters,
thiamine inducible promoters, chimeric promoters derived therefrom, yeast promoters,
mammalian promoters, insect promoters, plant promoters, reptile promoters, amphibian
promoters, viral promoters, and avian promoters.
Said eukaryotic cell may be a diploid, tetraploid cell, or polyploid.
The method may further comprise purifying said multi-subunit complex from said
eukaryotic cells or from the culture medium.
Said multi-subunit complex may be purified from an intracellular component,
cytoplasm, nucleoplasm, or a membrane of said eukaryotic cells.
Said eukaryotic cells secrete said multi-subunit complex into the culture medium.
Said multi-subunit complex may be purified from said culture medium.
Said multi-subunit complex may comprise a monospecific or bispecific antibody.
Said multi-subunit complex may comprise a human antibody or a humanized
antibody or fragment thereof.
Said humanized antibody may be of mouse, rat, rabbit, goat, sheep, or cow origin.
Said humanized antibody may be of rabbit origin.
Said multi-subunit complex may comprise a monovalent, bivalent, or multivalent
antibody.
Said antibody may be purified from said culture by protein A and/or protein G
affinity.
At least one of the genes that provide for expression of a subunit of said multi-
subunit complex in at least one of said eukaryotic cells in said panel may be optimized for
expression in said eukaryotic cell.
Said multi-subunit complex may comprise an antibody and the purity of said
antibody may be assessed by measuring the fraction of the antibody produced by said eukaryotic
cell that may be contained in antibody complexes having the expected apparent hydrodynamic
radius, may be contained in antibody complexes having the expected molecular weight, and / or
specifically binds a target of said antibody.
Said multi-subunit complex may comprise an antibody and the yield of said
antibody may be assessed by determining the amount of antibody produced by said eukaryotic
cell discounting any product-associated variants that may be abnormally glycosylated, contained
in antibody complexes other than complexes having the expected apparent hydrodynamic radius,
contained in antibody complexes having the expected molecular weight, and / or that fail to
specifically bind to the target of said antibody.
The molecular weight of said antibody complexes may be determined by non-
reducing SDS-PAGE.
Said multi-subunit complex may comprise an antibody, said method may further
comprise purifying said antibody.
Said culture cell may produce a supernatant antibody titer of at least 100 mg / L,
at least 150 mg / L, at least 200 mg / L, at least 250 mg / L, at least 300 mg / L, between 100 and
300 mg / L, between 100 and 500 mg / L, between 100 and 1000 mg / L, at least 1000 mg / L, at
least 1250 mg/liter, at least 1500 mg/liter, at least about 1750 mg/liter, at least about 2000
mg/liter, at least about 10000 mg/liter, or more.
One or more subunits of said multi-subunit complex may be expressed from more
than one gene copy.
Said multi-subunit complex may comprise an antibody which may be expressed
from between 1-10 copies of a gene encoding the light chain of said antibody and from 1-10
copies of a gene encoding the heavy chain of said antibody.
The genes that provide for expression of said multi-subunit complex may be
integrated into genome of said cells.
The genes that provide for expression of said multi-subunit complex may be
contained on an extrachromosomal element, plasmid, or artificial chromosome.
Said cells may comprise more copies of the gene that provide for the expression
of the light chain of said antibody than copies of the gene that provide for expression of the
heavy chain of said antibody.
The respective number of copies of the gene encoding the heavy chain of said
antibody and the number of copies of the gene encoding the light chain of said antibody in said
cells may be: 2 and 2, 2 and 3, 3 and 3, 3 and 4, 3 and 5, 4 and 3, 4 and 4, 4 and 5, 4 and 6, 5 and
4, 5 and 5, 5 and 6, or 5 and 7.
The respective number of copies of the gene encoding the heavy chain of said
antibody and the number of copies of the gene encoding the light chain of said antibody in said
cells may be: 2 and 1, 3 and 1, 4 and 1, 5 and 1, 6 and 1, 7 and 1, 8 and 1, 9 and 1, 10 and 1, 1
and 2, 2 and 2, 3 and 2, 4 and 2, 5 and 2, 6 and 2, 7 and 2, 8 and 2, 9 and 2, 10 and 2, 1 and 3, 2
and 3, 3 and 3, 4 and 3, 5 and 3, 6 and 3, 7 and 3, 8 and 3, 9 and 3, 10 and 3, 1 and 4, 2 and 4, 3
and 4, 4 and 4, 5 and 4, 6 and 4, 7 and 4, 8 and 4, 9 and 4, 10 and 4, 1 and 5, 2 and 5, 3 and 5, 4
and 5, 5 and 5, 6 and 5, 7 and 5, 8 and 5, 9 and 5, 10 and 5, 1 and 6, 2 and 6, 3 and 6, 4 and 6, 5
and 6, 6 and 6, 7 and 6, 8 and 6, 9 and 6, 10 and 6, 1 and 7, 2 and 7, 3 and 7, 4 and 7, 5 and 7, 6
and 7, 7 and 7, 8 and 7, 9 and 7, 10 and 7, 1 and 8, 2 and 8, 3 and 8, 4 and 8, 5 and 8, 6 and 8, 7
and 8, 8 and 8, 9 and 8, 10 and 8, 1 and 9, 2 and 9, 3 and 9, 4 and 9, 5 and 9, 6 and 9, 7 and 9, 8
and 9, 9 and 9, 10 and 9, 1 and 10, 2 and 10, 3 and 10, 4 and 10, 5 and 10, 6 and 10, 7 and 10, 8
and 10, 9 and 10, 10 and 10.
The culture of step (c) may be grown in a production medium.
Said production medium may be a minimal medium.
Said minimal medium lacks selective agents.
Said minimal medium lacks pre-formed amino acids or other complex
biomolecules.
The production medium may be a complex medium.
The complex medium may comprise one or more of yeast extract, soy peptones,
and other plant peptones.
The culture of step (c) may be grown to a high cell density.
Said high cell density may be at least 50 g/L.
Said high cell density may be at least 100 g/L.
Said high cell density may be at least 300 g/L.
Said high cell density may be at least 400 g/L.
Said high cell density may be at least 500 g/L.
Said high cell density may be at least 750 g/L.
The yeast cells may be cultured for at least 20 doublings and maintain high levels
of expression of said multi-subunit complex after said at least 20 doublings.
The cells of step (c) may be cultured for at least 50 doublings and maintain high
levels of expression of said multi-subunit complex after said at least 50 doublings.
The cells of step (c) may be cultured for at least 100 doublings and maintain high
levels of expression of said multi-subunit complex after said at least 100 doublings.
At least one subunit of said multi-subunit complex may comprise a secretion
signal.
Said multi-subunit complex may comprise an antibody.
The secretion signal may comprise one or more polypeptides selected from the
group consisting of: SEQ ID NOS: 414 to 437 and any combination thereof.
Said multi-subunit complex may be not any of the antibodies disclosed in U.S.
Provisional Application No. 61/418,832, filed December 1, 2010, PCT/US11/62963, filed
December 1, 2011, U.S. Ser. No. 13/309,295, filed December 1, 2011, U.S. Ser. No. 13/309,153,
filed December 1, 2011, U.S. Ser. No. 13/308,665 filed on December 1, 2011, and U.S. Ser. No.
13/308,831, filed December 1, 2011.
Said multi-subunit complex may not be Ab1-NGF, Ab2-NGF, Ab3-NGF, Ab4-
NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, Ab10-NGF, Ab11-NGF, Ab12-
NGF, Ab13-NGF, Ab14-NGF, Ab15-NGF, Ab16-NGF, Ab17-NGF, Ab18-NGF, Ab19-NGF,
Ab20-NGF, and Ab21-NGF, or an Fab2 or Fab1 fragment thereof.
Said multi-subunit complex may not contain at least one, at least two, at least
three, at least four, at least five, or at least all six of the complementarity determining regions
(CDRs) contained in any of the following antibodies: Ab1-NGF, Ab2-NGF, Ab3-NGF, Ab4-
NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, Ab10-NGF, Ab11-NGF, Ab12-
NGF, Ab13-NGF, Ab14-NGF, Ab15-NGF, Ab16-NGF, Ab17-NGF, Ab18-NGF, Ab19-NGF,
Ab20-NGF, or Ab21-NGF and optionally having binding specificity for NGF.
Said multi-subunit complex may not comprise or consist of the light and heavy
chain polypeptide sequences of SEQ ID NOs: 51 and 401, respectively, SEQ ID NOs: 53 and
402, respectively, SEQ ID NOs: 405 and 406, respectively, and SEQ ID NOs: 407 and 408,
respectively.
Said multi-subunit complex may not comprise an antibody containing at least one,
at least two, at least three, at least four, at least five, or at least all six of the CDRs of SEQ ID
NOs: 55, 56, 57, 58, 59, and 60, and optionally having binding specificity for NGF.
Said multi-subunit complex may not comprise any of the antibodies or antibody
coding sequences disclosed herein in the sections entitled “Anti-NGF Antibodies and Binding
Fragments Thereof Having Binding Activity for NGF” and “Polynucleotides Encoding Anti-
NGF Antibody Polypeptides.”In one aspect, the present disclosure provides methods of
producing a multi-subunit complex, which may comprise: culturing a host cell providing a
culture comprising eukaryotic cells that expresses said multi-subunit complex, adding a bolus
7of ethanol to said culture, and culturing said culture to produce said multi-subunit complex.
The multi-subunit complex may comprise one or more disulfide bonds, and may be an antibody.
The ethanol bolus concentration (expressed as % w/v) may be between about
0.1% and about 5%, such as at least about 0.1%, at least about 0.2%, at least about 0.3%, at least
about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at
least about 0.9%, at least about 1%, up to about 1%, up to about 1.1%, up to about 1.2%, up to
about 1.3%, up to about 1.4%, up to about 1.5%, up to about 1.6%, up to about 1.7%, up to about
1.8%, up to about 1.9%, up to about 2%, up to about 3%, up to about 4%, or up to about 5%,
such as between about 0.1% and about 1.9%, between about 0.2% and about 1.8%, between
about 0.3% and about 1.7%, between about 0.4% and about 1.6%, between about 0.5% and about
1.5%, between about 0.6% and about 1.4%, between about 0.7% and about 1.3%, between about
0.8% and about 1.2%, or between about 0.9% and about 1.1%, such as about 0.1%, about 0.2%,
about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about
1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%,
about 1.8%, about 1.9%, about 2%, about 2.5%, about 3%, about 4%, or about 5%.
The method may further comprise purification of said desired multi-subunit
complex.
In exemplary embodiments the ethanol concentration may be controlled
subsequent to addition of the ethanol bolus, which may be used to maintain the ethanol
concentration at a desired set point or within a desired set point range. The set point (expressed
as % w/v) may be between about 0.1% and about 4%, at least about 0.01%, at least about 0.02%,
at least about 0.04%, at least about 0.06%, at least about 0.08%, at least about 0.1%, at least
about 0.15%, at least about 0.2%, at least about 0.25%, at least about 0.3%, at least about 0.35%,
at least about 0.4%, at least about 0.45%, at least about 0.5%, at least about 0.6%, at least about
0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 1.2%, at least
about 1.4%, at least about 1.6%, at least about 1.8%, at least about 2%, up to about 4%, up to
about 3.75%, up to about 3.5%, up to about 3.25%, up to about 3%, up to about 2.75%, up to
about 2.5%, up to about 2.25%, up to about 2%, up to about 1.75%, up to about 1.5%, up to
about 1.25%, up to about 1%, between about 0.01% and about 4%, between about 0.02% and
about 3.75%, between about 0.04% and about 3.5%, between about 0.08% and about 3.25%,
between about 0.1% and about 3%, between about 0.2% and about 2.75%, between about 0.3%
and about 2.5%, between about 0.4% and about 2.25%, between about 0.5% and about 2%,
between about 0.6% and about 1.75%, between about 0.7% and about 1.5%, or between about
0.8% and about 1.25%. For example, the set point may be the same as the bolus concentration or
within plus or minus 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 60%,
70%, 80%, 90% or 100% of the bolus concentration.
The ethanol concentration set point may be maintained by controlling ethanol
production by yeast cells during fermentation. For example, the ethanol concentration may be
increased by increasing the concentration of glucose (e.g., increasing the rate of glucose feed),
decreasing the availability of oxygen, by decreasing the intensity of agitation (e.g., lowering the
fermenter input power), decreasing the gas pressure in the fermenter, decreasing the flow rate of
supplied air or other gas mixture, increasing the viscosity of the culture, or decreasing the
concentration of oxygen in the supplied air or other gas mixture (e.g., if oxygen supplementation
is being used). Ethanol production may also be increased by increasing the fermentation
temperature. Likewise, the ethanol concentration may be decreased by decreasing the glucose
concentration (e.g., decreasing the rate of glucose feed), decreased by increasing the availability
of oxygen, by increasing the intensity of agitation (e.g., increasing the fermenter input power),
increasing the gas pressure in the fermenter, increasing the flow rate of supplied air or other gas
mixture, decreasing the viscosity of the culture, or increasing the concentration of oxygen in the
supplied air or other gas mixture (e.g., if oxygen supplementation is being used). Ethanol
production may also be decreased by decreasing the fermentation temperature.
Using the methods of the present disclosure, the relative abundance of undesired
side-product(s) may be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 80% , at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or down to undetectable levels compared to initial
abundance levels, relative to conventional methods. Exemplary undesired side-products whose
relative abundance may be so decreased may include one or more species having a different
apparent molecular weight than the desired multi-subunit complex. For example, apparent
molecular weight may be affected by differences in stoichiometry, folding, complex assembly,
and/or glycosylation. For example, such undesired side products may be detected using size
exclusion chromatography and/or gel electrophoresis, and may have a higher or lower apparent
molecular weight than the desired multi-subunit complex. In exemplary embodiments, the
undesired side-products may be detected under reducing conditions. In other exemplary
embodiments, the undesired side-products may be detected under non-reducing conditions.
In exemplary embodiments, described are improved methods and compositions of
matter that provide for the recombinant production of antibodies and other multi-subunit
complexes, with a higher yield. In exemplary embodiments, the yield may be increased by at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or more (relative
to conventional methods) using the methods disclosed herein.
In exemplary embodiments, the host cell in which the multi-subunit proteins may
be produced may be a yeast, for example in a Pichia species such as P. pastoris or another
methylotrophic yeast, or in a Saccharomyces species such as S. cerevisiae, or another yeast such
as a Schizosaccharomyces (e.g., S. pombe). Other examples of methylotrophic yeast which may
be utilized include Pichia angusta (also known in the art as Hansenula polymorpha), Pichia
guillermordii, Pichia methanolica, Pichia inositovera, Ogataea nitratoaversa, and Candida
boidnii.
The host cell may be a eukaryotic cell, such as a yeast cell, such as a
methylotrophic yeast, such as a yeast of the genus Pichia. Exemplary methylotrophic yeasts of
the genus Pichia include Pichia pastoris, Pichia angusta, Pichia guillermordii, Pichia
methanolica, and Pichia inositovera. The host cell may be produced by mating, e.g., by mating
two haploid yeast cells that each contain one or more copies of at least one gene encoding a
subunit of the multi-subunit complex.
In a preferred embodiment, the methylotrophic yeasts of the genus Pichia is
Pichia pastoris. The host cell may be a diploid or tetraploid cell.
At least one of said genes encoding said subunits of the desired multi-subunit
complex, such as said desired antibody light chain and / or heavy chain, may be expressed under
control of an inducible or constitutive promoter, such as CUP1 (induced by the level of copper in
the medium; see Koller et al., Yeast 2000; 16: 651-656.), tetracycline inducible promoters (see,
e.g., Staib et al., Antimicrobial Agents And Chemotherapy, Jan. 2008, p. 146–156), thiamine
inducible promoters, AOX1, ICL1, glyceraldehydephosphate dehydrogenase (GAP), FLD1,
ADH1, alcohol dehydrogenase II, GAL4, PHO3, PHO5, and Pyk promoters, chimeric promoters
derived therefrom, yeast promoters, mammalian promoters, insect promoters, plant promoters,
reptile promoters, amphibian promoters, viral promoters, and avian promoters.
The host cell may secrete said desired multi-subunit complex into the culture
medium. Alternatively or in addition, said desired multi-subunit complex may be retained in
said host cell and may be isolated therefrom.
The desired multi-subunit complex may comprise an antibody, such as a
monospecific or bispecific antibody. The antibody may be an antibody that specifically binds
any antigen.
The desired multi-subunit complex may be an antibody other than any of the
antibodies (e.g., an antibody other than any of the anti-NGF antibodies) disclosed in U.S.
Provisional Application No. 61/418,832, filed December 1, 2010, PCT/US11/62963, filed
December 1, 2011, U.S. Ser. No. 13/309,295, filed December 1, 2011, U.S. Ser. No. 13/309,153,
filed December 1, 2011, U.S. Ser. No. 13/308,665 filed on December 1, 2011, and U.S. Ser. No.
13/308,831, filed December 1, 2011. In an exemplary embodiment, the desired multi-subunit
complex may not be any of the following antibodies: Ab1-NGF, Ab2-NGF, Ab3-NGF, Ab4-
NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, Ab10-NGF, Ab11-NGF, Ab12-
NGF, Ab13-NGF, Ab14-NGF, Ab15-NGF, Ab16-NGF, Ab17-NGF, Ab18-NGF, Ab19-NGF,
Ab20-NGF, and Ab21-NGF. In a further exemplary embodiment, the desired multi-subunit
complex may not be an Fab2 fragment of any of the following antibodies: Ab1-NGF, Ab2-NGF,
Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, Ab10-NGF,
Ab11-NGF, Ab12-NGF, Ab13-NGF, Ab14-NGF, Ab15-NGF, Ab16-NGF, Ab17-NGF, Ab18-
NGF, Ab19-NGF, Ab20-NGF, and Ab21-NGF. In a further exemplary embodiment, the desired
multi-subunit complex may not be an Fab1 fragment of any of the following antibodies: Ab1-
NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF,
Ab10-NGF, Ab11-NGF, Ab12-NGF, Ab13-NGF, Ab14-NGF, Ab15-NGF, Ab16-NGF, Ab17-
NGF, Ab18-NGF, Ab19-NGF, Ab20-NGF, and Ab21-NGF. In a further exemplary
embodiment, the desired multi-subunit complex may not comprise an antibody containing at
least one, at least two, at least three, at least four, at least five, or at least all six of the
complementarity determining regions (CDRs) contained in any of the following antibodies: Ab1-
NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF,
Ab10-NGF, Ab11-NGF, Ab12-NGF, Ab13-NGF, Ab14-NGF, Ab15-NGF, Ab16-NGF, Ab17-
NGF, Ab18-NGF, Ab19-NGF, Ab20-NGF, or Ab21-NGF and optionally having binding
specificity for NGF. For example, the desired multi-subunit complex may not comprise or may
not consist of the light and heavy chain polypeptide sequences SEQ ID NOs: 51 and 401,
respectively, and/or SEQ ID NOs: 53 and 402, respectively, and/or SEQ ID NOs: 405 and 406,
respectively, and/or SEQ ID NOs: 407 and 408, respectively. As a further example, the desired
multi-subunit complex may not comprise an antibody containing at least one, at least two, at
least three, at least four, at least five, or at least all six of the CDRs of SEQ ID NOs: 55, 56, 57,
58, 59, and 60, and optionally having binding specificity for NGF.
The desired multi-subunit complex may comprise an antibody of any type.
Exemplary antibody types include antibodies of any mammalian species, e.g., human, mouse,
rat, rabbit, goat, sheep, cow, etc. Preferably, the antibody is a human antibody or a humanized
antibody that may be of rabbit origin. The desired antibody may be a monovalent, bivalent, or
multivalent antibody.
At least one of said genes that provide for expression of a subunit of the desired
multi-subunit complex, such as the light chain and/or heavy chain of a desired antibody, in at
least one of said host cells in said panel may be optimized for expression in said host cell (e.g.,
by selecting preferred codons and/or altering the percentage AT through codon selection).
The purity of said desired multi-subunit complex, such as a desired antibody, may
be assessed by measuring the fraction of the desired multi-subunit complex produced by said
host cell that is non-glycosylated, is contained in complexes having the expected apparent
hydrodynamic radius and/or apparent molecular weight (e.g., measured by size exclusion
chromatography), has the expected electrophoretic mobility (e.g., detected by gel
electrophoresis, such as SDS-PAGE, and optionally Western blotting), and / or by measuring the
specific activity of the multi-subunit complex (e.g., specific binding a target of a desired
antibody).
The desired multi-subunit complex may be an antibody, and yield of said
antibody may be assessed by determining the amount of desired antibody produced by said host
cell discounting any product-associated variants that are glycosylated, contained in antibody
complexes other than complexes having the expected apparent molecular weight or
hydrodynamic radius, and / or that fail to specifically bind to the target of said desired antibody.
The subject methods may produce a supernatant antibody titer of at least 100 mg /
L, at least 150 mg / L, at least 200 mg / L, at least 250 mg / L, at least 300 mg / L, between 100
and 300 mg / L, between 100 and 500 mg / L, between 100 and 1000 mg / L or in excess of 1000
mg/L e.g., as high as 1200 mg/L, as high as 10,000 mg / L, or higher.
In another embodiment described, the host cell that produces a desired multi-
subunit complex may be a diploid or tetraploid cell of the genus Pichia, such as a Pichia pastoris
cell. The genes that provide for expression of the subunits of said desired multi-subunit
complex, such as the light chain and heavy chain of a desired antibody, may be integrated into
genome of said host cell, and/or may be contained on an extrachromosomal element, plasmid, or
artificial chromosome.
In another embodiment described, the host cell that produces a desired multi-
subunit complex may be engineered to increase yield and / or purity for example as further
described in U.S. Provisional Application Ser. No. 61/525,307 (Atty. Docket No.
67858.730200), filed August 31, 2011, which is incorporated by reference herein in its entirety.
As described therein, yield and purity of an antibody or other multi-subunit complex can be
greatly improved by altering the number of copies per cell of the genes encoding each subunit.
For example, where the desired multi-subunit complex is an antibody, the host cell may comprise
more copies of the gene that provide for the expression of the light chain than copies of the gene
that provide for expression of the heavy chain. In exemplary embodiments, the host cell may
comprise from 1-10 copies of a gene encoding the light chain and from 1-10 copies of a gene
encoding the heavy chain. The respective number of copies of the gene encoding the heavy chain
and the number of copies of the gene encoding the light chain in said host cell may be: 2 and 2, 2
and 3, 3 and 3, 3 and 4, 3 and 5, 4 and 3, 4 and 4, 4 and 5, 4 and 6, 5 and 4, 5 and 5, 5 and 6, or 5
and 7, respectively. Additional exemplary combinations of heavy and light chain gene copy
numbers include any combination of up to ten copies of the heavy and/or light chain gene, such
as H2xL1, H3xL1, H4xL1, H5xL1, H6xL1, H7xL1, H8xL1, H9xL1, H10xL1, H1xL2, H2xL2,
H3xL2, H4xL2, H5xL2, H6xL2, H7xL2, H8xL2, H9xL2, H10xL2, H1xL3, H2xL3, H3xL3,
H4xL3, H5xL3, H6xL3, H7xL3, H8xL3, H9xL3, H10xL3, H1xL4, H2xL4, H3xL4, H4xL4,
H5xL4, H6xL4, H7xL4, H8xL4, H9xL4, H10xL4, H1xL5, H2xL5, H3xL5, H4xL5, H5xL5,
H6xL5, H7xL5, H8xL5, H9xL5, H10xL5, H1xL6, H2xL6, H3xL6, H4xL6, H5xL6, H6xL6,
H7xL6, H8xL6, H9xL6, H10xL6, H1xL7, H2xL7, H3xL7, H4xL7, H5xL7, H6xL7, H7xL7,
H8xL7, H9xL7, H10xL7, H1xL8, H2xL8, H3xL8, H4xL8, H5xL8, H6xL8, H7xL8, H8xL8,
H9xL8, H10xL8, H1xL9, H2xL9, H3xL9, H4xL9, H5xL9, H6xL9, H7xL9, H8xL9, H9xL9,
H10xL9, H1xL10, H2xL10, H3xL10, H4xL10, H5xL10, H6xL10, H7xL10, H8xL10, H9xL10,
H10xL10, where the number following the “H” identifies the number of copies of the heavy
chain gene, and the number following the “L” identifies the number of copies of the light chain
gene. For example, the specified number of heavy and light chain gene copies may be tandemly
integrated into a single locus, or into multiple loci (any or all of which may contain more than
one copy). Optionally, each genomic locus may contain no more than three or four tandemly
integrated gene copies, thereby promoting copy number stability during propagation and/or
antibody production.
Culturing most typically involves proving cells with an energy source, oxygen,
and nutrients. Methods are also known in the literature for design and optimization of P.
pastoris fermentations for expression of recombinant proteins, including optimization of the cell
density, broth volume, substrate feed rate, and the length of each phase of the reaction. See
Zhang et al., “Rational Design and Optimization of Fed-Batch and Continuous Fermentations” in
Cregg, J. M., Ed., 2007, Pichia Protocols (2nd edition), Methods in Molecular Biology, vol. 389,
Humana Press, Totowa, N.J., pgs. 43-63. The culture may be provided with a gas mixture
comprising oxygen, such as air with or without oxygen supplementation. The yeast culture may
be cultured in a culture medium which may be a minimal medium, may lack selective agents,
and / or may lack pre-formed amino acids or other complex biomolecules. The culture medium
may also be a complex medium (e.g., containing yeast extract and/or plant peptone(s)). The
medium may include a nitrogen source (e.g., methylamine chloride, NH4SO4, yeast extract, soy
peptone, other plant peptones, etc.). Exemplary minimal media include minimal dextrose
medium (MD) (1.34% yeast nitrogen base (YNB) (w/o amino acids), 4 × 10 % biotin, and 2%
glucose.), buffered minimal glycerol complex medium (BMGY) (1% yeast extract, 2% peptone,
1% glycerol, 1.34% YNB (w/o amino acids), 4 × 10–5% biotin and 100 mM potassium
phosphate (pH 6.0)). Media may include one or more salts (such as sodium chloride, calcium,
magnesium, and phosphate), buffers (such as potassium phosphate, Tris, or HEPES), nucleosides
(such as adenosine and thymidine), antibiotics (e.g., added to inhibit growth of contaminants
and/or for maintenance of a selectable marker), trace elements, and glucose or another energy
source. Any supplements and substitutions may also be included at appropriate concentrations
that would be known to those skilled in the art.
The culture may be grown to a high cell density, such as at least 50 g/L, at least
100 g/L, at least 300 g/L, at least 400 g/L, at least 500 g/L, or at least 700 g/L. These culture
densities are illustrative rather than limiting, and suitable culture densities may be readily
determined by those of ordinary skill in the art.
The yeast cells may be cultured for at least 20 doublings and maintain high levels
of expression of said antibody after said at least 20 doublings.
The yeast cells may be cultured for at least 50 doublings and maintain high levels
of expression of said antibody after said at least 50 doublings.
The yeast cells may be cultured for at least 100 doublings and maintain high
levels of expression of said antibody after said at least 100 doublings.
Also described is a culture medium containing a stable diploid Pichia yeast
culture produced according to any of the foregoing methods, wherein the culture medium may
comprise expression levels of said desired antibody which may be at least about 50 mg/liter, 100
mg/liter, 500 mg/liter, 750 mg/liter, 1000 mg/liter, 1250 mg/liter, 1500 mg/liter, 1750 mg/liter,
2000 mg/liter, or more. These yield values are illustrative rather than limiting. Optionally, yield
may be optimized, for example using the methods and general approach described in Zhang et al.
(2007), supra. For example, yield may be optimized by varying temperature, pH, media
composition (e.g., carbon source, carbon source concentration, mixture of two or more carbon
sources, nitrogen source and concentration, concentration of salts and nutrients including
KH PO , K HPO , MgSO potassium sulfate, sodium citrate, potassium sulfate, sodium citrate,
2 4 2 4 4,
trace metals such as cobalt chloride, cupric sulfate, sodium iodide, manganese sulfate, sodium
molybdate, boric acid, zinc chloride, ferrous sulfate, vitamins such as biotin, inositol, thiamine,
peptone, yeast extract, casamino acids, urea, ammonium phosphate or other ammonium ions, L-
arginine-hydrochloride), time, culture density, oxygenation, and other factors that influence
yield. For example, yield, expression, and/or purity of the desired multi-subunit complex may in
some instances be improved by maintaining the temperature at a desired set point, e.g., a set
point between about 15ºC and about 30 ºC, such as between about 17 ºC and about 25 ºC).
Without intent to be limited by theory, it is hypothesized that controlling the temperature may
assist intracellular trafficking through the folding and post-translational processing pathways,
and/or may decrease the activity of cellular proteases. Likewise, yield, expression, and/or purity
of the desired multi-subunit complex may in some instances be improved by maintaining the pH
of the culture medium at a desired set point, e.g., a set point between pH 3 to pH 8, such as
between pH 4 and pH 7.
Also described is a culture medium containing a stable diploid Pichia pastoris
yeast culture produced according to any of the foregoing methods that expresses said desired
antibody into a culture medium wherein the cell density of said diploid cells in said culture may
be at least about 50 g/L, 100 g/L, 300 g/L, 400 g/L, 500 g/L, 700 g/L or more. These culture
densities are illustrative rather than limiting, and suitable culture densities may be readily
determined by those of ordinary skill in the art.
At least one subunit of said antibody or other multi-subunit protein may comprise
a secretion signal, such as the S. chicken lysozyme (CLY) signal peptide; CLY-L8; S. cerevisiae
invertase (SUC2) signal peptide; MF-alpha (Prepro); MF-alpha (Pre)-apv; MF-alpha (Pre)-apv-
SLEKR; MF-alpha (Prepro)-(EA)3; αF signal peptide; KILM1 signal peptide; repressible acid
phosphatase (PHO1) signal peptide; A. niger GOX signal peptide; Schwanniomyces occidentalis
glucoamylase gene (GAM1) signal peptide; human serum albumin (HSA) signal peptide without
pro-sequence; human serum albumin (HSA) signal peptide with pro-sequence; ISN signal
peptide; IFN signal peptide; HGH signal peptide; phytohaemagglutinin (PHA); Silkworm
lysozyme; Human lysozyme (LYZ1); activin receptor type-1; activin type II receptor; P. pastoris
immunoglobulin binding protein (PpBiP); human antibody 3D6 light chain leader; and any
combination thereof.
The host cell may be produced by mating two haploid yeast cells that each contain
one or more copies of a gene encoding one or more subunits of said antibody or other multi-
subunit protein.
BRIEF DESCRIPTION OF THE DRAWINGS
-B. Purity of a recombinantly produced Ab-A was improved by a bolus
addition of ethanol prior to the start of a glucose feed in yeast cultures from which the antibodies
were produced. Antibodies were harvested after 97 hours of culture and purified by protein-A
affinity, then purity was assessed by SDS-PAGE using a non-reduced gel () to resolve
the desired full antibody (arrow, “Full Ab (H2L2)”) from undesired product-associated variants.
Complexes having aberrant stoichiometry were identified based on their molecular weight,
affinity for protein A, and additional studies further described below, as a “half antibody” species
containing one heavy and one light chain (arrow, “H1L1”) and a complex containing two heavy
chains and one light chain (“H2L1”). The relative abundance of the H2L1 and H1L1 complexes
was greatly decreased by the bolus addition of ethanol during antibody production. Compare
, lanes 2-3 (no bolus) to lane 5 (with bolus). shows the same samples
processed under reducing conditions, which separated each of the full antibody, H1L1, and H2L1
complexes into individual heavy and light chains, confirming that the H1L1 and H2L1
complexes are composed of full-length heavy and light chains. Lane order in FIGS. 1A-B: Lane
1: molecular weight marker; lanes 2 and 3: control samples prepared from fermentation cultures
without a bolus addition of ethanol; lane 4: no sample; lane 5: sample prepared from
fermentation cultures with a bolus addition of ethanol.
FIGS. 1C-E show the gel band density plotted along the length of the non-
reduced gel (, lanes 2, 3, and 5, respectively); arrows identify the peaks corresponding to
the H1L1 species. tabulates the area contained in the H1L1 peaks shown in FIGS. 1C-
E, demonstrating approximately 90% reduction in the relative abundance of the H1L1
complexes. H2L1 complex abundance was not quantified due to the incomplete resolution from
the full antibody peaks.
FIGS. 2A-B and 3A-B demonstrate reproducibility of the improvement in purity
of Ab-A by a bolus addition of ethanol to the yeast cultures. Antibodies were harvested after 87
or 86 hours of culture (FIGs. 2 and 3, respectively) and purified by protein-A affinity, then purity
was assessed by SDS-PAGE using a non-reduced gel. The abundance of the H1L1 and H2L1
complexes (arrows) were again decreased by the bolus addition of ethanol. Compare ,
lane 3 (no bolus) to lane 2 (with bolus), and , lanes 4-6 (no bolus) with lanes 2 and 4
(with bolus). shows the same samples as in processed under reducing
conditions, again confirming that the observed product-associated variants are composed of full-
length heavy and light chains. Lane order in FIGS. 2A-B: Lane 1: molecular weight marker;
lane 2: sample prepared from a fermentation culture with a bolus addition of ethanol; lane 3:
control sample prepared from a fermentation culture with no bolus addition of ethanol. Lane
order in : Lane 1: molecular weight marker; lanes 2 and 4: samples prepared from
fermentation cultures with a bolus addition of ethanol; lane 3: no sample; lanes 5-7: control
samples prepared from fermentation cultures with no bolus addition of ethanol.
FIGS. 2C and 2D show the gel band density plotted along the length of the non-
reduced gel (, lanes 2, and 3, respectively); arrows identify the peaks corresponding to
the H1L1 species. FIGS. 2E and 3B tabulate the area contained in the H1L1 peaks shown in
FIGS. 2C and , demonstrating approximately 85% reduction in the relative abundance
of H1L1 complexes in and approximately 87% average reduction in the relative
abundance of H1L1 complexes in .
-D. Purity of a second recombinant antibody (”Ab-B”) was also
improved by a bolus addition of ethanol prior to the production phase of a fermentation process.
Samples of fermentation culture broth were harvested after 67 hours (“T67) or 87 hours (“T87”)
of culture (FIGs. 4A-B and 4C-D, respectively) and antibodies were purified by protein-A
affinity. Purity was then assessed by SDS-PAGE using non-reduced gels (FIGS. 4A and 4C).
At both assessed time points, the abundance of the half-antibody species (H1L1) and the H2L1
complex was greatly decreased in fermentation cultures prepared that received a bolus addition
of ethanol, relative to control cultures that did not receive a bolus addition of ethanol. Compare
, lanes 2-3 (no bolus) to lanes 6-7 (with bolus), and , lanes 2-3 (no bolus) to
lanes 6-7 (with bolus). FIGs. 4B and 4D shows the same samples processed under reducing
conditions. Lane order in FIGS. 4A-D: Lane 1: molecular weight marker; lanes 2-3: control
sample prepared from fermentation cultures with no bolus addition of ethanol; lanes 4-5: no
sample; lanes 6-7: samples prepared from fermentation cultures with a bolus addition of ethanol.
FIGS. 4E and 4F tabulate the area contained in the H1L1 peaks shown in FIGS.
4A (T67) and 4C (T87), respectively, demonstrating that the bolus addition of ethanol produced
about a 73% reduction in the relative abundance of H1L1 complexes at the earlier time point
shown and about a 34% average reduction in the relative abundance of H1L1
complexes at the later time point shown in .
-B. Purity of a third recombinant antibody (Ab-C) was also improved by
a bolus addition of ethanol prior to the production phase of fermentation. Antibodies were
harvested after 86 hours of culture and purified by protein-A affinity, then purity was assessed by
SDS-PAGE using a non-reduced gel (FIGS. 5A). The H1L1 and H2L1 complexes were less
abundant in the Ab-C product even without the addition of a bolus of ethanol, leaving less room
for improvement. Nonetheless, the abundance of the half-antibody species (H1L1) and the H2L1
complex was noticeably decreased in fermentation cultures that received a bolus addition of
ethanol, relative to control cultures that did not receive a bolus addition of ethanol. Compare
, lanes 5-6 (no bolus) to lane 3 (with bolus). shows the same samples
processed under reducing conditions. Lane order in FIGS. 5A-B: Lane 1: molecular weight
marker; lane 2: no sample; lane 3: sample prepared from fermentation cultures that received a
bolus addition of ethanol; lane 4: no sample; lanes 5-6: control sample prepared from
fermentation cultures that did not receive a bolus addition of ethanol.
tabulates the area contained in the H1L1 peaks shown in FIGS. 5A,
demonstrating about a 61% average reduction in the relative abundance of H1L1 complexes by
the bolus addition of ethanol.
-F shows assessment of the relative purity of the Ab-A preparations
shown in FIGS. 1-3 by size exclusion chromatography. In each panel, the main peak contains
the full antibody containing two heavy and two light chains (H2L2). The H1L1 species was not
resolved from the main peak by this method (thought to be due H1L1 dimers forming by non-
covalent association which is retained under the conditions used). However, other undesired
product-associated variants were detected, including higher molecular weight species (left of
main peak) and lower molecular weight species (right of main peak). A prominent peak though
to correspond to antibody dimers containing two full antibodies (H4L4) was detected (arrow)
and the relative abundance of these was decreased in samples prepared from fermentation
cultures that received a bolus addition of ethanol. Compare FIGS. 6A, 6C, and 6E (no bolus) to
FIGS. 6B, 6D, and 6F (with bolus).
summarizes quantitation of the amount of product-associated variants
detected by SEC for the six Ab-A samples shown in and five additional samples. For
each identified sample (col. 1), the run set number (col. 2, identifying fermentation runs that
were conducted in parallel), bolus added (either 10g/L or none, col. 3), and elapsed culture time
before culture samples were taken and processed (col. 4) are shown, together with the fraction of
protein detected in the main peak (“SEC Main Peak %,” col. 5). The bolus addition of ethanol at
the end of the growth phase increased the average percentage contained in the main peak, from
80.3% up to 90.6%.
summarizes quantitation of the amount of product-associated variants
detected by SEC for Ab-B antibody samples shown in For each fermentation run (col.
1), the bolus added at the end of the growth phase (either 10g/L or none, col. 2), and elapsed
culture time before culture samples were taken and processed (col. 3) are shown, together with
the fraction of protein detected in the main peak (“SEC Main Peak %,” col. 4). Overall purity
was increased, with the main peak increasing from 76% to 79% at T67 and from 60% to 73% at
T87.
summarizes quantitation of the amount of product-associated variants
detected by SEC for the Ab-C antibody samples shown in For each identified
fermentation run (col. 1), the bolus added (either 10g/L or none, col. 2), and elapsed culture time
before the sample was taken and processed (col. 3) are shown, together with the fraction of
protein detected in the main peak (“SEC Main Peak %,” col. 4). There was little difference in
overall purity as detected by this method, with about 89% of product contained in main peak
with or without the ethanol bolus. This was apparently due to the high initial purity of the Ab-C
antibody even without the bolus addition. Additionally, SEC did not resolve the H1L1 species
from the full antibody and accordingly the decreased production of this species due to the
ethanol bolus was not reflected in SEC results.
summarizes the results of mass spectrometry measurement of the quantity
of a free heavy chain (lacking a disulfide bond to a second heavy chain) in Ab-A antibody
samples containing high or low amounts of the H1L1 band. As expected, the amount of free
heavy chain correlated with the amount of the H1L1 band, confirming the identity thereof as
containing one heavy and one light chain and lacking a disulfide bond to a second heavy chain.
FIGS. 11-13 show the correlation between addition of an ethanol bolus and cell
viability. The addition of an ethanol bolus generally improved cell viability and antibody purity
for the Ab-A antibody () and the Ab-B antibody (). These results suggest that the
improvement in cell viability may account for at least part of the improvement in antibody purity
from the ethanol bolus addition. Consistent with these results, the Ab-C antibody culture
exhibited greater antibody purity and cell viability than the Ab-A and Ab-B cultures ().
Apparently because the viability of the Ab-C antibody-producing cultures was already high in
these experiments, there was little room for improvement and the cultures exhibited little
improvement in viability from the bolus addition of ethanol. In FIGS. 11-13, filled bars indicate
no bolus, while open bars indicate a bolus addition of ethanol. Viability was determined from
fermentation cultures sampled with 1.5 hours of the time at which samples were collected for
purity analyses (as identified in the preceding slides).
shows that a wide range of ethanol bolus concentrations can produce the
same improvement in antibody purity. Ab-A was produced with a bolus addition of ethanol
between 5 g/L (0.5% w/v) and 15 g/L (1.5% w/v) and purified by protein A affinity, then the
purity was analyzed by non-reduced SDS-PAGE. Each culture exhibited similarly low levels of
the H2L1 and H1L1 complexes at 63 hours (A) and 86 hours (B). Lane order in
FIGS. 14A-B: lane 1: molecular weight markers; lanes 2 and 7: 5 g/L bolus; lanes 3 and 5: 10
g/L bolus; lanes 4 and 6: 15 g/L bolus.
shows that the time elapsed between the dissolved oxygen spike and the
ethanol bolus addition can vary considerably while giving similar improvement in antibody
purity. Ab-A was produced with a bolus addition of ethanol of 10 g/L (1% w/v) and purified by
protein A affinity, then the purity was analyzed by non-reduced SDS-PAGE (A). The
“starvation period,” the time between the dissolved oxygen spike (indicating exhaustion of the
carbon source in the culture) and the bolus addition of ethanol, was varied between 0 and 3
hours. Each culture exhibited similarly low levels of the H2L1 and H1L1 complexes irrespective
of the duration of the starvation period, indicating that antibody purity is relatively insensitive to
absence of a starvation period or a starvation period of at least up to three hours. The same
samples were analyzed on a reduced gel (B). Lane order in FIGS. 15A-B: lane 1:
molecular weight markers; lanes 2-4: no sample; lane 5: 0 hours starvation period; lane 6: 3
hours starvation period.
shows the effect of the equilibration period (the time between ethanol
bolus addition and feed start) on antibody purity. The Ab-B antibody was produced with a bolus
addition of ethanol of 10 g/L (1% w/v) and purified by protein A affinity, then the purity was
analyzed by non-reduced SDS-PAGE (A). The duration of the equilibration period was
either 0, 30, or 60 minutes. The 60 minute equilibration period resulted in a lower antibody
purity (higher abundance of the H2L1 and H1L1 complexes). Culture viability was also
markedly lower with a 60 minute equilibration period, particularly earlier in the culture (at 23
hours, B); viability had improved somewhat by the end of the culture (at 85 hours, C). Lane order in A: lane 1: molecular weight markers; lanes 2 and 4: no sample; lane
3: 30 minutes equilibration; lanes 5 and 6: 60 minutes equilibration time; lanes 7 and 8: 0
minutes equilibration time. In FIGS. 16B-C, filled bars indicate an equilibration period of zero
minutes, hatched bars indicate an equilibration period of 30 minutes and open bars indicate an
equilibration period of 60 minutes.
DETAILED DESCRIPTION
Applicants have unexpectedly discovered that purity of multi-subunit complexes
expressed from yeast can be greatly improved by addition of a bolus of ethanol to the culture
media. A single bolus addition of ethanol was demonstrated to improve purity over a sustained
period of production, for up to at least 97 hours.
Described are improved methods and compositions of matter that provide for the
recombinant production of antibodies and other multi-subunit complexes with increased purity
and decreased production of one or more undesired side-products. In exemplary embodiments,
relative to the desired multi-subunit complex the undesired side product(s) may exhibit one or
more of: altered stoichiometry, aberrant glycosylation, differences in apparent molecular weight,
differences in disulfide bonds, differences in hydrodynamic radius, fragments and/or truncated
forms of one or more subunits. Undesired side-products may exhibit one or more additional
differences as well. Undesired side-products may also be detected by their effects on a
preparation, e.g., alteration in the level of specific activity, immunogenicity, or other effects on
physical constitution and/or function of the desired multi-subunit complex.
For example, when the desired multi-subunit complex is an antibody, the
undesired side products may include an H1L1 or “half antibody” species (i.e., containing a heavy
chain and a light chain, wherein the heavy chain is not linked by a disulfide bond to another
heavy chain), and/or a H2L1 species (i.e., containing two heavy chains and one light chain, but
lacking a second light chain).
Though not intending to be limited by theory, it is hypothesized that a rapid
increase in ethanol concentration (which can be brought about by a bolus addition of ethanol)
can cause sustained changes in gene expression which confer a lasting improvement in the
production of properly folded and assembled multi-subunit complexes and/or increases
processing of improperly folded or misassembled multi-subunit complexes, leading to improved
purity in the multi-subunit complex. Additionally, it was demonstrated that the improved
antibody purity correlated with improved viability of the yeast in the culture, and based thereon
Applicants hypothesize that the improved viability may account (at least in part) for the
improved purity, though this theory is not intended to be limiting.
In a preferred embodiment, the heterologous multi-subunit complex is an
antibody or antibody fragment, such as a humanized antibody, comprised of two heavy chain
subunits and two light chain subunits. Preferred host cells include yeasts, and particularly
preferred yeasts include methylotrophic yeast strains, e.g., Pichia pastoris, Hansenula
polymorpha (Pichia angusta), Pichia guillermordii, Pichia methanolica, Pichia inositovera, and
others (see, e.g., U.S. Patent 4,812,405, 4,818,700, 4,929,555, 5,736,383, 5,955,349, 5,888,768,
and 6,258,559 each of which is incorporated by reference in its entirety). The host cell may be
produced by methods known in the art, such as transformation, mating, sporulation, etc.
In a preferred embodiment, the host cell may comprise more than one copy of one
or more of the genes encoding the heterologous protein subunits. For example, multiple copies
of a subunit gene may be integrated in tandem into one or more chromosomal loci. Tandemly
integrated gene copies are preferably retained in a stable number of copies during culture for the
production of the multi-subunit complex. For example, in the examples described below, gene
copy numbers were generally stable for P. pastoris strains containing three to four tandemly
integrated copies of light and heavy chain antibody genes.
One or more of the genes encoding the heterologous protein subunits are
preferably integrated into one or more chromosomal loci of a host cell. Any suitable
chromosomal locus may be utilized for integration, including intergenic sequences, promoters
sequences, coding sequences, termination sequences, regulatory sequences, etc. Exemplary
chromosomal loci that may be used in P. pastoris include PpURA5; OCH1; AOX1; HIS4; and
GAP. The encoding genes may also be integrated into one or more random chromosomal loci
rather than being targeted. In preferred embodiments, the chromosomal loci are selected from
the group consisting of the pGAP locus, 3’ AOX TT, and the HIS4 TT locus. In additional
exemplary embodiments, the genes encoding the heterologous protein subunits may be contained
in one or more extrachromosomal elements, for example one or more plasmids or artificial
chromosomes.
In exemplary embodiments, the multi-subunit protein may comprise two, three,
four, five, six, or more non-identical subunits. Additionally, each subunit may be present one or
more times in each multi-subunit protein. For example, the multi-subunit protein may be a
multi-specific antibody such as a bi-specific antibody comprising two non-identical light chains
and two non-identical heavy chains.
The subunits may be expressed from monocistronic genes, polycistronic genes, or
any combination thereof. Each polycistronic gene may comprise multiple copies of the same
subunit, or may comprise one or more copies of each different subunit.
Exemplary methods that may be used for manipulation of Pichia pastoris
(including methods of culturing, transforming, and mating) are disclosed in Published
Applications including U.S. 20080003643, U.S. 20070298500, and U.S. 20060270045, and in
Higgins, D. R., and Cregg, J. M., Eds. 1998. Pichia Protocols. Methods in Molecular Biology.
Humana Press, Totowa, N.J., and Cregg, J. M., Ed., 2007, Pichia Protocols (2nd edition),
Methods in Molecular Biology. Humana Press, Totowa, N.J., each of which is incorporated by
reference in its entirety.
An exemplary expression cassette that may be utilized is composed of the
glyceraldehyde dehydrogenase gene (GAP gene) promoter, fused to sequences encoding a
secretion signal, followed by the sequence of the gene to be expressed, followed by sequences
encoding a P. pastoris transcriptional termination signal from the P. pastoris alcohol oxidase I
gene (AOX1). The Zeocin resistance marker gene may provide a means of enrichment for strains
that contain multiple integrated copies of an expression vector in a strain by selecting for
transformants that are resistant to higher levels of Zeocin. Similarly, G418 or Kanamycin
resistance marker genes may be used to provide a means of enrichment for strains that contain
multiple integrated copies of an expression vector in a strain by selecting for transformants that
are resistant to higher levels of Geneticin or Kanamycin.
Host strains that may be utilized include auxotrophic P. pastoris or other Pichia
strains, for example, strains having mutations in met1, lys3, ura3 and ade1 or other auxotrophy-
associated genes. Preferred mutations are incapable of giving rise to revertants at any appreciable
frequency and are preferably partial or even more preferably full deletion mutants. Preferably,
prototrophic diploid or tetraploid strains are produced by mating a complementing sets of
auxotrophic strains.
Transformation of haploid P. pastoris strains and genetic manipulation of the P.
pastoris sexual cycle may be performed as described in Pichia Protocols (1998, 2007), supra.
Prior to transformation, each expression vector may be linearized by restriction
enzyme cleavage within a region homologous to the target genomic locus (e.g., the GAP
promoter sequence) to direct the integration of the vectors into the target locus in the host cell.
Samples of each vector may then be individually transformed into cultures of the desired strains
by electroporation or other methods, and successful transformants may be selected by means of a
selectable marker, e.g., antibiotic resistance or complementation of an auxotrophy. Isolates may
be picked, streaked for single colonies under selective conditions and then examined to confirm
the number of copies of the gene encoding the subunit of the multi-subunit complex (e.g., a
desired antibody) by Southern Blot or PCR assay on genomic DNA extracted from each strain.
Optionally, expression of the expected subunit gene product may be confirmed, e.g., by FACS,
Western Blot, colony lift and immunoblot, and other means known in the art. Optionally,
haploid isolates are transformed additional times to introduce additional heterologous genes, e.g.,
additional copies of the same subunit integrated at a different locus, and / or copies of a different
subunit. The haploid strains are then mated to generate diploid strains (or strains of higher
ploidy) able to synthesize the multi-protein complex. Presence of each expected subunit gene
may be confirmed by Southern blotting, PCR, and other detection means known in the art.
Where the desired multi-protein complex is an antibody, its expression may also be confirmed by
a colony lift/immunoblot method (Wung et al. Biotechniques 21 808-812 (1996) and / or by
FACS.
This transformation protocol is optionally repeated to target a heterologous gene
into a second locus, which may be the same gene or a different gene than was targeted into the
first locus. When the construct to be integrated into the second locus encodes a protein that is
the same as or highly similar to the sequence encoded by the first locus, its sequence may be
varied to decrease the likelihood of undesired integration into the first locus. For example, the
sequence to be integrated into the second locus may have differences in the promoter sequence,
termination sequence, codon usage, and/or other tolerable sequence differences relative to the
sequence integrated into the first locus.
To mate P. pastoris haploid strains, each strain to be crossed can be patched
together onto mating plates. For example, multiple matings can be conveniently performed at
the same time by streaking each strain to be mated across a plate suitable for its growth, and the
mating partners may be streaked across a second plate (preferably the plates are rich media such
as YPD). Typically, after one or two days incubation at 30º C., cells from the two plates can be
replica plated in a crisscross fashion onto a mating plate, resulting in a cross-hatched pattern with
each pair of strains being co-plated and having the opportunity to mate at the intersection of a
pair of the original streak lines. The mating plate can then be incubated (e.g., at 30º C.) to
stimulate the initiation of mating between strains. After about two days, the cells on the mating
plates can be streaked, patched, or replica plated onto media selective for the desired diploid
strains (e.g., where the mated strains have complementary autotrophies, drop-out or minimal
medium plates may be used). These plates can be incubated (e.g., at 30º C.) for a suitable
duration (e.g., about three days) to allow for the selective growth of the desired diploid strains.
Colonies that arise can be picked and streaked for single colonies to isolate and purify each
diploid strain.
Expression vectors for use in the methods of the invention may further include
yeast specific sequences, including a selectable auxotrophic or drug marker for identifying
transformed yeast strains. A drug marker may further be used to amplify copy number of the
vector in a yeast host cell, e.g., by culturing a population of cells in an elevated concentration of
the drug, thereby selecting transformants that express elevated levels of the resistance gene.
In an exemplary embodiment, one or more of the genes encoding the heterologous
protein subunits are coupled to an inducible promoter. Suitable exemplary promoters include the
alcohol oxidase 1 gene promoter, formaldehyde dehydrogenase genes (FLD; see U.S. Pub. No.
2007/0298500), and other inducible promoters known in the art. The alcohol oxidase 1 gene
promoter, is tightly repressed during growth of the yeast on most common carbon sources, such
as glucose, glycerol, or ethanol, but is highly induced during growth on methanol (Tschopp et al.,
1987; U.S. Pat. No. 4,855,231 to Stroman, D. W., et al). For production of foreign proteins,
strains may be initially grown on a repressing carbon source to generate biomass and then shifted
to methanol as the sole (or main) carbon and energy source to induce expression of the foreign
gene. One advantage of this regulatory system is that P. pastoris strains transformed with foreign
genes whose expression products are toxic to the cells can be maintained by growing under
repressing conditions.
In another exemplary embodiment, one or more of the heterologous genes may be
coupled to a regulated promoter, whose expression level can be upregulated under appropriate
conditions. Exemplary regulated promoters include the CUP1 promoter (induced by the level of
copper in the medium), tetracycline inducible promoters, thiamine inducible promoters, the
AOX1 promoter, and the FLD1 promoter.
Though much of the present disclosure describes production of antibodies, the
methods described herein are readily adapted to other multi-subunit complexes as well. Without
intent to be limited by theory, it is believed that the yield and purity of multi-subunit complexes
can be greatly influenced by the concentration and stoichiometry of the subunits, which are in
turn influenced by the level of expression of the genes responsible for production of each
subunit. The methods disclosed herein may readily be utilized to improve the yield and / or
purity of any recombinant multi-subunit complex comprising two or more different subunits.
Additionally, the methods described are not limited to production of multi-protein complexes but
may also be readily adapted for use with ribonucleoprotein (RNP) complexes including
telomerase, hnRNPs, Ribosomes, snRNPs, signal recognition particles, prokaryotic and
eukaryotic RNase P complexes, and any other complexes that contain multiple distinct protein
and / or RNA subunits. The host cell that expresses the multi-subunit complex may be produced
by methods known in the art. For example, a panel of diploid or tetraploid yeast cells containing
differing combinations of gene copy numbers may be generated by mating cells containing
varying numbers of copies of the individual subunit genes (which numbers of copies preferably
are known in advance of mating).
Definitions
[00180A] The term “comprising” as used in this specification means “consisting at least in
part of”. When interpreting each statement in this specification that includes the term
“comprising”, features other than that or those prefaced by the term may also be present. Related
terms such as “comprise” and “comprises” are to be interpreted in the same manner
It is to be understood that this invention is not limited to the particular
methodology, protocols, cell lines, animal species or genera, and reagents described, as such may
vary. It is also to be understood that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope of the present invention
which will be limited only by the appended claims.
As used herein the singular forms "a", "and", and "the" include plural referents
unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a
plurality of such cells and reference to "the protein" includes reference to one or more proteins
and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific
terms used herein have the same meaning as commonly understood to one of ordinary skill in the
art to which this invention belongs unless clearly indicated otherwise.
Bolus addition: In the present disclosure, “bolus addition” generally refers to
rapid change in concentration of a substance (such as ethanol) in contact with cultured cells (for
example, in a culture medium). For example, the substance may be added to the cultured cells in
a single addition, a succession of more than one addition, and/or infused over a period of time
(e.g., over about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 90, or 120 minutes). The
substance may also be added by replacing the culture medium in part or in full, for example by
concentrating the cells (using centrifugation, filtration, settling, or other methods), removing part
or all of the medium, and adding the substance, or by adding the cells to a medium containing the
substance. The substance may be admixed with a carrier (e.g., culture media, water, saline, etc.).
For example, a bolus addition of ethanol may comprise the addition of pure or concentrated
ethanol (e.g., 100%, 95%, 70%, 50%, 60%, 40%, 30%, 20%, etc.) to the culture medium in an
amount sufficient to produce the desired concentration. As another example, the cells may be
added to a medium containing ethanol, e.g., by adding an inoculum containing the cells to a
medium containing ethanol.
Bolus concentration: In the present disclosure, “bolus concentration” generally
refers to the concentration that results from a bolus addition of a substance (e.g., ethanol).
Mating competent yeast species: In the present invention this is intended to
broadly encompass any diploid or tetraploid yeast which can be grown in culture. Such species
of yeast may exist in a haploid, diploid, or other polyploid form. The cells of a given ploidy
may, under appropriate conditions, proliferate for an indefinite number of generations in that
form. Diploid cells can also sporulate to form haploid cells. Sequential mating can result in
tetraploid strains through further mating or fusion of diploid strains. The present description,
contemplates the use of haploid yeast, as well as diploid or other polyploid yeast cells produced,
for example, by mating or fusion (e.g., spheroplast fusion).
In one embodiment described, the mating competent yeast is a member of the
Saccharomycetaceae family, which includes the genera Arxiozyma; Ascobotryozyma;
Citeromyces; Debaryomyces; Dekkera; Eremothecium; Issatchenkia; Kazachstania;
Kluyveromyces; Kodamaea; Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora;
Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces. Other types of yeast
described as potentially useful herein include Yarrowia; Rhodosporidium; Candida; Hansenula;
Filobasium; Sporidiobolus; Bullera; Leucosporidium and Filobasidella.
In a preferred embodiment described, the mating competent yeast is a member of
the genus Pichia or is another methylotroph. In a further preferred embodiment described, the
mating competent yeast of the genus Pichia is one of the following species: Pichia pastoris,
Pichia methanolica, and Hansenula polymorpha (Pichia angusta). In a particularly preferred
embodiment of the invention, the mating competent yeast of the genus Pichia is the species
Pichia pastoris.
Haploid Yeast Cell: A cell having a single copy of each gene of its normal
genomic (chromosomal) complement.
Polyploid Yeast Cell: A cell having more than one copy of its normal genomic
(chromosomal) complement.
Diploid Yeast Cell: A cell having two copies (alleles) of essentially every gene of
its normal genomic complement, typically formed by the process of fusion (mating) of two
haploid cells.
Tetraploid Yeast Cell: A cell having four copies (alleles) of essentially every gene
of its normal genomic complement, typically formed by the process of fusion (mating) of two
diploid cells. Tetraploids may carry two, three, four, or more different expression cassettes. Such
tetraploids might be obtained in S. cerevisiae by selective mating homozygotic heterothallic a/a
and alpha/alpha diploids and in Pichia by sequential mating of haploids to obtain auxotrophic
diploids. For example, a [met his] haploid can be mated with [ade his] haploid to obtain diploid
[his]; and a [met arg] haploid can be mated with [ade arg] haploid to obtain diploid [arg]; then
the diploid [his] can be mated with the diploid [arg] to obtain a tetraploid prototroph. It will be
understood by those of skill in the art that reference to the benefits and uses of diploid cells may
also apply to tetraploid cells.
Yeast Mating: The process by which two yeast cells fuse to form a single yeast
cell. The fused cells may be haploid cells or cells of higher ploidy (e.g., mating two diploid cells
to produce a tetraploid cell).
Meiosis: The process by which a diploid yeast cell undergoes reductive division
to form four haploid spore products. Each spore may then germinate and form a haploid
vegetatively growing cell line.
Selectable Marker: A selectable marker is a gene or gene fragment that confers a
growth phenotype (physical growth characteristic) on a cell receiving that gene as, for example
through a transformation event. The selectable marker allows that cell to survive and grow in a
selective growth medium under conditions in which cells that do not receive that selectable
marker gene cannot grow. Selectable marker genes generally fall into several types, including
positive selectable marker genes such as a gene that confers on a cell resistance to an antibiotic
or other drug, temperature when two temperature sensitive (“ts”) mutants are crossed or a ts
mutant is transformed; negative selectable marker genes such as a biosynthetic gene that confers
on a cell the ability to grow in a medium without a specific nutrient needed by all cells that do
not have that biosynthetic gene, or a mutagenized biosynthetic gene that confers on a cell
inability to grow by cells that do not have the wild type gene; and the like. Suitable markers
include but are not limited to: ZEO; NEO (G418); LYS3; MET1; MET3a; ADE1; ADE3; URA3;
and the like.
Integrated: A genetic element (typically a heterologous genetic element) that are
covalently joined into a chromosome of an organism.
Tandemly integrated: Two or more copies of a genetic element that are integrated
in adjacent locations in a chromosome. The two or more copies do not necessarily have the
orientation; e.g., for transcribed genes, some copies may be transcribed from the Watson strand
and others from the Crick strand.
Host cell: In the context of the present disclosure, the term host cell refers to a
cell (e.g., a eukaryotic cell, such as a Pichia cell) which contains a heterologous gene. For
example, the heterologous gene may provide for the expression of a subunit of a desired multi-
subunit complex, a gene involved in protein folding (e.g., a chaperone), expression, or secretion,
and/or another desired gene. The heterologous gene may be integrated into the genome of the
eukaryotic cell or contained in extrachromosomal element such as a plasmid or artificial
chromosome.
Expression Vector: These DNA vectors contain elements that facilitate
manipulation for the expression of a foreign protein within the target host cell. Conveniently,
manipulation of sequences and production of DNA for transformation is first performed in a
bacterial host, e.g. E. coli, and usually vectors will include sequences to facilitate such
manipulations, including a bacterial origin of replication and appropriate bacterial selection
marker. Selection markers encode proteins necessary for the survival or growth of transformed
host cells grown in a selective culture medium. Host cells not transformed with the vector
containing the selection gene will not survive in the culture medium. Typical selection genes
encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement
auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
Exemplary vectors and methods for transformation of yeast are described, for example, in Burke,
D., Dawson, D., & Stearns, T. (2000). Methods in yeast genetics: a Cold Spring Harbor
Laboratory course manual. Plainview, N.Y.: Cold Spring Harbor Laboratory Press, which is
incorporated by reference herein in its entirety.
Expression vectors for use in the methods of the invention may further include
yeast specific sequences, including a selectable auxotrophic or drug marker for identifying
transformed yeast strains. A drug marker may further be used to select for amplification of copy
number of the vector in a yeast host cell.
The polypeptide coding sequence of interest is typically operably linked to
transcriptional and translational regulatory sequences that provide for expression of the
polypeptide in yeast cells. These vector components may include, but are not limited to, one or
more of the following: an enhancer element, a promoter, and a transcription termination
sequence. Sequences for the secretion of the polypeptide may also be included, e.g. a signal
sequence, and the like. A yeast origin of replication is optional, as expression vectors are often
integrated into the yeast genome.
Though optional, in one embodiment described, one or more subunit of the multi-
subunit complex is operably linked, or fused, to a secretion sequence that provides for secretion
of the expressed polypeptide into the culture media, which can facilitate harvesting and
purification of the heterologous multi-subunit complex. Even more preferably, the secretion
sequences provide for optimized secretion of the polypeptide from the host cells (e.g., yeast
diploid cells), such as through selecting preferred codons and/or altering the percentage AT
through codon selection. It is known in the art that secretion efficiency and / or stability can be
affected by the choice of secretion sequence and the optimal secretion sequence can vary
between different proteins (see, e.g., Koganesawa et al., Protein Eng. 2001 Sep;14(9):705-10,
which is incorporated by reference herein in its entirety). Many potentially suitable secretion
signals are known in the art and can readily be tested for their effect upon yield and/or purity of a
particular heterologous multi-subunit complex. Any secretion sequences may potentially be
used, including those present in secreted proteins of yeasts and other species, as well as
engineered secretion sequences. Exemplary secretion sequences that may be utilized include:
chicken lysozyme (CLY) signal peptide (MRSLLILVLCFLPLAALG (SEQ ID NO:414)), CLY-
L8 (MRLLLLLLLLPLAALG (SEQ ID NO:415)), S. cerevisiae invertase (SUC2) signal peptide
(MLLQAFLFLLAGFAAKISA (SEQ ID NO:416)), MF-alpha (Prepro)
(MRFPSIFTAVLFAASSALA-APVNTTTE-EGVSLEKR (SEQ ID NO:417)), MF-alpha (Pre)-
apv (MRFPSIFTAVLFAASSALA-APV (SEQ ID NO:418)), MF-alpha (Pre)-apv-SLEKR
(MRFPSIFTAVLFAASSALA-APVSLEKR (SEQ ID NO:419)), MF-alpha (Prepro)-(EA)3
(MRFPSIFTAVLFAASSALA-APVNTTTE-EGVSLEKR-EAEAEA (SEQ ID NO:420)), αF
signal peptide (MRFPSIFTAVLFAASSALA-APVNTTTE-
DETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKE-EGVSLEKR
(SEQ ID NO:421)), KILM1 signal peptide
(MTKPTQVLVRSVSILFFITLLHLVVALNDVAGPAETAPVSLLPR (SEQ ID NO:422)),
repressible acid phosphatase (PHO1) signal peptide (MFSPILSLEIILALATLQSVFA (SEQ ID
NO:423)), A. niger GOX signal peptide (MQTLLVSSLVVSLAAALPHYIR (SEQ ID NO:424)),
Schwanniomyces occidentalis glucoamylase gene (GAM1) signal peptide
(MIFLKLIKSIVIGLGLVSAIQA (SEQ ID NO:425)), human serum albumin (HSA) signal
peptide with pro-sequence (MKWVTFISLLFLFSSAYSRGVFRR (SEQ ID NO:426)), human
serum albumin (HSA) signal peptide without pro-sequence (MKWVTFISLLFLFSSAYS (SEQ
ID NO:427)), ISN signal peptide (MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO:428)),
IFN signal peptide (MKYTSYILAFQLCIVLGSLGCDLP (SEQ ID NO:429)), HGH signal
peptide (MAADSQTPWLLTFSLLCLLWPQEPGA (SEQ ID NO:430)), phytohaemagglutinin
(PHA) (MKKNRMMMMIWSVGVVWMLLLVGGSYG (SEQ ID NO:431)), Silkworm
lysozyme (MQKLIIFALVVLCVGSEA (SEQ ID NO:432)), Human lysozyme (LYZ1)
(MKALIVLGLVLLSVTVQG (SEQ ID NO:433)), activin receptor type-1
(MVDGVMILPVLIMIALPSPS (SEQ ID NO:434)), activin type II receptor
(MGAAAKLAFAVFLISCSSG (SEQ ID NO:435)), P. pastoris immunoglobulin binding protein
(PpBiP) (MLSLKPSWLTLAALMYAMLLVVVPFAKPVRA (SEQ ID NO:436)), and human
antibody 3D6 light chain leader (MDMRVPAQLLGLLLLWLPGAKC (SEQ ID NO:437)). See
Hashimoto et al., Protein Engineering vol. 11 no. 2 pp.75–77, 1998; Oka et al., Biosci Biotechnol
Biochem. 1999 Nov; 63(11):1977-83; Gellissen et al., FEMS Yeast Research 5 (2005) 1079–
1096; Ma et al., Hepatology. 2005 Dec;42(6):1355-63; Raemaekers et al., Eur J Biochem. 1999
Oct 1;265(1):394-403; Koganesawa et al., Protein Eng. (2001) 14 (9): 705-710; Daly et al.,
Protein Expr Purif. 2006 Apr;46(2):456-67 ; Damasceno et al., Appl Microbiol Biotechnol
(2007) 74:381–389; and Felgenhauer et al., Nucleic Acids Res. 1990 Aug 25;18(16):4927, each
of which is incorporated by reference herein in its entirety). The multi-subunit complex may
also be secreted into the culture media without being operably linked or fused to a secretion
signal. For example, it has been demonstrated that some heterologous polypeptides are secreted
into the culture media when expressed in P. pastoris even without being linked or fused to a
secretion signal. Additionally, the multi-subunit complex may be purified from host cells
(which, for example, may be preferable if the complex is poorly secreted) using methods known
in the art.
Media or cells comprising a desired multi-subunit complex may be recovered
from the culture. Optionally, the secreted proteins may be purified. For example, cells
comprising a desired multi-subunit complex may be lysed using mechanical, chemical,
enzymatic, and/or osmotic methods (e.g., freezing with liquid nitrogen, using a homogenizer,
spheroplasting, sonication, agitation in the presence of glass beads, using detergents, etc.). The
desired multi-subunit complex may be concentrated, filtered, dialyzed, etc., using methods
known in the art. The desired multi-subunit complex may be purified based on, for example, its
molecular mass (e.g., size exclusion chromatography), isoelectric point (e.g., isoelectric
focusing), electrophoretic mobility (e.g., gel electrophoresis), hydrophobic interaction
chromatography (e.g., HPLC), charge (e.g., ion exchange chromatography), affinity (e.g., in the
case of an antibody, binding to protein A, protein G, and/or an epitope to which the desired
antibody binds), and/or glycosylation state (e.g., detected by lectin binding affinity). Multiple
purification steps may be performed to obtain the desired level of purity. In an exemplary
embodiment, the desired multi-subunit complex may be comprise an immunoglobulin constant
domain and may be purified using protein A or protein G affinity, size exclusion
chromatography, and lack of binding to lectin (to remove glycosylated forms). Optionally the A
protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) may be added to inhibit
proteolytic degradation during purification.
Nucleic acids are "operably linked" when placed into a functional relationship
with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked
to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the
polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the
transcription of the sequence. Generally, "operably linked" means that the DNA sequences being
linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame.
However, enhancers do not have to be contiguous. Linking may be accomplished by ligation at
convenient restriction sites or alternatively via a PCR/recombination method familiar to those
skilled in the art (Gateway® Technology; Invitrogen, Carlsbad Calif.). If such sites do not exist,
the synthetic oligonucleotide adapters or linkers may be used in accordance with conventional
practice. Desired nucleic acids (including nucleic acids comprising operably linked sequences)
may also be produced by chemical synthesis.
Promoters are untranslated sequences located upstream (5') to the start codon of a
structural gene (generally within about 100 to 1000 bp) that control the transcription and
translation of particular nucleic acid sequences to which they are operably linked. Such
promoters fall into several classes: inducible, constitutive, and repressible promoters (that
increase levels of transcription in response to absence of a repressor). Inducible promoters may
initiate increased levels of transcription from DNA under their control in response to some
change in culture conditions, e.g., the presence or absence of a nutrient or a change in
temperature.
The yeast promoter fragment may also serve as the site for homologous
recombination and integration of the expression vector into the same site in the yeast genome;
alternatively a selectable marker is used as the site for homologous recombination. Pichia
transformation is described in Cregg et al. (1985) Mol. Cell. Biol. 5:3376-3385, which is
incorporated by reference herein in its entirety.
Examples of suitable promoters from Pichia include the CUP1 (induced by the
level of copper in the medium), tetracycline inducible promoters, thiamine inducible promoters,
AOX1 promoter (Cregg et al. (1989) Mol. Cell. Biol. 9:1316-1323); ICL1 promoter (Menendez
et al. (2003) Yeast 20(13):1097-108); glyceraldehydephosphate dehydrogenase promoter
(GAP) (Waterham et al. (1997) Gene 186(1):37-44); and FLD1 promoter (Shen et al. (1998)
Gene 216(1):93-102). The GAP promoter is a strong constitutive promoter and the CUP1, AOX
and FLD1 promoters are inducible. Each foregoing reference is incorporated by reference herein
in its entirety.
Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4, PHO3,
PHO5, Pyk, and chimeric promoters derived therefrom. Additionally, non-yeast promoters may
be used in the invention such as mammalian, insect, plant, reptile, amphibian, viral, and avian
promoters. Most typically the promoter will comprise a mammalian promoter (potentially
endogenous to the expressed genes) or will comprise a yeast or viral promoter that provides for
efficient transcription in yeast systems.
The polypeptides of interest may be produced recombinantly not only directly, but
also as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or other
polypeptide having a specific cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of the vector, or it may be a
part of the polypeptide coding sequence that is inserted into the vector. The heterologous signal
sequence selected preferably is one that is recognized and processed through one of the standard
pathways available within the host cell. The S. cerevisiae alpha factor pre-pro signal has proven
effective in the secretion of a variety of recombinant proteins from P. pastoris. Other yeast signal
sequences include the alpha mating factor signal sequence, the invertase signal sequence, and
signal sequences derived from other secreted yeast polypeptides. Additionally, these signal
peptide sequences may be engineered to provide for enhanced secretion in diploid yeast
expression systems. Other secretion signals of interest also include mammalian signal sequences,
which may be heterologous to the protein being secreted, or may be a native sequence for the
protein being secreted. Signal sequences include pre-peptide sequences, and in some instances
may include propeptide sequences. Many such signal sequences are known in the art, including
the signal sequences found on immunoglobulin chains, e.g., K28 preprotoxin sequence, PHA-E,
FACE, human MCP-1, human serum albumin signal sequences, human Ig heavy chain, human
Ig light chain, and the like. For example, see Hashimoto et. al. Protein Eng 11(2) 75 (1998); and
Kobayashi et. al. Therapeutic Apheresis 2(4) 257 (1998), each of which is incorporated by
reference herein in its entirety.
Transcription may be increased by inserting a transcriptional activator sequence
into the vector. These activators are cis-acting elements of DNA, usually about from 10 to 300
bp, which act on a promoter to increase its transcription. Transcriptional enhancers are relatively
orientation and position independent, having been found 5' and 3' to the transcription unit, within
an intron, as well as within the coding sequence itself. The enhancer may be spliced into the
expression vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5'
from the promoter.
Expression vectors used in eukaryotic host cells may also contain sequences
necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are
commonly available from 3' to the translation termination codon, in untranslated regions of
eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA.
Construction of suitable vectors containing one or more of the above-listed
components employs standard ligation techniques or PCR/recombination methods. Isolated
plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form desired to generate
the plasmids required or via recombination methods. For analysis to confirm correct sequences in
plasmids constructed, the ligation mixtures are used to transform host cells, and successful
transformants selected by antibiotic resistance (e.g. ampicillin or Zeocin) where appropriate.
Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion
and/or sequenced.
As an alternative to restriction and ligation of fragments, recombination methods
based on att sites and recombination enzymes may be used to insert DNA sequences into a
vector. Such methods are described, for example, by Landy (1989) Ann. Rev. Biochem. 58:913-
949; and are known to those of skill in the art. Such methods utilize intermolecular DNA
recombination that is mediated by a mixture of lambda and E. coli-encoded recombination
proteins. Recombination occurs between specific attachment (att) sites on the interacting DNA
molecules. For a description of att sites see Weisberg and Landy (1983) Site-Specific
Recombination in Phage Lambda, in Lambda II, Weisberg, ed. (Cold Spring Harbor, N.Y.: Cold
Spring Harbor Press), pp. 211-250. The DNA segments flanking the recombination sites are
switched, such that after recombination, the att sites are hybrid sequences comprised of
sequences donated by each parental vector. The recombination can occur between DNAs of any
topology. Each foregoing reference is incorporated by reference herein in its entirety.
Att sites may be introduced into a sequence of interest by ligating the sequence of
interest into an appropriate vector; generating a PCR product containing att B sites through the
use of specific primers; generating a cDNA library cloned into an appropriate vector containing
att sites; and the like.
Monocistronic and polycistronic genes. A monocistronic gene encodes an RNA
that contains the genetic information to translate only a single protein. A polycistronic gene
encodes an mRNA that contains the genetic information to translate more than one protein. The
proteins encoded in a polycistronic gene may have the same or different sequences or a
combination thereof. Dicistronic or bicistronic refers to a polycistronic gene that encodes two
proteins. Polycistronic genes optionally include one or more internal ribosome entry site (IRES)
elements to facilitate cap-independent initiation of translation, which may be situated at a
location that can drive translation of the downstream protein coding region independently of the
'-cap structure bound to the 5' end of the mRNA molecule. Any known IRES sequence (e.g.,
viral, eukaryotic, or artificial in origin) may be used. For example, the cricket paralysis virus
IRES sequence in the intergenic region (IGR) may be used, as described in Thompson et al.
(2001) PNAS 98:12972-12977. Optionally, IRES function may be potentiated by genetic
alteration, e.g., by causing constitutive expression of eIF2 kinase GCN2 or disrupting two
initiator tRNA(met) genes disrupted (id.).
Folding, as used herein, refers to the three-dimensional structure of polypeptides
and proteins, where interactions between amino acid residues act to stabilize the structure. While
non-covalent interactions are important in determining structure, usually the proteins of interest
will have intra- and/or intermolecular covalent disulfide bonds formed by two cysteine residues.
For naturally occurring proteins and polypeptides or derivatives and variants thereof, the proper
folding is typically the arrangement that results in optimal biological activity, and can
conveniently be monitored by assays for activity, e.g. ligand binding, enzymatic activity, etc.
In some instances, for example where the desired product is of synthetic origin,
assays based on biological activity will be less meaningful. The proper folding of such molecules
may be determined on the basis of physical properties, energetic considerations, modeling
studies, and the like.
The expression host may be further modified by the introduction of sequences
encoding one or more enzymes that enhance folding and disulfide bond formation, i.e. foldases,
chaperoning, etc. Such sequences may be constitutively or inducibly expressed in the yeast host
cell, using vectors, markers, etc. as known in the art. Preferably the sequences, including
transcriptional regulatory elements sufficient for the desired pattern of expression, are stably
integrated in the yeast genome through a targeted methodology.
For example, the eukaryotic PDI is not only an efficient catalyst of protein
cysteine oxidation and disulfide bond isomerization, but also exhibits chaperone activity. Co-
expression of PDI can facilitate the production of active proteins having multiple disulfide
bonds. Also of interest is the expression of BIP (immunoglobulin heavy chain binding protein);
cyclophilin; and the like. In one embodiment of the invention, the multi-subunit complex may be
expressed from a yeast strain produced by mating, wherein each of the haploid parental strains
expresses a distinct folding enzyme, e.g. one strain may express BIP, and the other strain may
express PDI or combinations thereof.
The terms "desired protein" or "target protein" are used interchangeably and refer
generally to a heterologous multi-subunit protein such as an antibody (e.g., a humanized
antibody) or a binding portion thereof described herein.
The term "antibody" includes any polypeptide chain-containing molecular
structure with a specific shape that fits to and recognizes an epitope, where one or more non-
covalent binding interactions stabilize the complex between the molecular structure and the
epitope. The archetypal antibody molecule is the immunoglobulin, and all types of
immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit,
cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be
"antibodies." A preferred source for producing antibodies useful as starting material according to
the invention is rabbits. Numerous antibody coding sequences have been described; and others
may be raised by methods well-known in the art. Examples thereof include chimeric antibodies,
human antibodies and other non-human mammalian antibodies, humanized antibodies, single
chain antibodies such as scFvs, camelbodies, nanobodies, IgNAR (single-chain antibodies
derived from sharks), small-modular immunopharmaceuticals (SMIPs), and antibody fragments
such as Fabs, Fab', F(ab')2 and the like. See Streltsov V A, et al., Structure of a shark IgNAR
antibody variable domain and modeling of an early-developmental isotype, Protein Sci. 2005
November; 14(11):2901-9. Epub 2005 Sep. 30; Greenberg A S, et al., A new antigen receptor
gene family that undergoes rearrangement and extensive somatic diversification in sharks,
Nature. 1995 Mar. 9; 374(6518):168-73; Nuttall S D, et al., Isolation of the new antigen receptor
from wobbegong sharks, and use as a scaffold for the display of protein loop libraries, Mol
Immunol. 2001 August; 38(4):313-26; Hamers-Casterman C, et al., Naturally occurring
antibodies devoid of light chains, Nature. 1993 Jun. 3; 363(6428):446-8; Gill D S, et al.,
Biopharmaceutical drug discovery using novel protein scaffolds, Curr Opin Biotechnol. 2006
December; 17(6):653-8. Epub 2006 Oct. 19. Each foregoing reference is incorporated by
reference herein in its entirety.
For example, antibodies or antigen binding fragments may be produced by genetic
engineering. In this technique, as with other methods, antibody-producing cells are sensitized to
the desired antigen or immunogen. The messenger RNA isolated from antibody producing cells
is used as a template to make cDNA using PCR amplification. A library of vectors, each
containing one heavy chain gene and one light chain gene retaining the initial antigen specificity,
is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the
expression vectors. A combinatorial library is constructed by combining the heavy chain gene
library with the light chain gene library. This results in a library of clones which co-express a
heavy and light chain (resembling the Fab fragment or antigen binding fragment of an antibody
molecule). The vectors that carry these genes are co-transfected into a host cell. When antibody
gene synthesis is induced in the transfected host, the heavy and light chain proteins self-assemble
to produce active antibodies that can be detected by screening with the antigen or immunogen.
Antibody coding sequences of interest include those encoded by native sequences,
as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in
sequence to the disclosed nucleic acids, and variants thereof. Variant polypeptides can include
amino acid (aa) substitutions, additions or deletions. The amino acid substitutions can be
conservative amino acid substitutions or substitutions to eliminate non-essential amino acids,
such as to alter a glycosylation site, or to minimize misfolding by substitution or deletion of one
or more cysteine residues that are not necessary for function. Variants can be designed so as to
retain or have enhanced biological activity of a particular region of the protein (e.g., a functional
domain, catalytic amino acid residues, etc). Variants also include fragments of the polypeptides
disclosed herein, particularly biologically active fragments and/or fragments corresponding to
functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also
described are polypeptides that have been modified using ordinary molecular biological
techniques so as to improve their resistance to proteolytic degradation or to optimize solubility
properties or to render them more suitable as a therapeutic agent.
Chimeric antibodies may be made by recombinant means by combining the
variable light and heavy chain regions (V and V ), obtained from antibody producing cells of
one species with the constant light and heavy chain regions from another. Typically chimeric
antibodies utilize rodent or rabbit variable regions and human constant regions, in order to
produce an antibody with predominantly human domains. The production of such chimeric
antibodies is well known in the art, and may be achieved by standard means (as described, e.g.,
in U.S. Pat. No. 5,624,659, incorporated herein by reference in its entirety). It is further
contemplated that the human constant regions of chimeric antibodies of the invention may be
selected from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12,
IgG13, IgG14, IgG15, IgG16, IgG17, IgG18 or IgG19 constant regions.
Humanized antibodies are engineered to contain even more human-like
immunoglobulin domains, and incorporate only the complementarity-determining regions of the
animal-derived antibody. This is accomplished by carefully examining the sequence of the
hyper-variable loops of the variable regions of the monoclonal antibody, and fitting them to the
structure of the human antibody chains. Although facially complex, the process is
straightforward in practice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully herein by
reference. Methods of humanizing antibodies have been described previously in issued U.S.
Patent No. 7935340, the disclosure of which is incorporated herein by reference in its entirety.
In some instances, a determination of whether additional rabbit framework residues are required
to maintain activity is necessary. In some instances the humanized antibodies still requires some
critical rabbit framework residues to be retained to minimize loss of affinity or activity. In these
cases, it is necessary to change single or multiple framework amino acids from human germline
sequences back to the original rabbit amino acids in order to have desired activity. These
changes are determined experimentally to identify which rabbit residues are necessary to
preserve affinity and activity.
In addition to entire immunoglobulins (or their recombinant counterparts),
immunoglobulin fragments comprising the epitope binding site (e.g., Fab', F(ab') , or other
fragments) may be synthesized. "Fragment," or minimal immunoglobulins may be designed
utilizing recombinant immunoglobulin techniques. For instance "Fv" immunoglobulins may be
produced by synthesizing a fused variable light chain region and a variable heavy chain region.
Combinations of antibodies are also of interest, e.g. diabodies, which comprise two distinct Fv
specificities. In another embodiment, SMIPs (small molecule immunopharmaceuticals),
camelbodies, nanobodies, and IgNAR are encompassed by immunoglobulin fragments.
Immunoglobulins and fragments thereof may be modified post-translationally,
e.g. to add effector moieties such as chemical linkers, detectable moieties, such as fluorescent
dyes, enzymes, toxins, substrates, bioluminescent materials, radioactive materials,
chemiluminescent moieties and the like, or specific binding moieties, such as streptavidin,
avidin, or biotin, and the like may be utilized in the methods and compositions described.
Examples of additional effector molecules are provided infra.
Product-associated variant: a product other than the desired product (e.g., the
desired multi-subunit complex) which is present in a preparation of the desired product and
related to the desired product. Exemplary product-associated variants include truncated or
elongated peptides, products having different glycosylation than the desired glycosylation (e.g.,
if an aglycosylated product is desired then any glycosylated product would be considered to be a
product-associated variant), complexes having abnormal stoichiometry, improper assembly,
abnormal disulfide linkages, abnormal or incomplete folding, aggregation, protease cleavage, or
other abnormalities. Exemplary product-associated variants may exhibit alterations in one or
more of molecular mass (e.g., detected by size exclusion chromatography), isoelectric point (e.g.,
detected by isoelectric focusing), electrophoretic mobility (e.g., detected by gel electrophoresis),
phosphorylation state (e.g., detected by mass spectrometry), charge to mass ratio (e.g., detected
by mass spectrometry), mass or identity of proteolytic fragments (e.g., detected by mass
spectrometry or gel electrophoresis), hydrophobicity (e.g., detected by HPLC) , charge (e.g.,
detected by ion exchange chromatography), affinity (e.g., in the case of an antibody, detected by
binding to protein A, protein G, and/or an epitope to which the desired antibody binds), and
glycosylation state (e.g., detected by lectin binding affinity). Where the desired protein is an
antibody, the term product-associate variant may include a glyco-heavy variant and/or half
antibody species (described below).
Exemplary product-associated variants include variant forms that contain aberrant
disulfide bonds. For example, most IgG1 antibody molecules are stabilized by a total of 16 intra-
chain and inter-chain disulfide bridges, which stabilize the folding of the IgG domains in both
heavy and light chains, while the inter-chain disulfide bridges stabilize the association between
heavy and light chains. Other antibody types likewise contain characteristic stabilizing intra-
chain and inter-chain disulfide bonds. Further, some antibodies (including Ab-A and Ab-B
disclosed herein) contain additional disulfide bonds referred to as non-canonical disulfide bonds.
Thus, aberrant inter-chain disulfide bonds may result in abnormal complex stoichiometry, due to
the absence of a stabilizing covalent linkage, and/or disulfide linkages to additional subunits.
Additionally, aberrant disulfide bonds (whether inter-chain or intra-chain) may decrease
structural stability of the antibody, which may result in decreased activity, decreased stability,
increased propensity to form aggregates, and/or increased immunogenicity. Product-associated
variants containing aberrant disulfide bonds may be detected in a variety of ways, including non-
reduced denaturing SDS-PAGE, capillary electrophoresis, cIEX, mass spectrometry (optionally
with chemical modification to produce a mass shift in free cysteines), size exclusion
chromatography, HPLC, changes in light scattering, and any other suitable methods known in the
art. See, e.g., The Protein Protocols Handbook 2002, Part V, 581-583, DOI: 10.1385/1
169-8:581;
Half antibody, half-antibody species, or H1L1 refer to a protein complex that
includes a single heavy and single light antibody chain, but lacks a covalent linkage to a second
heavy and light antibody chain. Two half antibodies may remain non-covalently associated
under some conditions (which may give behavior similar to a full antibody, e.g., apparent
molecular weight determined by size exclusion chromatography). Similarly, H2L1 refers to a
protein complex that includes two heavy antibody chains and single light antibody chain, but
lacks a covalent linkage to a second light antibody chain; these complexes may also non-
covalently associate with another light antibody chain (and likewise give similar behavior to a
full antibody). Like full antibodies, half antibody species and H2L1 species can dissociate under
reducing conditions into individual heavy and light chains. Half antibody species and H2L1
species can be detected on a non-reduced SDS-PAGE gel as a species migrating at a lower
apparent molecular weight than the full antibody, e.g., H1L1 migrates at approximately half the
apparent molecular weight of the full antibody (e.g., about 75 kDa).
Glyco-heavy variant refers to a glycosylated product-associated variant
sometimes present in antibody preparations and which contains at least a partial Fc sequence.
The glyco-heavy variant is characterized by decreased electrophoretic mobility observable by
SDS-PAGE (relative to a normal heavy chain), lectin binding affinity, binding to an anti-Fc
antibody, and apparent higher molecular weight of antibody complexes containing the glyco-
heavy variant as determined by size exclusion chromatography. See U.S. Provisional
Application Ser. No. 61/525,307 (Atty. Docket No. 67858.730200), filed August 31, 2011 which
is incorporated by reference herein in its entirety.
The term "polyploid yeast that stably expresses or expresses a desired secreted
heterologous polypeptide for prolonged time" refers to a yeast culture that secretes said
polypeptide for at least several days to a week, more preferably at least a month, still more
preferably at least 1-6 months, and even more preferably for more than a year at threshold
expression levels, typically at least 50-500 mg/liter (after about 90 hours in culture) and
preferably substantially greater.
The term "polyploidal yeast culture that secretes desired amounts of recombinant
polypeptide" refers to cultures that stably or for prolonged periods secrete at least at least 50-500
mg/liter, and most preferably 500-1000 mg/liter or more.
A polynucleotide sequence "corresponds" to a polypeptide sequence if translation
of the polynucleotide sequence in accordance with the genetic code yields the polypeptide
sequence (i.e., the polynucleotide sequence "encodes" the polypeptide sequence), one
polynucleotide sequence "corresponds" to another polynucleotide sequence if the two sequences
encode the same polypeptide sequence.
A "heterologous" region or domain of a DNA construct is an identifiable segment
of DNA within a larger DNA molecule that is not found in association with the larger molecule
in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually
be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the
source organism. Another example of a heterologous region is a construct where the coding
sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains
introns, or synthetic sequences having codons different than the native gene). Allelic variations
or naturally-occurring mutational events do not give rise to a heterologous region of DNA as
defined herein.
A "coding sequence" is an in-frame sequence of codons that (in view of the
genetic code) correspond to or encode a protein or peptide sequence. Two coding sequences
correspond to each other if the sequences or their complementary sequences encode the same
amino acid sequences. A coding sequence in association with appropriate regulatory sequences
may be transcribed and translated into a polypeptide. A polyadenylation signal and transcription
termination sequence will usually be located 3' to the coding sequence. A "promoter sequence" is
a DNA regulatory region capable of binding RNA polymerase in a cell and initiating
transcription of a downstream (3' direction) coding sequence. Promoter sequences typically
contain additional sites for binding of regulatory molecules (e.g., transcription factors) which
affect the transcription of the coding sequence. A coding sequence is "under the control" of the
promoter sequence or "operatively linked" to the promoter when RNA polymerase binds the
promoter sequence in a cell and transcribes the coding sequence into mRNA, which is then in
turn translated into the protein encoded by the coding sequence.
Vectors are used to introduce a foreign substance, such as DNA, RNA or protein,
into an organism or host cell. Typical vectors include recombinant viruses (for polynucleotides)
and liposomes (for polypeptides). A "DNA vector" is a replicon, such as plasmid, phage or
cosmid, to which another polynucleotide segment may be attached so as to bring about the
replication of the attached segment. An "expression vector" is a DNA vector which contains
regulatory sequences which will direct polypeptide synthesis by an appropriate host cell. This
usually means a promoter to bind RNA polymerase and initiate transcription of mRNA, as well
as ribosome binding sites and initiation signals to direct translation of the mRNA into a
polypeptide(s). Incorporation of a polynucleotide sequence into an expression vector at the
proper site and in correct reading frame, followed by transformation of an appropriate host cell
by the vector, enables the production of a polypeptide encoded by said polynucleotide sequence.
"Amplification" of polynucleotide sequences is the in vitro production of multiple
copies of a particular nucleic acid sequence. The amplified sequence is usually in the form of
DNA. A variety of techniques for carrying out such amplification are described in the following
review articles, each of which is incorporated by reference herein in its entirety: Van Brunt 1990,
Bio/Technol., 8(4):291-294; and Gill and Ghaemi, Nucleosides Nucleotides Nucleic Acids. 2008
Mar;27(3):224-43. Polymerase chain reaction or PCR is a prototype of nucleic acid
amplification, and use of PCR herein should be considered exemplary of other suitable
amplification techniques.
The general structure of antibodies in most vertebrates (including mammals) is
now well understood (Edelman, G. M., Ann. N.Y. Acad. Sci., 190: 5 (1971)). Conventional
antibodies consist of two identical light polypeptide chains of molecular weight approximately
23,000 daltons (the "light chain"), and two identical heavy chains of molecular weight 53,000-
70,000 (the "heavy chain"). The four chains are joined by disulfide bonds in a "Y" configuration
wherein the light chains bracket the heavy chains starting at the mouth of the "Y" configuration.
The "branch" portion of the "Y" configuration is designated the F region; the stem portion of
the "Y" configuration is designated the F region. The amino acid sequence orientation runs
from the N-terminal end at the top of the "Y" configuration to the C-terminal end at the bottom
of each chain. The N-terminal end possesses the variable region having specificity for the
antigen that elicited it, and is approximately 100 amino acids in length, there being slight
variations between light and heavy chain and from antibody to antibody.
The variable region is linked in each chain to a constant region that extends the
remaining length of the chain and that within a particular class of antibody does not vary with the
specificity of the antibody (i.e., the antigen eliciting it). There are five known major classes of
constant regions that determine the class of the immunoglobulin molecule (IgG, IgM, IgA, IgD,
and IgE corresponding to gamma, mu, alpha, delta, and epsilon heavy chain constant regions).
The constant region or class determines subsequent effector function of the antibody, including
activation of complement (Kabat, E. A., Structural Concepts in Immunology and
Immunochemistry, 2nd Ed., p. 413-436, Holt, Rinehart, Winston (1976)), and other cellular
responses (Andrews, D. W., et al., Clinical Immunobiology, pp 1-18, W. B. Sanders (1980);
Kohl, S., et al., Immunology, 48: 187 (1983)); while the variable region determines the antigen
with which it will react. Light chains are classified as either kappa or lambda. Each heavy chain
class can be paired with either kappa or lambda light chain. The light and heavy chains are
covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to
each other by covalent disulfide linkages when the immunoglobulins are generated either by
hybridomas or by B cells.
The expression "variable region" or "VR" refers to the domains within each pair
of light and heavy chains in an antibody that are involved directly in binding the antibody to the
antigen. Each heavy chain has at one end a variable domain (V ) followed by a number of
constant domains. Each light chain has a variable domain (V ) at one end and a constant domain
at its other end; the constant domain of the light chain is aligned with the first constant domain of
the heavy chain, and the light chain variable domain is aligned with the variable domain of the
heavy chain.
The expressions "complementarity determining region," "hypervariable region,"
or "CDR" refer to one or more of the hyper-variable or complementarity determining regions
(CDRs) found in the variable regions of light or heavy chains of an antibody (See Kabat, E. A. et
al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda,
Md., (1987)). These expressions include the hypervariable regions as defined by Kabat et al.
("Sequences of Proteins of Immunological Interest," Kabat E., et al., US Dept. of Health and
Human Services, 1983) or the hypervariable loops in 3-dimensional structures of antibodies
(Chothia and Lesk, J Mol. Biol. 196 901-917 (1987)). The CDRs in each chain are held in close
proximity by framework regions and, with the CDRs from the other chain, contribute to the
formation of the antigen binding site. Within the CDRs there are select amino acids that have
been described as the selectivity determining regions (SDRs) which represent the critical contact
residues used by the CDR in the antibody-antigen interaction (Kashmiri, S., Methods, 36:25-34
(2005)).
The expressions "framework region" or "FR" refer to one or more of the
framework regions within the variable regions of the light and heavy chains of an antibody (See
Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of
Health, Bethesda, Md., (1987)). These expressions include those amino acid sequence regions
interposed between the CDRs within the variable regions of the light and heavy chains of an
antibody.
Anti-NGF Antibodies and Binding Fragments Thereof Having Binding Activity
for NGF
Antibody Ab1
Described are methods of treating pain and the specific pain associated disorders
using antibody Ab1 or fragments thereof, or an antibody or antibody fragment that binds to the
same or overlapping epitope as Ab1, for example as set forth below, alone or is association with
another active agent, e.g., an NSAID or opioid analgesic, in a therapeutically effective amount
which inhibits the association of NGF with TrkA and the association of NGF with p75 and/or
inhibits or prevents pain. In one embodiment, described are chimeric antibodies having binding
specificity to NGF and possessing a variable light chain sequence comprising the sequence set
forth below:
ALVMTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQRPGQRPKLLIYGASNLDAGV
PSRFRGSGSGTEYTLTISDLECDDVGTYYCQSAFDSDSTENTFGGGTEVVVKR (SEQ ID
NO: 1).
Also described are methods of treating pain and the specific pain associated
disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic,
wherein the antibodies include chimeric antibodies having binding specificity to NGF and
possessing a light chain sequence comprising the sequence set forth below:
ALVMTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQRPGQRPKLLIYGASNLDAGV
PSRFRGSGSGTEYTLTISDLECDDVGTYYCQSAFDSDSTENTFGGGTEVVVKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).
Also described are methods of treating pain and the specific pain associated
disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic,
wherein the antibodies include chimeric antibodies having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGVI
TSIGSTVYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGYDDYDEMTYFNIWGQ
GTLVTVSS (SEQ ID NO: 3).
Also described are methods of treating pain and the specific pain associated
disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic,
wherein the antibodies include chimeric antibodies having binding specificity to NGF and
possessing a heavy chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGVI
TSIGSTVYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGYDDYDEMTYFNIWGQ
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4).
Also described are methods of treating pain and the specific pain associated
disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic,
wherein the antibodies comprise one or more of the polypeptide sequences of SEQ ID NO: 5;
SEQ ID NO: 6; and SEQ ID NO: 7 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 1
or the light chain sequence of SEQ ID NO: 2, and/or one or more of the polypeptide sequences
of SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 3 or the heavy chain sequence of SEQ ID NO: 4, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
Also described are methods of treating pain and the specific pain associated
disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic,
wherein the antibody is a fragment having binding specificity to NGF. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 3 or SEQ ID NO:
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain comprise, or alternatively consist of, one
or more of the polypeptide sequences of SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7
which correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO:
Also described are methods of treating pain and the specific pain associated
disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic,
wherein the antibodies include fragments having binding specificity to NGF comprise, or
alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 8; SEQ ID
NO: 9; and SEQ ID NO: 10 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3 or the
heavy chain sequence of SEQ ID NO: 4.
Also described are antibody fragments which include one or more of the antibody
fragments described herein. In one embodiment described, fragments of the antibodies having
binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following antibody fragments: the variable light chain region of SEQ ID NO:
1; the variable heavy chain region of SEQ ID NO: 3; the complementarity-determining regions
(SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7) of the variable light chain region of SEQ
ID NO: 1; and the complementarity-determining regions (SEQ ID NO: 8; SEQ ID NO: 9; and
SEQ ID NO: 10) of the variable heavy chain region of SEQ ID NO: 3.
In a particularly preferred optional embodiment described, the chimeric anti-NGF
antibody is Ab1, comprising, or alternatively consisting of, SEQ ID NO: 2 and SEQ ID NO: 4,
and having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for use herein comprise, or alternatively consist of, Fab (fragment antigen binding)
fragments having binding specificity for NGF or another Fab or monovalent antibody fragment
that binds to the same or overlapping epitope as Ab1. With respect to antibody Ab1, the Fab
fragment includes the variable light chain sequence of SEQ ID NO: 1 and the variable heavy
chain sequence of SEQ ID NO: 3. This embodiment described further contemplates additions,
deletions, and variants of SEQ ID NO: 1 and/or SEQ ID NO: 3 in said Fab while retaining
binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments may be
produced by enzymatic digestion (e.g., papain) of Ab1. In another embodiment described, anti-
NGF antibodies such as Ab1 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast
strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab2
Also described are methods of treating pain and the specific pain associated
disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic,
wherein the antibodies include chimeric or humanized antibodies having binding specificity to
NGF wherein the antibody is antibody Ab2 or fragments thereof, or an antibody or antibody
fragment that binds to the same or overlapping epitope as Ab2, for example as set forth below, in
a therapeutically effective amount which inhibits the association of NGF with TrkA and the
association of NGF with p75. In one embodiment, described are chimeric or humanized
antibodies having binding specificity to NGF and possessing a variable light chain sequence
comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQASQNIYSNLAWYQQKPGKAPKLLIYGASNLDAGVP
SRFSGSGSGTEYTLTISSLQPDDFATYYCQSAFDSDSTENTFGGGTKVEIKR (SEQ ID NO:
11).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQASQNIYSNLAWYQQKPGKAPKLLIYGASNLDAGVP
SRFSGSGSGTEYTLTISSLQPDDFATYYCQSAFDSDSTENTFGGGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).
Further described are chimeric or humanized antibodies having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions and
possessing a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSYAMSWVRQAPGKGLEWVGVITSIGSTV
YASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYDDYDEMTYFNIWGQGT
LVTVSS (SEQ ID NO: 13).
Also described are chimeric or humanized antibodies having binding specificity to
NGF for treatment or prevention of pain and pain associated conditions and possessing a heavy
chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSYAMSWVRQAPGKGLEWVGVITSIGSTV
YASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYDDYDEMTYFNIWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 15;
SEQ ID NO: 16; and SEQ ID NO: 17 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 11
or the light chain sequence of SEQ ID NO: 12, and/or one or more of the polypeptide sequences
of SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 11 or SEQ ID NO: 12. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 13 or SEQ ID
NO: 14.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions optionally
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
; SEQ ID NO: 16; and SEQ ID NO: 17 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 11
or the light chain sequence of SEQ ID NO: 12.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
18; SEQ ID NO: 19; and SEQ ID NO: 20 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO:
13 or the heavy chain sequence of SEQ ID NO: 14.
Also described are antibody fragments which include one or more of the antibody
fragments described herein. In one embodiment described, fragments of the antibodies having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 11; the variable heavy chain
region of SEQ ID NO: 13; the complementarity-determining regions (SEQ ID NO: 15; SEQ ID
NO: 16; and SEQ ID NO: 17) of the variable light chain region of SEQ ID NO: 11; and the
complementarity-determining regions (SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20)
of the variable heavy chain region of SEQ ID NO: 13.
In a particularly preferred optional embodiment described, the chimeric or
humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions
optionally is Ab2, comprising, or alternatively consisting of, SEQ ID NO: 12 and SEQ ID NO:
14, and having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF or a Fab or other monovalent antibody fragment that binds to the same or overlapping
epitope as Ab2,. With respect to antibody Ab2, the Fab fragment includes the variable light
chain sequence of SEQ ID NO: 11 and the variable heavy chain sequence of SEQ ID NO: 13.
This embodiment described further contemplates additions, deletions, and variants of SEQ ID
NO: 11 and/or SEQ ID NO: 13 in said Fab while retaining binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments may be
produced by enzymatic digestion (e.g., papain) of Ab2. In another embodiment described, anti-
NGF antibodies such as Ab2 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast
strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab3
Also described are methods of treating pain and the specific pain associated
disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic,
wherein the antibodies include chimeric or humanized antibodies having binding specificity to
NGF wherein the antibody is Ab3 or fragments thereof, or another antibody or antibody
fragment that binds to the same or overlapping epitope as Ab3, for example as set forth below, in
a therapeutically effective amount which inhibits the association of NGF with TrkA without
appreciably inhibiting the association of NGF with p75. In one embodiment, described are
chimeric antibodies having binding specificity to NGF and possessing a variable light chain
sequence comprising the sequence set forth below:
AVLTQTPSPVSAAMGDTVTIKCQSSQSVYKNNYLSWYQQKPGQPPRLLIYDASNLPSGV
PSRFSGSGSGTQFTLTISGVQCDDAATYYCLGDYDDDADNAFGGGTEVVVKR (SEQ ID
NO: 21).
Also described are chimeric antibodies or treatment or prevention of pain and pain
associated conditions having binding specificity to NGF and possessing a light chain sequence
comprising the sequence set forth below:
AVLTQTPSPVSAAMGDTVTIKCQSSQSVYKNNYLSWYQQKPGQPPRLLIYDASNLPSGV
PSRFSGSGSGTQFTLTISGVQCDDAATYYCLGDYDDDADNAFGGGTEVVVKRTVAAPS
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22).
Further described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a variable heavy
chain sequence comprising the sequence set forth below:
QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYVMIWVRQAPGKGLEYIGITWSAGTYYAS
WAKGRFTISKTSSTTVDLKITSPTTEDTATYFCAGGGGSIYDIWGPGTLVTVSS (SEQ ID
NO: 23).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYVMIWVRQAPGKGLEYIGITWSAGTYYAS
WAKGRFTISKTSSTTVDLKITSPTTEDTATYFCAGGGGSIYDIWGPGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 25;
SEQ ID NO: 26; and SEQ ID NO: 27 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 21
or the light chain sequence of SEQ ID NO: 22, and/or one or more of the polypeptide sequences
of SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
Also described are fragments of the antibody for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 21 or SEQ ID NO: 22. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 23 or SEQ ID
NO: 24.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
; SEQ ID NO: 26; and SEQ ID NO: 27 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 21
or the light chain sequence of SEQ ID NO: 22.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 28; SEQ ID
NO: 29; and SEQ ID NO: 30 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 23 or the
heavy chain sequence of SEQ ID NO: 24.
Also described are antibody fragments which include one or more of the antibody
fragments described herein for treatment or prevention of pain and pain associated conditions. In
one embodiment described, fragments of the antibodies having binding specificity to NGF
comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 21; the variable heavy chain
region of SEQ ID NO: 23; the complementarity-determining regions (SEQ ID NO: 25; SEQ ID
NO: 26; and SEQ ID NO: 27) of the variable light chain region of SEQ ID NO: 21; and the
complementarity-determining regions (SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30)
of the variable heavy chain region of SEQ ID NO: 23.
In a particularly preferred optional embodiment described, the chimeric anti-NGF
antibody for treatment or prevention of pain and pain associated conditions is Ab3, comprising,
or alternatively consisting of, SEQ ID NO: 22 and SEQ ID NO: 24, and having at least one of the
biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF or another Fab or monovalent antibody fragment that binds to the same or overlapping
epitope as Ab3. With respect to antibody Ab3, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 21 and the variable heavy chain sequence of SEQ ID NO: 23. This
embodiment further contemplates additions, deletions, and variants of SEQ ID NO: 21 and/or
SEQ ID NO: 23 in said Fab while retaining binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments may be
produced by enzymatic digestion (e.g., papain) of Ab3. In another embodiment described, anti-
NGF antibodies such as Ab3 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast
strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab4
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include chimeric or humanized antibodies having binding specificity to NGF wherein
the antibody is antibody Ab4 or fragments thereof, or another antibody or antibody fragment that
binds to the same or overlapping epitope as Ab4, for example as set forth below, in a
therapeutically effective amount which inhibits the association of NGF with TrkA without
appreciably inhibiting the association of NGF with p75 and/or for preventing or effectively
treating pain. In one embodiment, described are humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable light chain sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQSSQSVYKNNYLSWYQQKPGKAPKLLI
YDASNLPSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCLGDYDDDADNAFGGGTKVEI
KR (SEQ ID NO: 31).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQSSQSVYKNNYLSWYQQKPGKAPKLLIYDASNLPSG
VPSRFSGSGSGTEFTLTISSLQPDDFATYYCLGDYDDDADNAFGGGTKVEIKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 32).
Further described are humanized antibodies for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF and possessing a variable
heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSYVMIWVRQAPGKGLEYIGITWSAGTYY
ASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGGGSIYDIWGQGTLVTVSS
(SEQ ID NO: 33).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSYVMIWVRQAPGKGLEYIGITWSAGTYY
ASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGGGSIYDIWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 35;
SEQ ID NO: 36; and SEQ ID NO: 37 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 31
or the light chain sequence of SEQ ID NO: 32, and/or one or more of the polypeptide sequences
of SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 31 or SEQ ID NO: 32. In another embodiment described, antibody fragments of
the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 33
or SEQ ID NO: 34.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 35; SEQ ID
NO: 36; and SEQ ID NO: 37 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 31 or the
light chain sequence of SEQ ID NO: 32.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 38; SEQ ID
NO: 39; and SEQ ID NO: 40 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 33 or the
heavy chain sequence of SEQ ID NO: 34.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 31; the variable heavy chain
region of SEQ ID NO: 33; the complementarity-determining regions (SEQ ID NO: 35; SEQ ID
NO: 36; and SEQ ID NO: 37) of the variable light chain region of SEQ ID NO: 31; and the
complementarity-determining regions (SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40)
of the variable heavy chain region of SEQ ID NO: 33.
In a particularly preferred embodiment described, the chimeric or humanized anti-
NGF antibody for treatment or prevention of pain and pain associated conditions is Ab4,
comprising, or alternatively consisting of, SEQ ID NO: 32 and SEQ ID NO: 34, and having at
least one of the biological activities set forth herein.
In a further particularly preferred embodiment described, antibody fragments for
treatment or prevention of pain and pain associated conditions comprise, or alternatively consist
of, Fab (fragment antigen binding) fragments having binding specificity for NGF or another Fab
or monovalent antibody fragment that binds to the same or overlapping epitope as Ab14. With
respect to antibody Ab4, the Fab fragment includes the variable light chain sequence of SEQ ID
NO: 31 and the variable heavy chain sequence of SEQ ID NO: 33. This embodiment described
further contemplates additions, deletions, and variants of SEQ ID NO: 31 and/or SEQ ID NO: 33
in said Fab while retaining binding specificity for NGF.
In one embodiment described herein (infra), Fab fragments for treatment or
prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g.,
papain) of Ab4. In another embodiment described, anti-NGF antibodies such as Ab4 or Fab
fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab5
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies optionally include Ab5 or fragments thereof, or another antibody or antibody
fragment that binds to the same or overlapping epitope as Ab5, for example as set forth below, in
a therapeutically effective amount which inhibits the association of NGF with TrkA and the
association of NGF with p75. In one embodiment, described are chimeric antibodies having
binding specificity to NGF and possessing a variable light chain sequence comprising the
sequence set forth below:
AYDMTQTPASVEVAVGGTVTIKCQASQSIYSNLAWYQQRPGQPPKLLIYDASTLESGVP
SRFKGSGSGTEYTLTISGVECADAASYYCQQGFTVSDIDNAFGGGTEVVVKR (SEQ ID
NO: 41).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a light chain
sequence comprising the sequence set forth below:
AYDMTQTPASVEVAVGGTVTIKCQASQSIYSNLAWYQQRPGQPPKLLIYDASTLESGVP
SRFKGSGSGTEYTLTISGVECADAASYYCQQGFTVSDIDNAFGGGTEVVVKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42).
Further described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a variable heavy
chain sequence comprising the sequence set forth below:
QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYAVGWVRQAPGKGLEWIGIIGRNGNTWYA
SWARGRFTISKTSTTVDLKITSPTSEDTATYFCARGYGRSVAYYVFNIWGPGTLVTVSS
(SEQ ID NO: 43).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYAVGWVRQAPGKGLEWIGIIGRNGNTWYA
SWARGRFTISKTSTTVDLKITSPTSEDTATYFCARGYGRSVAYYVFNIWGPGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 45;
SEQ ID NO: 46; and SEQ ID NO: 47 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 41
or the light chain sequence of SEQ ID NO: 42, and/or one or more of the polypeptide sequences
of SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
described or fragments thereof optionally comprise, or alternatively consist of, combinations of
one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy
and light chain sequences set forth above, including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 41 or SEQ ID NO: 42. In another optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 43 or SEQ ID NO: 44.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide
sequences of SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable light
chain sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
optionally comprise, or alternatively consist of, one or more of the polypeptide sequences of
SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 41; the variable heavy chain
region of SEQ ID NO: 43; the complementarity-determining regions (SEQ ID NO: 45; SEQ ID
NO: 46; and SEQ ID NO: 47) of the variable light chain region of SEQ ID NO: 41; and the
complementarity-determining regions (SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50)
of the variable heavy chain region of SEQ ID NO: 43.
In a particularly preferred optional embodiment described, the optionally included
chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is
Ab5, comprising, or alternatively consisting of, SEQ ID NO: 42 and SEQ ID NO: 44, and having
at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF or another Fab or antibody fragment that binds to the same or overlapping epitope as Ab5.
With respect to antibody Ab5, the Fab fragment includes the variable light chain sequence of
SEQ ID NO: 41 and the variable heavy chain sequence of SEQ ID NO: 43. This embodiment
described further contemplates additions, deletions, and variants of SEQ ID NO: 41 and/or SEQ
ID NO: 43 in said Fab while retaining binding specificity for NGF.
In one embodiment described herein (infra), Fab fragments for treatment or
prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g.,
papain) of Ab5. In another embodiment described, anti-NGF antibodies such as Ab5 or Fab
fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab6
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include Ab6 or fragments thereof, or another antibody or antibody fragment that binds
to the same or overlapping epitope as Ab6, for example as set forth below, in a therapeutically
effective amount which inhibits the association of NGF with TrkA and the association of NGF
with p75. In one embodiment, described are chimeric or humanized antibodies having binding
specificity to NGF and possessing a variable light chain sequence comprising the sequence set
forth below:
DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS
RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKR (SEQ ID NO:
51).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS
RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 52).
Further described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAVGWVRQAPGKGLEWVGIIGRNGNT
WYASSARGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYGRSVAYYVFNIWGPGT
LVTVSS (SEQ ID NO: 53).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAVGWVRQAPGKGLEWVGIIGRNGNT
WYASSARGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYGRSVAYYVFNIWGPGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 55;
SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51
or the light chain sequence of SEQ ID NO: 52, and/or one or more of the polypeptide sequences
of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54, or
combinations of these polypeptide sequences. In another optional embodiment described, the
antibodies or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light
chain sequences set forth above, including all of them.
Also described are fragments of the antibody for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 51 or SEQ ID NO: 52. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 53 or SEQ ID
NO: 54.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide
sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable light
chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO:
53 or the heavy chain sequence of SEQ ID NO: 54.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 51; the variable heavy chain
region of SEQ ID NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID
NO: 56; and SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51; and the
complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60)
of the variable heavy chain region of SEQ ID NO: 53.
In a particularly preferred optional embodiment described is the chimeric or
humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions
is Ab6, comprising, or alternatively consisting of, SEQ ID NO: 52 and SEQ ID NO: 54, and
having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described are, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF or another Fab or monovalent antibody fragment that binds to the same or overlapping
epitope as Ab6. With respect to antibody Ab6, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 51 and the variable heavy chain sequence of SEQ ID NO: 53. This
optional embodiment described further contemplates additions, deletions, and variants of SEQ
ID NO: 51 and/or SEQ ID NO: 53 in said Fab while retaining binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab6. In another embodiment described, anti-NGF antibodies such as Ab6 or
Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab7
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include Ab7 or fragments thereof, or another antibody or antibody fragment that binds
to the same or overlapping epitope as Ab7, for example as set forth below, in a therapeutically
effective amount which inhibits the association of NGF with TrkA and the association of NGF
with p75. In one embodiment, described are chimeric antibodies for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF and possessing a variable
light chain sequence comprising the sequence set forth below:
ADVVMTQTPASVSQPVGGTVTIKCQASEDIYNLLAWYQQKPGQPPKLLIYSASTLASGV
PSRFKGSGSGTEYTLTISGLECADAATYYCQNNYLVTTYGVAFGGGTEVVVKR (SEQ
ID NO: 61).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a light chain
sequence comprising the sequence set forth below:
ADVVMTQTPASVSQPVGGTVTIKCQASEDIYNLLAWYQQKPGQPPKLLIYSASTLASGV
PSRFKGSGSGTEYTLTISGLECADAATYYCQNNYLVTTYGVAFGGGTEVVVKRTVAAPS
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 62).
Further described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a variable heavy
chain sequence comprising the sequence set forth below:
QEQLKESGGRLVTPGTPLTLTCTVSGFSLSSYAMIWVRQAPGKGLEYIGYIDTDTSAYYA
SWVKGRFTISRTSTTVDLKITSPTTEDTATYFCARSYAAYGGYPATFDPWGPGTLVTVSS
(SEQ ID NO: 63).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
QEQLKESGGRLVTPGTPLTLTCTVSGFSLSSYAMIWVRQAPGKGLEYIGYIDTDTSAYYA
SWVKGRFTISRTSTTVDLKITSPTTEDTATYFCARSYAAYGGYPATFDPWGPGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 64).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 65;
SEQ ID NO: 66; and SEQ ID NO: 67 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 61
or the light chain sequence of SEQ ID NO: 62, and/or one or more of the polypeptide sequences
of SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
Also described are fragments of the antibody for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 61 or SEQ ID NO: 62. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 63 or SEQ ID
NO: 64.
In a further optional embodiment described, fragments of the antibody for
treatment or prevention of pain and pain associated conditions having binding specificity to NGF
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
65; SEQ ID NO: 66; and SEQ ID NO: 67 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 61
or the light chain sequence of SEQ ID NO: 62.
In a further optional embodiment described, fragments of the antibody for
treatment or prevention of pain and pain associated conditions having binding specificity to NGF
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
68; SEQ ID NO: 69; and SEQ ID NO: 70 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO:
63 or the heavy chain sequence of SEQ ID NO: 64.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 61; the variable heavy chain
region of SEQ ID NO: 63; the complementarity-determining regions (SEQ ID NO: 65; SEQ ID
NO: 66; and SEQ ID NO: 67) of the variable light chain region of SEQ ID NO: 61; and the
complementarity-determining regions (SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70)
of the variable heavy chain region of SEQ ID NO: 63.
In a particularly preferred optional embodiment described is, the chimeric anti-
NGF antibody for treatment or prevention of pain and pain associated conditions is Ab7,
comprising, or alternatively consisting of, SEQ ID NO: 62 and SEQ ID NO: 64, and having at
least one of the biological activities set forth herein.
In a further particularly optionally preferred embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF or another Fab or monovalent antibody fragment that binds to the same or overlapping
epitope as Ab7. With respect to antibody Ab7, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 61 and the variable heavy chain sequence of SEQ ID NO: 63. This
embodiment further contemplates additions, deletions, and variants of SEQ ID NO: 61 and/or
SEQ ID NO: 63 in said Fab while retaining binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab7. In another embodiment described, anti-NGF antibodies such as Ab7 or
Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab8
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include Ab8 or fragments thereof, or another antibody or fragment that binds to the
same or overlapping epitope as Ab8, for example as set forth below, in a therapeutically effective
amount which inhibits the association of NGF with TrkA and the association of NGF with p75.
In one embodiment, described are chimeric or humanized antibodies having binding specificity
to NGF and possessing a variable light chain sequence comprising the sequence set forth below:
DIQMTQSPSSLSASVGDRVTITCQASEDIYNLLAWYQQKPGKVPKLLIYSASTLASGVPS
RFSGSGSGTDYTLTISSLQPEDVATYYCQNNYLVTTYGVAFGGGTKVEIKR (SEQ ID
NO: 71).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
DIQMTQSPSSLSASVGDRVTITCQASEDIYNLLAWYQQKPGKVPKLLIYSASTLASGVPS
RFSGSGSGTDYTLTISSLQPEDVATYYCQNNYLVTTYGVAFGGGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 72).
Further described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMIWVRQAPGKGLEYIGYIDTDTSAYY
ASSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSYAAYGGYPATFDPWGQGTL
VTVSS (SEQ ID NO: 73).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMIWVRQAPGKGLEYIGYIDTDTSAYY
ASSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSYAAYGGYPATFDPWGQGTL
VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 75;
SEQ ID NO: 76; and SEQ ID NO: 77 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 71
or the light chain sequence of SEQ ID NO: 72, and/or one or more of the polypeptide sequences
of SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID NO: 80 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74, or
combinations of these polypeptide sequences. In another optional embodiment described, the
antibodies or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light
chain sequences set forth above, including all of them.
Also described are fragments of the antibody for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 71 or SEQ ID NO: 72. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 73 or SEQ ID
NO: 74.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
75; SEQ ID NO: 76; and SEQ ID NO: 77 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 71
or the light chain sequence of SEQ ID NO: 72.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
78; SEQ ID NO: 79; and SEQ ID NO: 80 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO:
73 or the heavy chain sequence of SEQ ID NO: 74.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described are , fragments of the antibodies having binding specificity
to NGF comprise, or alternatively consist of, one, two, three or more, including all of the
following antibody fragments: the variable light chain region of SEQ ID NO: 71; the variable
heavy chain region of SEQ ID NO: 73; the complementarity-determining regions (SEQ ID NO:
75; SEQ ID NO: 76; and SEQ ID NO: 77) of the variable light chain region of SEQ ID NO: 71;
and the complementarity-determining regions (SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID
NO: 80) of the variable heavy chain region of SEQ ID NO: 73.
In a particularly preferred optional embodiment described is, the chimeric or
humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions
is Ab8, comprising, or alternatively consisting of, SEQ ID NO: 72 and SEQ ID NO: 74, and
having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab8, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 71 and the variable heavy chain sequence of SEQ ID NO: 73 or another Fab or
monovalent antibody fragment that binds to the same or overlapping epitope as Ab8. This
embodiment described further contemplates additions, deletions, and variants of SEQ ID NO: 71
and/or SEQ ID NO: 73 in said Fab while retaining binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab8. In another embodiment described, anti-NGF antibodies such as Ab8 or
Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab9
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include Ab9 or fragments thereof, or another antibody or antibody fragment that binds
to the same or overlapping epitope as Ab9, for example as set forth below, in a therapeutically
effective amount which inhibits the association of NGF with TrkA and the association of NGF
with p75. In one optional embodiment, described are chimeric antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable light chain sequence comprising the sequence set forth below:
AYDMTQTPASVSAAVGGTVTIKCQASENIGSYLAWYQQKPGQPPELLIYRASTLASGVP
SRFKGSGSGTQFTLTISGVECADAATYYCQQGYNSENLDNAFGGGTEVVVKR (SEQ ID
NO: 81).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a light chain
sequence comprising the sequence set forth below:
AYDMTQTPASVSAAVGGTVTIKCQASENIGSYLAWYQQKPGQPPELLIYRASTLASGVP
SRFKGSGSGTQFTLTISGVECADAATYYCQQGYNSENLDNAFGGGTEVVVKRTVAAPS
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 82).
Further described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a variable heavy
chain sequence comprising the sequence set forth below:
QSVEESGGRLVTPGTPLTLTCTVSGIDLSMYSMGWVRQAPGKGLEYIGWISYGGTAYYA
SWAKGRFTISKTSTTVELKITSPTIEDTATYFCARETPVNYYLDIWGQGTLVTVSS (SEQ
ID NO: 83).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
QSVEESGGRLVTPGTPLTLTCTVSGIDLSMYSMGWVRQAPGKGLEYIGWISYGGTAYYA
SWAKGRFTISKTSTTVELKITSPTIEDTATYFCARETPVNYYLDIWGQGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 84).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 85;
SEQ ID NO: 86; and SEQ ID NO: 87 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 81
or the light chain sequence of SEQ ID NO: 82, and/or one or more of the polypeptide sequences
of SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84, or
combinations of these polypeptide sequences. In another optional embodiment described, the
antibodies or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light
chain sequences set forth above, including all of them.
Also described are fragments of the antibody for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 81 or SEQ ID NO: 82. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 83 or SEQ ID
NO: 84.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
85; SEQ ID NO: 86; and SEQ ID NO: 87 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 81
or the light chain sequence of SEQ ID NO: 82.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
88; SEQ ID NO: 89; and SEQ ID NO: 90 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO:
83 or the heavy chain sequence of SEQ ID NO: 84.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 81; the variable heavy chain
region of SEQ ID NO: 83; the complementarity-determining regions (SEQ ID NO: 85; SEQ ID
NO: 86; and SEQ ID NO: 87) of the variable light chain region of SEQ ID NO: 81; and the
complementarity-determining regions (SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90)
of the variable heavy chain region of SEQ ID NO: 83.
In a particularly preferred optional embodiment described, the chimeric anti-NGF
antibody for treatment or prevention of pain and pain associated conditions, is Ab9, comprising,
or alternatively consisting of, SEQ ID NO: 82 and SEQ ID NO: 84, and having at least one of the
biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab9, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 81 and the variable heavy chain sequence of SEQ ID NO: 83 or another Fab or
monovalent antibody fragment that binds to the same or overlapping epitope as Ab9. This
embodiment described further contemplates additions, deletions, and variants of SEQ ID NO: 81
and/or SEQ ID NO: 83 in said Fab while retaining binding specificity for NGF.
In one embodiment described herein (infra), Fab fragments for treatment or
prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g.,
papain) of Ab9. In another optional embodiment described, anti-NGF antibodies such as Ab9 or
Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab10
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include Ab10 or fragments thereof, or another antibody or antibody fragment that
binds to the same or overlapping epitope as Ab10, for example as set forth below, in a
therapeutically effective amount which inhibits the association of NGF with TrkA and the
association of NGF with p75. In one embodiment, described are chimeric or humanized
antibodies for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF and possessing a variable light chain sequence comprising the sequence set
forth below:
AYDMTQSPSSLSASVGDRVTITCQASENIGSYLAWYQQKPGKVPKLLIYRASTLASGVPS
RFSGSGSGTDFTLTISSLQPEDVATYYCQQGYNSENLDNAFGGGTKVEIKR (SEQ ID
NO: 91).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
AYDMTQSPSSLSASVGDRVTITCQASENIGSYLAWYQQKPGKVPKLLIYRASTLASGVPS
RFSGSGSGTDFTLTISSLQPEDVATYYCQQGYNSENLDNAFGGGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 92).
Further described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSMYSMGWVRQAPGKGLEYIGWISYGGTAY
YASSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARETPVNYYLDIWGQGTLVTV
SS (SEQ ID NO: 93).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSMYSMGWVRQAPGKGLEYIGWISYGGTAY
YASSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARETPVNYYLDIWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 94).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 95;
SEQ ID NO: 96; and SEQ ID NO: 97 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 91
or the light chain sequence of SEQ ID NO: 92, and/or one or more of the polypeptide sequences
of SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
Also described are fragments of the antibody for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 91 or SEQ ID NO: 92. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 93 or SEQ ID
NO: 94.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions optionally
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
95; SEQ ID NO: 96; and SEQ ID NO: 97 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 91
or the light chain sequence of SEQ ID NO: 92.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
98; SEQ ID NO: 99; and SEQ ID NO: 100 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 91; the variable heavy chain region of SEQ ID NO:
93; the complementarity-determining regions (SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID
NO: 97) of the variable light chain region of SEQ ID NO: 91; and the complementarity-
determining regions (SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100) of the variable
heavy chain region of SEQ ID NO: 93.
In a particularly preferred optional embodiment described is, the chimeric or
humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions
is Ab10, comprising, or alternatively consisting of, SEQ ID NO: 92 and SEQ ID NO: 94, and
having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab10, the Fab fragment for treatment or prevention of pain and
pain associated conditions includes the variable light chain sequence of SEQ ID NO: 91 and the
variable heavy chain sequence of SEQ ID NO: 93 or another Fab or monovalent antibody
fragment that binds to the same or overlapping epitope as Ab10. This embodiment described
further optionally contemplates additions, deletions, and variants of SEQ ID NO: 91 and/or SEQ
ID NO: 93 in said Fab while retaining binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab10. In another embodiment described, anti-NGF antibodies such as Ab10 or
Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab11
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include Ab11 or fragments thereof, or another antibody or antibody fragment that
binds to the same or overlapping epitope as Ab11, for example as set forth below, in a
therapeutically effective amount which inhibits the association of NGF with TrkA and the
association of NGF with p75. In one optional embodiment, described are chimeric antibodies
having binding specificity to NGF and possessing a variable light chain sequence comprising the
sequence set forth below:
AFELTQTPSSVEAAVGGTVTIKCQASQNIVTNLAWYQQKPGQPPKLLIYGASTLASGVSS
RFKGSGSGTQFTLTISDLECADAATYFCQSYDGFNSAGFGGGTEVVVKR (SEQ ID NO:
101).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a light chain
sequence comprising the sequence set forth below:
AFELTQTPSSVEAAVGGTVTIKCQASQNIVTNLAWYQQKPGQPPKLLIYGASTLASGVSS
RFKGSGSGTQFTLTISDLECADAATYFCQSYDGFNSAGFGGGTEVVVKRTVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 102).
Further described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a variable heavy
chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCTASGFSLSGYDMSWVRQAPGKGLEYIGLISYDGNTYYA
TWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARSLYAGPNAGIGPFNIWGQGTLVTVSS
(SEQ ID NO: 103).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCTASGFSLSGYDMSWVRQAPGKGLEYIGLISYDGNTYYA
TWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARSLYAGPNAGIGPFNIWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 104).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 105;
SEQ ID NO: 106; and SEQ ID NO: 107 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
101 or the light chain sequence of SEQ ID NO: 102, and/or one or more of the polypeptide
sequences of SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO: 110 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof optionally comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light
chain sequences set forth above, including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 101 or SEQ ID NO: 102. In another optional embodiment described, antibody
fragments comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 103 or
SEQ ID NO: 104.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
105; SEQ ID NO: 106; and SEQ ID NO: 107 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of
SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102.
In a further optional embodiment described, fragments of the antibody for
treatment or prevention of pain and pain associated conditions having binding specificity to NGF
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
108; SEQ ID NO: 109; and SEQ ID NO: 110 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 101; the variable heavy chain
region of SEQ ID NO: 103; the complementarity-determining regions (SEQ ID NO: 105; SEQ
ID NO: 106; and SEQ ID NO: 107) of the variable light chain region of SEQ ID NO: 101; and
the complementarity-determining regions (SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO:
110) of the variable heavy chain region of SEQ ID NO: 103.
In a particularly preferred optional embodiment described is, the chimeric anti-
NGF antibody for treatment or prevention of pain and pain associated conditions is Ab11,
comprising, or alternatively consisting of, SEQ ID NO: 102 and SEQ ID NO: 104, and having at
least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab11, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 101 and the variable heavy chain sequence of SEQ ID NO: 103 or
comprises another Fab or monovalent antibody fragment that binds to the same or overlapping
epitope as Ab11. This embodiment described further contemplates additions, deletions, and
variants of SEQ ID NO: 101 and/or SEQ ID NO: 103 in said Fab while retaining binding
specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments may for
treatment or prevention of pain and pain associated conditions be produced by enzymatic
digestion (e.g., papain) of Ab11. In another optional embodiment described, anti-NGF
antibodies such as Ab11 or Fab fragments thereof may be produced via expression in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast
strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab12
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include Ab12 or fragments thereof, f or another antibody or antibody fragment that
binds to the same or overlapping epitope as Ab12, or example as set forth below, in a
therapeutically effective amount which inhibits the association of NGF with TrkA and the
association of NGF with p75. In one embodiment, described are chimeric or humanized
antibodies for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF and possessing a variable light chain sequence comprising the sequence set
forth below:
AFQMTQSPSSLSASVGDRVTITCQASQNIVTNLAWYQQKPGKVPKLLIYGASTLASGVP
SRFSGSGSGTDFTLTISSLQPEDVATYYCQSYDGFNSAGFGGGTKVEIKR (SEQ ID NO:
111).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
AFQMTQSPSSLSASVGDRVTITCQASQNIVTNLAWYQQKPGKVPKLLIYGASTLASGVP
SRFSGSGSGTDFTLTISSLQPEDVATYYCQSYDGFNSAGFGGGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 112).
Further described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
QVQLVESGGGVVQPGRSLRLSCAASGFSLSGYDMSWVRQAPGKGLEWVGLISYDGNT
YYATSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSLYAGPNAGIGPFNIWGQG
TLVTVSS (SEQ ID NO: 113).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
QVQLVESGGGVVQPGRSLRLSCAASGFSLSGYDMSWVRQAPGKGLEWVGLISYDGNT
YYATSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSLYAGPNAGIGPFNIWGQG
TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 114).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 115;
SEQ ID NO: 116; and SEQ ID NO: 117 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
111 or the light chain sequence of SEQ ID NO: 112, and/or one or more of the polypeptide
sequences of SEQ ID NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO: 114, or
combinations of these polypeptide sequences. In another optional embodiment described, the
antibodies or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light
chain sequences set forth above, including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one optional embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 111 or SEQ ID NO: 112. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 113 or SEQ ID
NO: 114.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
115; SEQ ID NO: 116; and SEQ ID NO: 117 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of
SEQ ID NO: 111 or the light chain sequence of SEQ ID NO: 112.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
118; SEQ ID NO: 119; and SEQ ID NO: 120 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO: 114.
Also described are antibody fragments which include one or more of the antibody
fragments described herein. In one optional embodiment described, fragments of the antibodies
having binding specificity to NGF for treatment or prevention of pain and pain associated
conditions comprise, or alternatively consist of, one, two, three or more, including all of the
following antibody fragments: the variable light chain region of SEQ ID NO: 111; the variable
heavy chain region of SEQ ID NO: 113; the complementarity-determining regions (SEQ ID NO:
115; SEQ ID NO: 116; and SEQ ID NO: 117) of the variable light chain region of SEQ ID NO:
111; and the complementarity-determining regions (SEQ ID NO: 118; SEQ ID NO: 119; and
SEQ ID NO: 120) of the variable heavy chain region of SEQ ID NO: 113.
In a particularly preferred optional embodiment described is the chimeric or
humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions
is Ab12, comprising, or alternatively consisting of, SEQ ID NO: 112 and SEQ ID NO: 114, and
having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab12, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 111 and the variable heavy chain sequence of SEQ ID NO: 113 or
comprises another Fab or monovalent antibody fragment that binds to the same or overlapping
epitope as Ab12. This optional embodiment described further contemplates additions, deletions,
and variants of SEQ ID NO: 111 and/or SEQ ID NO: 113 in said Fab while retaining binding
specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab12. In another embodiment described, anti-NGF antibodies such as Ab12 or
Fab fragments thereof for treatment or prevention of pain and pain associated conditions may be
produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal,
insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid
Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab13
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include Ab13 or fragments thereof, or another antibody or antibody fragment that
binds to the same or overlapping epitope as Ab13, for example as set forth below, in a
therapeutically effective amount which inhibits the association of NGF with TrkA and the
association of NGF with p75. In one embodiment, described are chimeric antibodies for
treatment or prevention of pain and pain associated conditions having binding specificity to NGF
and possessing a variable light chain sequence comprising the sequence set forth below:
AAVLTQTPSPVSAAVGGTVSISCQSSQNVYKNNYLSWYQQKPGQPPKLLIYKASTLASG
VPSRFKGGGSGTDFTLTISDVQCDAAATYYCAGGYTSSSDNAFGGGTEVVVKR (SEQ
ID NO: 121).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a light chain
sequence comprising the sequence set forth below:
AAVLTQTPSPVSAAVGGTVSISCQSSQNVYKNNYLSWYQQKPGQPPKLLIYKASTLASG
VPSRFKGGGSGTDFTLTISDVQCDAAATYYCAGGYTSSSDNAFGGGTEVVVKRTVAAP
SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 122).
Further described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a variable heavy
chain sequence comprising the sequence set forth below:
QSVEASGGRLVTPGTPLTLTCTASGFSLSTYWMSWVRQAPGKGLEWIGDIYFSNEETNY
ASWAKGRFTISKTSTTVDLNVISPTTEDTATYFCARGSPDVDIGIDMWGPGTLVTVSS
(SEQ ID NO: 123).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
QSVEASGGRLVTPGTPLTLTCTASGFSLSTYWMSWVRQAPGKGLEWIGDIYFSNEETNY
ASWAKGRFTISKTSTTVDLNVISPTTEDTATYFCARGSPDVDIGIDMWGPGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 124).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 125;
SEQ ID NO: 126; and SEQ ID NO: 127 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
121 or the light chain sequence of SEQ ID NO: 122, and/or one or more of the polypeptide
sequences of SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124, or
combinations of these polypeptide sequences. In another optional embodiment described, the
antibodies or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light
chain sequences set forth above, including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 121 or SEQ ID NO: 122. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 123 or SEQ ID
NO: 124.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
125; SEQ ID NO: 126; and SEQ ID NO: 127 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of
SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
128; SEQ ID NO: 129; and SEQ ID NO: 130 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 121; the variable heavy chain
region of SEQ ID NO: 123; the complementarity-determining regions (SEQ ID NO: 125; SEQ
ID NO: 126; and SEQ ID NO: 127) of the variable light chain region of SEQ ID NO: 121; and
the complementarity-determining regions (SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID NO:
130) of the variable heavy chain region of SEQ ID NO: 123.
In a particularly preferred optional embodiment described is, the chimeric anti-
NGF antibody for treatment or prevention of pain and pain associated conditions is Ab13,
comprising, or alternatively consisting of, SEQ ID NO: 122 and SEQ ID NO: 124, and having at
least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab13, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 121 and the variable heavy chain sequence of SEQ ID NO: 123 or
comprises another Fab or monovalent antibody fragment that binds to the same or overlapping
epitope as Ab13. This optional embodiment described further contemplates additions, deletions,
and variants of SEQ ID NO: 121 and/or SEQ ID NO: 123 in said Fab while retaining binding
specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab13. In another embodiment described, anti-NGF antibodies such as Ab13 or
Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab14
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is
Ab14 or fragments thereof, or another antibody or fragment that binds to the same or overlapping
epitope as Ab14, for example as set forth below, in a therapeutically effective amount which
inhibits the association of NGF with TrkA and the association of NGF with p75. In one optional
embodiment, described are chimeric or humanized antibodies having binding specificity to NGF
and possessing a variable light chain sequence comprising the sequence set forth below:
DIQMTQSPSSLSASVGDRVTITCQSSQNVYKNNYLSWYQQKPGKVPKLLIYKASTLASG
VPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYTSSSDNAFGGGTKVEIKR (SEQ ID
NO: 131).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
DIQMTQSPSSLSASVGDRVTITCQSSQNVYKNNYLSWYQQKPGKVPKLLIYKASTLASG
VPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYTSSSDNAFGGGTKVEIKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 132).
Further described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSTYWMSWVRQAPGKGLEWVGDIYFSNEET
NYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSPDVDIGIDMWGPGTLV
TVSS (SEQ ID NO: 133).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSTYWMSWVRQAPGKGLEWVGDIYFSNEET
NYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSPDVDIGIDMWGPGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 134).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 135;
SEQ ID NO: 136; and SEQ ID NO: 137 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
131 or the light chain sequence of SEQ ID NO: 132, and/or one or more of the polypeptide
sequences of SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134, or
combinations of these polypeptide sequences. In another optional embodiment described, the
antibodies or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light
chain sequences set forth above, including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 131 or SEQ ID NO: 132. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 133 or SEQ ID
NO: 134.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
optionally comprise, or alternatively consist of, one or more of the polypeptide sequences of
SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable light
chain sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
138; SEQ ID NO: 139; and SEQ ID NO: 140 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 131; the variable heavy chain
region of SEQ ID NO: 133; the complementarity-determining regions (SEQ ID NO: 135; SEQ
ID NO: 136; and SEQ ID NO: 137) of the variable light chain region of SEQ ID NO: 131; and
the complementarity-determining regions (SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO:
140) of the variable heavy chain region of SEQ ID NO: 133.
In a particularly preferred optional embodiment described is, the chimeric or
humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions
is Ab14, comprising, or alternatively consisting of, SEQ ID NO: 132 and SEQ ID NO: 134, and
having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab14, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 131 and the variable heavy chain sequence of SEQ ID NO: 133 or
another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as
Ab14. This embodiment described further contemplates additions, deletions, and variants of
SEQ ID NO: 131 and/or SEQ ID NO: 133 in said Fab while retaining binding specificity for
NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab14. In another embodiment described, anti-NGF antibodies such as Ab14 or
Fab fragments thereof for treatment or prevention of pain and pain associated conditions may be
produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal,
insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid
Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab15
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is
Ab15 or fragments thereof, for example as set forth below, or comprises another antibody or
antibody fragment that binds to the same or overlapping epitope as Ab15, in a therapeutically
effective amount which inhibits the association of NGF with TrkA without appreciably inhibiting
the association of NGF with p75. In one embodiment, described are chimeric antibodies for
treatment or prevention of pain and pain associated conditions having binding specificity to NGF
and possessing a variable light chain sequence comprising the sequence set forth below:
AAVLTQTPSPVSAAVGDTVTIKCQSSQSVYKNNYLSWYQQKPGQPPKLLIYDASNLPSG
VPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGDYDDDTDNGFGGGTEVVVKR (SEQ
ID NO: 141).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a light chain
sequence comprising the sequence set forth below:
AAVLTQTPSPVSAAVGDTVTIKCQSSQSVYKNNYLSWYQQKPGQPPKLLIYDASNLPSG
VPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGDYDDDTDNGFGGGTEVVVKRTVAAP
SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 142).
Further described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a variable heavy
chain sequence comprising the sequence set forth below:
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMIWVRQAPGKGLEYIGIIWSGGTYYATW
AKGRFTISKTSTTVDLQITSPTTEDAATYFCAAGGGSIYDVWGPGTLVTVSS (SEQ ID
NO: 143).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMIWVRQAPGKGLEYIGIIWSGGTYYATW
AKGRFTISKTSTTVDLQITSPTTEDAATYFCAAGGGSIYDVWGPGTLVTVSSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 144).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 145;
SEQ ID NO: 146; and SEQ ID NO: 147 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
141 or the light chain sequence of SEQ ID NO: 142, and/or one or more of the polypeptide
sequences of SEQ ID NO: 148; SEQ ID NO: 149; and SEQ ID NO: 150 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 143 or the heavy chain sequence of SEQ ID NO: 144, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 141 or SEQ ID NO: 142. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 143 or SEQ ID
NO: 144.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 145; SEQ
ID NO: 146; and SEQ ID NO: 147 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
141 or the light chain sequence of SEQ ID NO: 142.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 148; SEQ
ID NO: 149; and SEQ ID NO: 150 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO:
143 or the heavy chain sequence of SEQ ID NO: 144.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, one, two, three or more, including all of the following antibody
fragments: the variable light chain region of SEQ ID NO: 141; the variable heavy chain region of
SEQ ID NO: 143; the complementarity-determining regions (SEQ ID NO: 145; SEQ ID NO:
146; and SEQ ID NO: 147) of the variable light chain region of SEQ ID NO: 141; and the
complementarity-determining regions (SEQ ID NO: 148; SEQ ID NO: 149; and SEQ ID NO:
150) of the variable heavy chain region of SEQ ID NO: 143.
In a particularly preferred embodiment described is, the chimeric anti-NGF
antibody for treatment or prevention of pain and pain associated conditions is Ab15, comprising,
or alternatively consisting of, SEQ ID NO: 142 and SEQ ID NO: 144, and having at least one of
the biological activities set forth herein.
In a further particularly preferred embodiment described, antibody fragments for
treatment or prevention of pain and pain associated conditions comprise, or alternatively consist
of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect
to antibody Ab15, the Fab fragment includes the variable light chain sequence of SEQ ID NO:
141 and the variable heavy chain sequence of SEQ ID NO: 143 or comprises another Fab that
binds to the same or overlapping epitope as Ab15,. This embodiment described further
contemplates additions, deletions, and variants of SEQ ID NO: 141 and/or SEQ ID NO: 143 in
said Fab while retaining binding specificity for NGF.
In one embodiment described herein (infra), Fab fragments for treatment or
prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g.,
papain) of Ab15. In another embodiment described, anti-NGF antibodies for treatment or
prevention of pain and pain associated conditions such as Ab15 or Fab fragments thereof may be
produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal,
insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid
Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab16
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is
Ab16 or fragments thereof, for example as set forth below, or comprises another antibody or
antibody fragment that binds to the same or overlapping epitope as Ab16, in a therapeutically
effective amount which inhibits the association of NGF with TrkA without appreciably inhibiting
the association of NGF with p75. In one embodiment, described are chimeric or humanized
antibodies for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF and possessing a variable light chain sequence comprising the sequence set
forth below:
ALVMTQTPSSTSEPVGGTVTINCQASQNIGNDLSWYQQKPGQPPELLIYSTSKLATGVPK
RFSGSRSGTQFTLTISDLECDDAATYYCLGVYSYISDDGNAFGGGTEVVVKR (SEQ ID
NO: 151).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
ALVMTQTPSSTSEPVGGTVTINCQASQNIGNDLSWYQQKPGQPPELLIYSTSKLATGVPK
RFSGSRSGTQFTLTISDLECDDAATYYCLGVYSYISDDGNAFGGGTEVVVKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 152).
Further described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
QSVEEFGGRLVTPGTPLTLTCTVSGFSLNNYAMTWVRQAPGKGLEWIGIIGSIGTTYYAS
WAKGRFFISKTSTTVDLKIISPTTEDTATYFCARDAGVTVDGYGYYFNIWGPGTLVTVSS
(SEQ ID NO: 153).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
QSVEEFGGRLVTPGTPLTLTCTVSGFSLNNYAMTWVRQAPGKGLEWIGIIGSIGTTYYAS
WAKGRFFISKTSTTVDLKIISPTTEDTATYFCARDAGVTVDGYGYYFNIWGPGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 154).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 155;
SEQ ID NO: 156; and SEQ ID NO: 157 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
151 or the light chain sequence of SEQ ID NO: 152, and/or one or more of the polypeptide
sequences of SEQ ID NO: 158; SEQ ID NO: 159; and SEQ ID NO: 160 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 153 or the heavy chain sequence of SEQ ID NO: 154, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 151 or SEQ ID NO: 152. In another embodiment described, antibody fragments for
treatment or prevention of pain and pain associated conditions comprise, or alternatively consist
of, the polypeptide sequence of SEQ ID NO: 153 or SEQ ID NO: 154.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 155; SEQ
ID NO: 156; and SEQ ID NO: 157 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
151 or the light chain sequence of SEQ ID NO: 152.
In a further embodiment described, fragments of the antibody having binding
specificity to NGF for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 158; SEQ
ID NO: 159; and SEQ ID NO: 160 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO:
153 or the heavy chain sequence of SEQ ID NO: 154.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 151; the variable heavy chain
region of SEQ ID NO: 153; the complementarity-determining regions (SEQ ID NO: 155; SEQ
ID NO: 156; and SEQ ID NO: 157) of the variable light chain region of SEQ ID NO: 151; and
the complementarity-determining regions (SEQ ID NO: 158; SEQ ID NO: 159; and SEQ ID NO:
160) of the variable heavy chain region of SEQ ID NO: 153.
In a particularly preferred embodiment described is, the chimeric or humanized
anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab16,
comprising, or alternatively consisting of, SEQ ID NO: 152 and SEQ ID NO: 154, and having at
least one of the biological activities set forth herein.
In a further particularly preferred embodiment described, antibody fragments for
treatment or prevention of pain and pain associated conditions comprise, or alternatively consist
of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect
to antibody Ab16, the Fab fragment includes the variable light chain sequence of SEQ ID NO:
151 and the variable heavy chain sequence of SEQ ID NO: 153 or comprises another Fab or
another bivalent or monovalent antibody fragment that binds to the same or overlapping epitope
as Ab16. This embodiment described further contemplates additions, deletions, and variants of
SEQ ID NO: 151 and/or SEQ ID NO: 153 in said Fab while retaining binding specificity for
NGF.
In one embodiment described herein (infra), Fab fragments for treatment or
prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g.,
papain) of Ab16. In another embodiment described, anti-NGF antibodies for treatment or
prevention of pain and pain associated conditions such as Ab16 or Fab fragments thereof may be
produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal,
insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid
Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab17
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is
Ab17 or fragments thereof, for example as set forth below, or comprises another antibody or
antibody fragment that binds to the same or overlapping epitope as Ab17, in a therapeutically
effective amount which inhibits the association of NGF with TrkA and the association of NGF
with p75. In one embodiment, described are chimeric antibodies for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF and possessing a variable
light chain sequence comprising the sequence set forth below:
AIEMTQTPFSVSAAVGGTVTIKCQASQTISNYLAWYQQKPGQPPKLLIYGASNLESGVPS
RFKGSGSGTQFTLTISDLECDDAATYYCQQGYTISNVDNNVFGGGTEVVVKR (SEQ ID
NO: 161).
Also described are includes chimeric antibodies for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
AIEMTQTPFSVSAAVGGTVTIKCQASQTISNYLAWYQQKPGQPPKLLIYGASNLESGVPS
RFKGSGSGTQFTLTISDLECDDAATYYCQQGYTISNVDNNVFGGGTEVVVKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 162).
Further described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a variable heavy
chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGGSLTLTCAASGFSLTGYNLVWVRQAPGKGLEWIGFISYGDTTYYA
SWAKGRFTISKTSTTVTLTITDLQPSDTGTYFCARETANTYDYGIWGPGTLVTVSS (SEQ
ID NO: 163).
Also described are includes chimeric antibodies for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGGSLTLTCAASGFSLTGYNLVWVRQAPGKGLEWIGFISYGDTTYYA
SWAKGRFTISKTSTTVTLTITDLQPSDTGTYFCARETANTYDYGIWGPGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 164).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 165;
SEQ ID NO: 166; and SEQ ID NO: 167 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
161 or the light chain sequence of SEQ ID NO: 162, and/or one or more of the polypeptide
sequences of SEQ ID NO: 168; SEQ ID NO: 169; and SEQ ID NO: 170 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 163 or the heavy chain sequence of SEQ ID NO: 164, or
combinations of these polypeptide sequences. In another optional embodiment described, the
antibodies or fragments thereof for treatment or prevention of pain and pain associated
conditions comprise, or alternatively consist of, combinations of one or more of the CDRs, the
variable heavy and variable light chain sequences, and the heavy and light chain sequences set
forth above, including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 161 or SEQ ID NO: 162. In another optional embodiment described, antibody
fragments comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 163 or
SEQ ID NO: 164.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
165; SEQ ID NO: 166; and SEQ ID NO: 167 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of
SEQ ID NO: 161 or the light chain sequence of SEQ ID NO: 162.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
168; SEQ ID NO: 169; and SEQ ID NO: 170 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 163 or the heavy chain sequence of SEQ ID NO: 164.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF comprise, or alternatively
consist of, one, two, three or more, including all of the following antibody fragments: the
variable light chain region of SEQ ID NO: 161; the variable heavy chain region of SEQ ID NO:
163; the complementarity-determining regions (SEQ ID NO: 165; SEQ ID NO: 166; and SEQ ID
NO: 167) of the variable light chain region of SEQ ID NO: 161; and the complementarity-
determining regions (SEQ ID NO: 168; SEQ ID NO: 169; and SEQ ID NO: 170) of the variable
heavy chain region of SEQ ID NO: 163.
In a particularly preferred optional embodiment described is, the chimeric anti-
NGF antibody for treatment or prevention of pain and pain associated conditions is Ab17,
comprising, or alternatively consisting of, SEQ ID NO: 162 and SEQ ID NO: 164, and having at
least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab17, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 161 and the variable heavy chain sequence of SEQ ID NO: 163 or
comprises another Fab or monovalent or bivalent antibody fragment that binds to the same or
overlapping epitope as Ab15,. This embodiment described further contemplates additions,
deletions, and variants of SEQ ID NO: 161 and/or SEQ ID NO: 163 in said Fab while retaining
binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab17. In another embodiment described, anti-NGF antibodies such as Ab17 or
Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab18
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is
Ab18 or fragments thereof, for example as set forth below, or comprises another antibody or
antibody fragment that binds to the same or overlapping epitope as Ab18, in a therapeutically
effective amount which inhibits the association of NGF with TrkA and the association of NGF
with p75. In one embodiment, described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable light chain sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQASQTISNYLAWYQQKPGKAPKLLIYGASNLESGVPS
RFSGSGSGTEFTLTISSLQPDDFATYYCQQGYTISNVDNNVFGGGTKVEIKR (SEQ ID
NO: 171).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQASQTISNYLAWYQQKPGKAPKLLIYGASNLESGVPS
RFSGSGSGTEFTLTISSLQPDDFATYYCQQGYTISNVDNNVFGGGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 172).
Further described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSGYNLVWVRQAPGKGLEWVGFISYGDTTY
YASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETANTYDYGIWGQGTLVTV
SS (SEQ ID NO: 173).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSGYNLVWVRQAPGKGLEWVGFISYGDTTY
YASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETANTYDYGIWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 174).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 175;
SEQ ID NO: 176; and SEQ ID NO: 177 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
171 or the light chain sequence of SEQ ID NO: 172, and/or one or more of the polypeptide
sequences of SEQ ID NO: 178; SEQ ID NO: 179; and SEQ ID NO: 180 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 173 or the heavy chain sequence of SEQ ID NO: 174, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
Also described are fragments of the antibody having binding specificity to NGF
for treatment or prevention of pain and pain associated conditions. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 171 or SEQ ID NO: 172. In another embodiment described, antibody fragments
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 173 or SEQ ID
NO: 174.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
175; SEQ ID NO: 176; and SEQ ID NO: 177 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of
SEQ ID NO: 171 or the light chain sequence of SEQ ID NO: 172.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
178; SEQ ID NO: 179; and SEQ ID NO: 180 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 173 or the heavy chain sequence of SEQ ID NO: 174.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, one, two, three or more, including all of the following antibody
fragments: the variable light chain region of SEQ ID NO: 171; the variable heavy chain region of
SEQ ID NO: 173; the complementarity-determining regions (SEQ ID NO: 175; SEQ ID NO:
176; and SEQ ID NO: 177) of the variable light chain region of SEQ ID NO: 171; and the
complementarity-determining regions (SEQ ID NO: 178; SEQ ID NO: 179; and SEQ ID NO:
180) of the variable heavy chain region of SEQ ID NO: 173.
In a particularly preferred optional embodiment described is, the chimeric or
humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions
is Ab18, comprising, or alternatively consisting of, SEQ ID NO: 172 and SEQ ID NO: 174, and
having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having
binding specificity for NGF. With respect to antibody Ab18, the Fab fragment includes the
variable light chain sequence of SEQ ID NO: 171 and the variable heavy chain sequence of SEQ
ID NO: 173 or comprise another Fab or antibody fragment that binds to the same or overlapping
epitope as Ab18. This embodiment described further contemplates additions, deletions, and
variants of SEQ ID NO: 171 and/or SEQ ID NO: 173 in said Fab while retaining binding
specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab18. In another embodiment described, anti-NGF antibodies such as Ab18 or
Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab19
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is
Ab19 or fragments thereof, for example as set forth below, or comprises another antibody or
antibody fragment that binds to the same or overlapping epitope as Ab19, in a therapeutically
effective amount which inhibits the association of NGF with TrkA and the association of NGF
with p75. In one embodiment, described are chimeric antibodies for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF and possessing a variable
light chain sequence comprising the sequence set forth below:
AAVLTQTPSPVSAAVGGTVSISCQSSQNVYKNNYLSWYQQKPGQPPKLLIYKASTLASG
VPSRFKGSGSGTDFTLTISDVQCDAAATYYCAGGYSSSSDNAFGGGTEVVVKR (SEQ ID
NO: 181).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a light chain
sequence comprising the sequence set forth below:
AAVLTQTPSPVSAAVGGTVSISCQSSQNVYKNNYLSWYQQKPGQPPKLLIYKASTLASG
VPSRFKGSGSGTDFTLTISDVQCDAAATYYCAGGYSSSSDNAFGGGTEVVVKRTVAAPS
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 182).
Further described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a variable heavy
chain sequence comprising the sequence set forth below:
QSVEASGGRLVMPGGSLTLTCTASGFSLSTYWMSWVRQAPGKGLEWIGDIYFSNEETN
YATWAKGRFTISKTSTTVDLNVISPTTEDTATYFCARGSPDVEIAIDMWGQGTLVTVSS
(SEQ ID NO: 183).
Also described are chimeric antibodies for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
QSVEASGGRLVMPGGSLTLTCTASGFSLSTYWMSWVRQAPGKGLEWIGDIYFSNEETN
YATWAKGRFTISKTSTTVDLNVISPTTEDTATYFCARGSPDVEIAIDMWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 184).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 185;
SEQ ID NO: 186; and SEQ ID NO: 187 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
181 or the light chain sequence of SEQ ID NO: 182, and/or one or more of the polypeptide
sequences of SEQ ID NO: 188; SEQ ID NO: 189; and SEQ ID NO: 190 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 183 or the heavy chain sequence of SEQ ID NO: 184, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
Also described fragments of the antibody having binding specificity to NGF for
treatment or prevention of pain and pain associated conditions. In one embodiment described,
antibody fragments comprise, or alternatively consist of, the polypeptide sequence of SEQ ID
NO: 181 or SEQ ID NO: 182. In another embodiment described, antibody fragments comprise,
or alternatively consist of, the polypeptide sequence of SEQ ID NO: 183 or SEQ ID NO: 184.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
185; SEQ ID NO: 186; and SEQ ID NO: 187 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of
SEQ ID NO: 181 or the light chain sequence of SEQ ID NO: 182.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
188; SEQ ID NO: 189; and SEQ ID NO: 190 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 183 or the heavy chain sequence of SEQ ID NO: 184.
Also described are antibody fragments which include one or more of the antibody
fragments described herein for treatment or prevention of pain and pain associated conditions. In
one optional embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 181; the variable heavy chain
region of SEQ ID NO: 183; the complementarity-determining regions (SEQ ID NO: 185; SEQ
ID NO: 186; and SEQ ID NO: 187) of the variable light chain region of SEQ ID NO: 181; and
the complementarity-determining regions (SEQ ID NO: 188; SEQ ID NO: 189; and SEQ ID NO:
190) of the variable heavy chain region of SEQ ID NO: 183.
In a particularly preferred optional embodiment described, the chimeric anti-NGF
antibody for treatment or prevention of pain and pain associated conditions is Ab19, comprising,
or alternatively consisting of, SEQ ID NO: 182 and SEQ ID NO: 184, and having at least one of
the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab19, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 181 and the variable heavy chain sequence of SEQ ID NO: 183 or
comprises another Fab or antibody fragment that binds to the same or overlapping epitope as
Ab19. This optional embodiment described further contemplates additions, deletions, and
variants of SEQ ID NO: 181 and/or SEQ ID NO: 183 in said Fab while retaining binding
specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab19. In another embodiment described, anti-NGF antibodies such as Ab19 or
Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab20
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is
Ab20 or fragments thereof, for example as set forth below, or comprises another antibody or
antibody fragment that binds to the same or overlapping epitope as Ab20, in a therapeutically
effective amount which inhibits the association of NGF with TrkA and the association of NGF
with p75. In one embodiment, described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable light chain sequence comprising the sequence set forth below:
DIQMTQSPSSLSASVGDRVTITCQSSQNVYKNNYLSWYQQKPGKVPKLLIYKASTLASG
VPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYTSSSDNAFGGGTKVEIKR (SEQ ID
NO: 191).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
DIQMTQSPSSLSASVGDRVTITCQSSQNVYKNNYLSWYQQKPGKVPKLLIYKASTLASG
VPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYTSSSDNAFGGGTKVEIKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 192).
Further described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSTYWMSWVRQAPGKGLEWVGDIYFSNEET
NYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSPDVEIAIDMWGQGTLV
TVSS (SEQ ID NO: 193).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSTYWMSWVRQAPGKGLEWVGDIYFSNEET
NYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSPDVEIAIDMWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 194).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 195;
SEQ ID NO: 196; and SEQ ID NO: 197 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO:
191 or the light chain sequence of SEQ ID NO: 192, and/or one or more of the polypeptide
sequences of SEQ ID NO: 198; SEQ ID NO: 199; and SEQ ID NO: 200 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 193 or the heavy chain sequence of SEQ ID NO: 194, or
combinations of these polypeptide sequences. In another embodiment described, the antibodies
or fragments thereof for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
Also described are fragments of the antibody having binding specificity to NGF.
In one embodiment described, antibody fragments for treatment or prevention of pain and pain
associated conditions comprise, or alternatively consist of, the polypeptide sequence of SEQ ID
NO: 191 or SEQ ID NO: 192. In another embodiment described, antibody fragments comprise,
or alternatively consist of, the polypeptide sequence of SEQ ID NO: 193 or SEQ ID NO: 194.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
195; SEQ ID NO: 196; and SEQ ID NO: 197 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of
SEQ ID NO: 191 or the light chain sequence of SEQ ID NO: 192.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide
sequences of SEQ ID NO: 198; SEQ ID NO: 199; and SEQ ID NO: 200 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 193 or the heavy chain sequence of SEQ ID NO: 194.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 191; the variable heavy chain
region of SEQ ID NO: 193; the complementarity-determining regions (SEQ ID NO: 195; SEQ
ID NO: 196; and SEQ ID NO: 197) of the variable light chain region of SEQ ID NO: 191; and
the complementarity-determining regions (SEQ ID NO: 198; SEQ ID NO: 199; and SEQ ID NO:
200) of the variable heavy chain region of SEQ ID NO: 193.
In a particularly preferred optional embodiment described, the chimeric or
humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions
is Ab20, comprising, or alternatively consisting of, SEQ ID NO: 192 and SEQ ID NO: 194, and
having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab20, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 191 and the variable heavy chain sequence of SEQ ID NO: 193. This
embodiment further contemplates additions, deletions, and variants of SEQ ID NO: 191 and/or
SEQ ID NO: 193 in said Fab while retaining binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab20. In another embodiment described, anti-NGF antibodies for treatment or
prevention of pain and pain associated conditions such as Ab20 or Fab fragments thereof may be
produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal,
insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid
Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody Ab21
Described are methods of treating pain and the specific pain associated disorders
alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the
antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is
Ab21 or fragments thereof, or another antibody or antibody fragment that binds to the same or
overlapping epitope as Ab5, for example as set forth below, in a therapeutically effective amount
which inhibits the association of NGF with TrkA and the association of NGF with p75. In one
embodiment, described are chimeric or humanized antibodies having binding specificity to NGF
and possessing a variable light chain sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS
RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKR (SEQ ID NO:
51).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a light
chain sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS
RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 401).
Further described are chimeric or humanized antibodies for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAVGWVRQAPGKGLEWVGIIGRNGNT
WYASSARGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYGRSVAYYVFNIWGPGT
LVTVSS (SEQ ID NO: 53).
Also described are chimeric or humanized antibodies for treatment or prevention
of pain and pain associated conditions having binding specificity to NGF and possessing a heavy
chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAVGWVRQAPGKGLEWVGIIGRNGNT
WYASSARGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYGRSVAYYVFNIWGPGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 402).
Further described are antibodies for treatment or prevention of pain and pain
associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 55;
SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51
or the light chain sequence of SEQ ID NO: 401, and/or one or more of the polypeptide sequences
of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 402, or
combinations of these polypeptide sequences. In another optional embodiment described, the
antibodies or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light
chain sequences set forth above, including all of them.
Also described are fragments of the antibody for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF. In one embodiment
described, antibody fragments for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 51 or SEQ ID
NO: 401. In another embodiment described, antibody fragments for treatment or prevention of
pain and pain associated conditions comprise, or alternatively consist of, the polypeptide
sequence of SEQ ID NO: 53 or SEQ ID NO: 402.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51
or the light chain sequence of SEQ ID NO: 401.
In a further optional embodiment described, fragments of the antibody having
binding specificity to NGF for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO:
58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO:
53 or the heavy chain sequence of SEQ ID NO: 402.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 51; the variable heavy chain
region of SEQ ID NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID
NO: 56; and SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51; and the
complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60)
of the variable heavy chain region of SEQ ID NO: 53.
In a particularly preferred optional embodiment described, the chimeric or
humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions
is Ab21, comprising, or alternatively consisting of, SEQ ID NO: 401 and SEQ ID NO: 402, and
having at least one of the biological activities set forth herein.
In a further particularly preferred optional embodiment described, antibody
fragments for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for
NGF. With respect to antibody Ab21, the Fab fragment includes the variable light chain
sequence of SEQ ID NO: 51 and the variable heavy chain sequence of SEQ ID NO: 53 or
another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as
Ab5,. This embodiment described further contemplates additions, deletions, and variants of SEQ
ID NO: 51 and/or SEQ ID NO: 53 in said Fab while retaining binding specificity for NGF.
In one optional embodiment described herein (infra), Fab fragments for treatment
or prevention of pain and pain associated conditions may be produced by enzymatic digestion
(e.g., papain) of Ab21. In another embodiment described, anti-NGF antibodies for treatment or
prevention of pain and pain associated conditions such as Ab21 or Fab fragments thereof may be
produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal,
insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid
Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia
pastoris.
Antibody fragment Fab1
Described are methods of treating pain using antibody fragment Fab1 or
fragments thereof, for example as set forth below, in a therapeutically effective amount which
inhibits the association of NGF with TrkA and the association of NGF with p75. In one
embodiment, the invention optionally includes Fab antibody fragments for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF and
possessing a light chain sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS
RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 405).
Further described are Fab antibody fragments for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAVGWVRQAPGKGLEWVGIIGRNGNT
WYASSARGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYGRSVAYYVFNIWGPGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTH (SEQ ID
NO: 406).
Further described are antibody fragments for treatment or prevention of pain and
pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO:
55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51
or the light chain sequence of SEQ ID NO: 405, and/or one or more of the polypeptide sequences
of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 406, or
combinations of these polypeptide sequences. In another optional embodiment described,
antibody fragments for treatment or prevention of pain and pain associated conditions comprise,
or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
Also described are fragments of the antibody for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF. In one embodiment
described, antibody fragments comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 51 or SEQ ID NO: 405. In another embodiment described, antibody fragments of
the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 53
or SEQ ID NO: 406.
In a further optional embodiment described, antibody fragments for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF comprise, or
alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID
NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the
light chain sequence of SEQ ID NO: 405.
In a further optional embodiment described, antibody fragments for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF comprise, or
alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID
NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the
heavy chain sequence of SEQ ID NO: 406.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF comprise, or alternatively consist of, one, two, three or more, including all of the following
antibody fragments: the variable light chain region of SEQ ID NO: 51; the variable heavy chain
region of SEQ ID NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID
NO: 56; and SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51; and the
complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60)
of the variable heavy chain region of SEQ ID NO: 53.
In a particularly preferred optional embodiment described, the anti-NGF antibody
fragment for treatment or prevention of pain and pain associated conditions is Fab1, comprising
SEQ ID NO: 405 and SEQ ID NO: 406, or another Fab or antibody fragment that binds to the
same or overlapping epitope as Fab1, and having at least one of the biological activities set forth
herein. In one embodiment described, antibody fragment Fab1 may be produced by enzymatic
digestion (e.g., papain) of Ab21.
Antibody fragment Fab2
Described are methods of treating pain using antibody fragment Fab2 or
fragments thereof, for example as set forth below, in a therapeutically effective amount which
inhibits the association of NGF with TrkA and the association of NGF with p75. In one
embodiment, the invention includes Fab antibody fragments for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF and possessing a light chain
sequence comprising the sequence set forth below:
DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS
RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 407).
Further described are Fab antibody fragments for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF and possessing a heavy chain
sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAVGWVRQAPGKGLEWVGIIGRNGNT
WYASSARGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYGRSVAYYVFNIWGPGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTH (SEQ ID
NO: 408).
Further described are antibody fragments for treatment or prevention of pain and
pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO:
55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51
or the light chain sequence of SEQ ID NO: 407, and/or one or more of the polypeptide sequences
of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 408, or
combinations of these polypeptide sequences. In another embodiment described are, antibody
fragments comprise, or alternatively consist of, combinations of one or more of the CDRs, the
variable heavy and variable light chain sequences, and the heavy and light chain sequences set
forth above, including all of them.
Also described are fragments of the antibody for treatment or prevention of pain
and pain associated conditions having binding specificity to NGF. In one embodiment
described, antibody fragments for treatment or prevention of pain and pain associated conditions
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 51 or SEQ ID
NO: 407. In another embodiment described, antibody fragments for treatment or prevention of
pain and pain associated conditions comprise, or alternatively consist of, the polypeptide
sequence of SEQ ID NO: 53 or SEQ ID NO: 408.
In a further optional embodiment described, antibody fragments for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF comprise, or
alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID
NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the
light chain sequence of SEQ ID NO: 407.
In a further optional embodiment described, antibody fragments for treatment or
prevention of pain and pain associated conditions having binding specificity to NGF comprise, or
alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID
NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the
heavy chain sequence of SEQ ID NO: 408.
Also described are antibody fragments for treatment or prevention of pain and
pain associated conditions which include one or more of the antibody fragments described
herein. In one embodiment described, fragments of the antibodies having binding specificity to
NGF for treatment or prevention of pain and pain associated conditions comprise, or
alternatively consist of, one, two, three or more, including all of the following antibody
fragments: the variable light chain region of SEQ ID NO: 51; the variable heavy chain region of
SEQ ID NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID NO: 56;
and SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51; and the
complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60)
of the variable heavy chain region of SEQ ID NO: 53.
In a particularly preferred optional embodiment described, the anti-NGF antibody
fragment for treatment or prevention of pain and pain associated conditions is Fab2, comprising
SEQ ID NO: 407 and SEQ ID NO: 408, or another Fab or antibody fragment that binds to the
same or overlapping epitope as Fab2, and having at least one of the biological activities set forth
herein.
In another optional embodiment described herein (infra), Fab fragments for
treatment or prevention of pain and pain associated conditions may be produced via expression
in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast
strains. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment of the invention, antibody fragment Fab2 may be produced by expression in Pichia
pastoris using protocols set forth herein in the examples.
In another embodiment, antibody fragments may be present in one or more of the
following non-limiting forms: Fab, Fab', F(ab') , Fv and single chain Fv antibody forms. In a
preferred embodiment, the anti-NGF antibodies described herein further comprises the kappa
constant light chain sequence comprising the sequence set forth below:
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ
ID NO: 412).
In another preferred optional embodiment, the anti-NGF antibodies described
herein for treatment or prevention of pain and pain associated conditions further comprises the
gamma-1 constant heavy chain polypeptide sequence comprising the sequence set forth below:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 413).
In another optional embodiment, described is an isolated anti-NGF antibody for
treatment or prevention of pain and pain associated conditions comprising a V polypeptide
sequence selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133,
143, 153, 163, 173, 183, 193, or 402, or a variant thereof; and further comprising a V
polypeptide sequence selected from: SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111,
121, 131, 141, 151, 161, 171, 181, 191, or 401, or a variant thereof, wherein one or more of the
framework residues (FR residues) in said V or V polypeptide has been substituted with
another amino acid residue resulting in an anti-NGF antibody that specifically binds NGF.
Described are humanized and chimeric forms of these antibodies for treatment or prevention of
pain and pain associated conditions. The chimeric antibodies may include an Fc derived from
IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14,
IgG15, IgG16, IgG17, IgG18 or IgG19 constant regions.
In one embodiment described, the antibodies or V or V polypeptides originate
or are selected from one or more rabbit B cell populations prior to initiation of the humanization
process referenced herein.
In another embodiment described, the anti-NGF antibodies and fragments thereof
for treatment or prevention of pain and pain associated conditions do not have binding specificity
for p75 or TrkA. In a further embodiment described, there is contemplated methods for treating
pain comprising using the anti-NGF antibodies and fragments thereof to inhibit the association of
NGF with p75 and/or TrkA. In another embodiment described, there is contemplated methods
for treating pain comprising using anti-NGF antibodies and fragments thereof to inhibit the
association of NGF with TrkA and/or multimers thereof and/or antagonizes the biological effects
thereof. In another embodiment described, there is contemplated methods for treating pain
comprising using anti-NGF antibodies and fragments thereof to inhibit the association of NGF
with p75 and/or multimers thereof and the association of NGF with TrkA and/or multimers
thereof, and antagonizes the biological effects of p75 and TrkA.
As stated supra, antibodies and fragments thereof may be modified post-
translationally to add effector moieties such as chemical linkers, detectable moieties such as for
example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials,
and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin,
biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
Regarding detectable moieties, further exemplary enzymes include, but are not
limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase
and luciferase. Further exemplary fluorescent materials include, but are not limited to,
rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine,
phycoerythrin and dansyl chloride. Further exemplary chemiluminescent moieties include, but
are not limited to, luminol. Further exemplary bioluminescent materials include, but are not
limited to, luciferin and aequorin. Further exemplary radioactive materials include, but are not
125 14 35 3
limited to, Iodine 125 ( I), Carbon 14 ( C), Sulfur 35 ( S), Tritium ( H) and Phosphorus 32
( P).
Regarding functional moieties, exemplary cytotoxic agents include, but are not
limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-
fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil,
melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea,
cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C,
cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines
include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin,
carminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics include
dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin
(AMC); and antimytotic agents such as the vinca alkaloids, vincristine and vinblastine. Other
cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine,
cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin,
dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids,
mytotane (O,P'-(DDD)), interferons, and mixtures of these cytotoxic agents.
Further cytotoxic agents include, but are not limited to, chemotherapeutic agents
such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-
fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin. Toxic
enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin may be
conjugated to the humanized or chimeric antibodies, or binding fragments thereof, to generate
cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980);
Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci.
USA 77:5419 (1980)).
Other cytotoxic agents include cytotoxic ribonucleases as described by
Goldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention also relate to
radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled
to the antibody, or binding fragments thereof, with or without the use of a complex-forming
32 47
agent. Such radionuclides include beta-emitters such asPhosphorus-32 ( P), Scandium-47 ( Sc),
67 67 88 90 125
Copper-67 ( Cu), Gallium-67 ( Ga), Yttrium-88 ( Y), Yttrium-90 ( Y), Iodine-125 ( I),
131 153 177 186
Iodine-131 ( I), Samarium-153 ( Sm), Lutetium-177 ( Lu), Rhenium-186 ( Re) or
188 211 212
Rhenium-188 ( Re), and alpha-emitters such as Astatine-211 ( At), Lead-212 ( Pb),
212 213 225
Bismuth-212 ( Bi) or -213 ( Bi) or Actinium-225 ( Ac).
Further exemplary radioactive materials include, but are not limited to, Iodine 125
125 14 35 3 32
( I), Carbon 14 ( C), Sulfur 35 ( S), Tritium ( H) and Phosphorus 32 ( P).
Regarding functional moieties, exemplary cytotoxic agents include, but are not
limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-
fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil,
melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea,
cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C,
cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines
include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin,
carminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics include
dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin
(AMC); and antimytotic agents such as the vinca alkaloids, vincristine and vinblastine. Other
cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine,
cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin,
dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids,
mytotane (O,P'-(DDD)), interferons, and mixtures of these cytotoxic agents.
Further cytotoxic agents include, but are not limited to, chemotherapeutic agents
such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-
fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin. Toxic
enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin may be
conjugated to the humanized or chimeric antibodies, or binding fragments thereof, to generate
cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980);
Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci.
USA 77:5419 (1980)).
Other cytotoxic agents include cytotoxic ribonucleases as described by
Goldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention also relate to
radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled
to the antibody, or binding fragments thereof, with or without the use of a complex-forming
32 47
agent. Such radionuclides include beta-emitters such asPhosphorus-32 ( P), Scandium-47 ( Sc),
67 67 88 90 125
Copper-67 ( Cu), Gallium-67 ( Ga), Yttrium-88 ( Y), Yttrium-90 ( Y), Iodine-125 ( I),
131 153 177 186
Iodine-131 ( I), Samarium-153 ( Sm), Lutetium-177 ( Lu), Rhenium-186 ( Re) or
188 211 212
Rhenium-188 ( Re), and alpha-emitters such as Astatine-211 ( At), Lead-212 ( Pb),
212 213 225
Bismuth-212 ( Bi) or -213 ( Bi) or Actinium-225 ( Ac). .
Methods are known in the art for conjugating an antibody or binding fragment
thereof to a detectable moiety and the like, such as for example those methods described by
Hunter et al, Nature 144:945 (1962); David et al, Biochemistry 13:1014 (1974); Pain et al, J.
Immunol. Meth. 40:219 (1981); and Nygren, J., Histochem. and Cytochem. 30:407 (1982).
Embodiments described herein further include variants and equivalents that are
substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs, camelbodies,
nanobodies, IgNAR, polypeptides, variable regions and CDRs set forth herein. These may
contain, e.g., conservative substitution mutations, (i.e., the substitution of one or more amino
acids by similar amino acids). For example, conservative substitution refers to the substitution of
an amino acid with another within the same general class, e.g., one acidic amino acid with
another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral
amino acid by another neutral amino acid. What is intended by a conservative amino acid
substitution is well known in the art.
In another embodiment, described are polypeptide sequences having at least 90%
or greater sequence homology to any one or more of the polypeptide sequences of antibody
fragments, variable regions and CDRs set forth herein. More preferably, described are
polypeptide sequences having at least 95% or greater sequence homology, even more preferably
at least 98% or greater sequence homology, and still more preferably at least 99% or greater
sequence homology to any one or more of the polypeptide sequences of antibody fragments,
variable regions and CDRs set forth herein. Methods for determining homology between nucleic
acid and amino acid sequences are well known to those of ordinary skill in the art.
In another embodiment, described are the above-recited polypeptide homologs of
the antibody fragments, variable regions and CDRs set forth herein further having anti-NGF
activity. Non-limiting examples of anti-NGF activity are set forth herein.
In another embodiment, described is the generation and use of anti-idiotypic
antibodies that bind any of the foregoing sequences. In an exemplary embodiment, such an anti-
idiotypic antibody could be administered to a subject who has received an anti-NGF antibody to
modulate, reduce, or neutralize, the effect of the anti-NGF antibody. Such anti-idiotypic
antibodies could also be useful for treatment of an autoimmune disease characterized by the
presence of anti-NGF antibodies. A further exemplary use of such anti-idiotypic antibodies is
for detection of the anti-NGF antibodies of the present invention, for example to monitor the
levels of the anti-NGF antibodies present in a subject’s blood or other bodily fluids.
Also described are anti-NGF antibodies comprising any of the polypeptide or
polynucleotide sequences described herein substituted for any of the other polynucleotide
sequences described herein. For example, without limitation thereto, described are antibodies
comprising the combination of any of the variable light chain and variable heavy chain
sequences described herein, and further described are antibodies resulting from substitution of
any of the CDR sequences described herein for any of the other CDR sequences described
herein.
Polynucleotides Encoding Anti-NGF Antibody Polypeptides
Antibody Ab1
Further described is the use of polynucleotides set forth below to produce
antibody Ab1 polypeptides for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF, which inhibit the association of NGF with TrkA and the
association of NGF with p75, in methods of treating pain in an individual comprising
administering to said individual antibody Ab1 polypeptides. In one embodiment described,
polynucleotides described comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 1:
GCCCTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGA
GGCACAGTCACCATCAATTGCCAGGCCAGTCAGAACATTTACAGCAATTTAGCCTGG
TATCAACAGAGACCAGGGCAGCGTCCCAAGCTCCTGATCTATGGTGCATCCAATCTG
GATGCTGGGGTCCCATCGCGGTTCAGAGGCAGTGGATCTGGGACAGAGTACACTCT
CACCATCAGCGACCTGGAGTGTGACGATGTTGGCACTTACTACTGTCAAAGTGCTTT
TGATAGTGATAGTACTGAAAATACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC
GT (SEQ ID NO: 201).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 2:
GCCCTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGA
GGCACAGTCACCATCAATTGCCAGGCCAGTCAGAACATTTACAGCAATTTAGCCTGG
TATCAACAGAGACCAGGGCAGCGTCCCAAGCTCCTGATCTATGGTGCATCCAATCTG
GATGCTGGGGTCCCATCGCGGTTCAGAGGCAGTGGATCTGGGACAGAGTACACTCT
CACCATCAGCGACCTGGAGTGTGACGATGTTGGCACTTACTACTGTCAAAGTGCTTT
TGATAGTGATAGTACTGAAAATACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC
GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT
CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA
AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA
GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 202).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 3:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGCTTCTCCCTCAGTAGCTATGCAATGAGCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTACTAGTATTG
GTAGCACAGTCTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT
CTGTGCCAGAGGCTACGATGACTATGATGAGATGACCTACTTTAACATCTGGGGCCA
GGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 203).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 4:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGCTTCTCCCTCAGTAGCTATGCAATGAGCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTACTAGTATTG
GTAGCACAGTCTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT
CTGTGCCAGAGGCTACGATGACTATGATGAGATGACCTACTTTAACATCTGGGGCCA
GGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCT
GGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT
CACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAA
AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC
ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG
TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGC
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA
TCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT
ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC
TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
TGA (SEQ ID NO: 204).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 205; SEQ ID NO: 206; and SEQ ID NO: 207 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 1 or the light chain sequence of
SEQ ID NO: 2.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 208; SEQ ID NO: 209; and SEQ ID NO: 210 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 3 or the heavy chain sequence of
SEQ ID NO: 4.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments having binding specificity to NGF comprise, or alternatively
consist of, one, two, three or more, including all of the following polynucleotides encoding
antibody fragments: the polynucleotide SEQ ID NO: 201 encoding the light chain variable
sequence of SEQ ID NO: 1; the polynucleotide SEQ ID NO: 202 encoding the light chain
sequence of SEQ ID NO: 2; the polynucleotide SEQ ID NO: 203 encoding the heavy chain
variable sequence of SEQ ID NO: 3; the polynucleotide SEQ ID NO: 204 encoding the heavy
chain sequence of SEQ ID NO: 4; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 205; SEQ ID NO: 206; and SEQ ID NO: 207) of the light chain variable
sequence of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2; and polynucleotides
encoding the complementarity-determining regions (SEQ ID NO: 208; SEQ ID NO: 209; and
SEQ ID NO: 210) of the heavy chain variable sequence of SEQ ID NO: 3 or the heavy chain
sequence of SEQ ID NO: 4.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity for NGF. With respect to antibody Ab1, the polynucleotides encoding the full length
Ab1 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 202
encoding the light chain sequence of SEQ ID NO: 2 and the polynucleotide SEQ ID NO: 204
encoding the heavy chain sequence of SEQ ID NO: 4.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments for treatment or prevention of pain and pain
associated conditions may be produced by enzymatic digestion (e.g., papain) of Ab1 following
expression of the full-length polynucleotides in a suitable host. In another embodiment
described, anti-NGF antibodies such as Ab1 or Fab fragments thereof may be produced via
expression of Ab1 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells,
fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as
diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to,
Pichia pastoris.
Antibody Ab2
Further described is the use of polynucleotides set forth below to produce
antibody Ab2 polypeptides which inhibit the association of NGF with TrkA and the association
of NGF with p75, for treatment or prevention of pain and pain associated conditions having
binding specificity to NGF in methods of treating pain in an individual comprising administering
to said individual antibody Ab2 polypeptides. Further described are polynucleotides encoding
antibody polypeptides having binding specificity to NGF for treatment or prevention of pain and
pain associated conditions. In one embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable light
chain polypeptide sequence of SEQ ID NO: 11:
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAACATTTACAGCAACTTAGCCTG
GTATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAATCT
GGATGCTGGAGTCCCATCAAGGTTCTCTGGCAGTGGATCTGGGACAGAGTACACTCT
CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAAAGTGCTTT
TGATAGTGATAGTACTGAAAACACTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC
GT (SEQ ID NO: 211).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 12:
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAACATTTACAGCAACTTAGCCTG
GTATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAATCT
GGATGCTGGAGTCCCATCAAGGTTCTCTGGCAGTGGATCTGGGACAGAGTACACTCT
CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAAAGTGCTTT
TGATAGTGATAGTACTGAAAACACTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC
GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT
CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA
AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA
GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 212).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 13:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCTATGCAATGAG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTACTAGTA
TTGGTAGCACAGTCTACGCGAGCAGCGCGAAAGGCCGATTCACCATCTCCAGAGAC
AATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCT
GTGTATTACTGTGCTAGAGGCTACGATGACTATGATGAGATGACCTACTTTAACATC
TGGGGCCAAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 213).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 14:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCTATGCAATGAG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTACTAGTA
TTGGTAGCACAGTCTACGCGAGCAGCGCGAAAGGCCGATTCACCATCTCCAGAGAC
AATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCT
GTGTATTACTGTGCTAGAGGCTACGATGACTATGATGAGATGACCTACTTTAACATC
TGGGGCCAAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG
CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCT
GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCT
CAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCA
ACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCC
TGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA
GCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
CTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT
CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT
ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC
TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT
CCGGGTAAATGA (SEQ ID NO: 214).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 215; SEQ ID NO: 216; and SEQ ID NO: 217 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 11 or the light chain sequence of
SEQ ID NO: 12.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 218; SEQ ID NO: 219; and SEQ ID NO: 220 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 13 or the heavy chain sequence of
SEQ ID NO: 14.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments having binding specificity to NGF comprise, or alternatively
consist of, one, two, three or more, including all of the following polynucleotides encoding
antibody fragments: the polynucleotide SEQ ID NO: 211 encoding the light chain variable
sequence of SEQ ID NO: 11; the polynucleotide SEQ ID NO: 212 encoding the light chain
sequence of SEQ ID NO: 12; the polynucleotide SEQ ID NO: 213 encoding the heavy chain
variable sequence of SEQ ID NO: 13; the polynucleotide SEQ ID NO: 214 encoding the heavy
chain sequence of SEQ ID NO: 14; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 215; SEQ ID NO: 216; and SEQ ID NO: 217) of the light chain variable
sequence of SEQ ID NO: 11 or the light chain sequence of SEQ ID NO: 12; and polynucleotides
encoding the complementarity-determining regions (SEQ ID NO: 218; SEQ ID NO: 219; and
SEQ ID NO: 220) of the heavy chain variable sequence of SEQ ID NO: 13 or the heavy chain
sequence of SEQ ID NO: 14.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity for NGF. With respect to antibody Ab2, the polynucleotides encoding the full length
Ab2 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 212
encoding the light chain sequence of SEQ ID NO: 12 and the polynucleotide SEQ ID NO: 214
encoding the heavy chain sequence of SEQ ID NO: 14.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab2 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab2 or Fab fragments thereof
may be produced via expression of Ab2 polynucleotides in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab3
Described is the use of polynucleotides set forth below to produce antibody Ab3
polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA
without appreciably inhibiting the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab3 polypeptides. Further
described are polynucleotides encoding antibody polypeptides having binding specificity to
NGF. In one embodiment described, polynucleotides described comprise, or alternatively
consist of, the following polynucleotide sequence encoding the variable light chain polypeptide
sequence of SEQ ID NO: 21:
GCAGCCGTGCTGACCCAGACACCATCGCCCGTGTCTGCAGCTATGGGA
GACACAGTCACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAGGCTCCTGATCTATGATGCATCC
AATCTGCCATCTGGGGTCCCATCACGGTTCAGCGGCAGTGGATCTGGGACACAGTTC
ACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGC
GATTATGATGATGATGCTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGT (SEQ ID NO: 221).
In one embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 22:
GCAGCCGTGCTGACCCAGACACCATCGCCCGTGTCTGCAGCTATGGGA
GACACAGTCACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAGGCTCCTGATCTATGATGCATCC
AATCTGCCATCTGGGGTCCCATCACGGTTCAGCGGCAGTGGATCTGGGACACAGTTC
ACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGC
GATTATGATGATGATGCTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 222).
In another embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain
polypeptide sequence of SEQ ID NO: 23:
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGTAATGATCTGG
GTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCACTTGGAGTGCTGG
TACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGA
CCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTC
TGTGCCGGAGGTGGTGGTAGTATTTATGATATTTGGGGCCCGGGCACCCTGGTCACC
GTCTCGAGC (SEQ ID NO: 223).
In one embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 24:
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGTAATGATCTGG
GTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCACTTGGAGTGCTGG
TACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGA
CCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTC
TGTGCCGGAGGTGGTGGTAGTATTTATGATATTTGGGGCCCGGGCACCCTGGTCACC
GTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA
ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT
CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAAC
ACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCC
ACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGG
ACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGT
GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC
AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT
GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA
TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO:
224).
In a further embodiment described, polynucleotides encoding antibody fragments
having binding specificity to NGF comprise, or alternatively consist of, one or more of the
polynucleotide sequences of SEQ ID NO: 225; SEQ ID NO: 226; and SEQ ID NO: 227 which
correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or
hypervariable regions) of the light chain variable sequence of SEQ ID NO: 21 or the light chain
sequence of SEQ ID NO: 22.
In a further embodiment described, polynucleotides encoding antibody fragments
for treatment or prevention of pain and pain associated conditions having binding specificity to
NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ
ID NO: 228; SEQ ID NO: 229; and SEQ ID NO: 230 which correspond to polynucleotides
encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the
heavy chain variable sequence of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO:
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments described herein for treatment or
prevention of pain and pain associated conditions. In one embodiment described,
polynucleotides encoding antibody fragments having binding specificity to NGF comprise, or
alternatively consist of, one, two, three or more, including all of the following polynucleotides
encoding antibody fragments: the polynucleotide SEQ ID NO: 221 encoding the light chain
variable sequence of SEQ ID NO: 21; the polynucleotide SEQ ID NO: 222 encoding the light
chain sequence of SEQ ID NO: 22; the polynucleotide SEQ ID NO: 223 encoding the heavy
chain variable sequence of SEQ ID NO: 23; the polynucleotide SEQ ID NO: 224 encoding the
heavy chain sequence of SEQ ID NO: 24; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 225; SEQ ID NO: 226; and SEQ ID NO: 227) of the light
chain variable sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 228; SEQ ID
NO: 229; and SEQ ID NO: 230) of the heavy chain variable sequence of SEQ ID NO: 23 or the
heavy chain sequence of SEQ ID NO: 24.
In a preferred embodiment described, polynucleotides described comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments for
treatment or prevention of pain and pain associated conditions having binding specificity for
NGF. With respect to antibody Ab3, the polynucleotides encoding the full length Ab3 antibody
comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 222 encoding the light
chain sequence of SEQ ID NO: 22 and the polynucleotide SEQ ID NO: 224 encoding the heavy
chain sequence of SEQ ID NO: 24.
Another embodiment described contemplates these polynucleotides incorporated
into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in
fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable
Pichia species include, but are not limited to, Pichia pastoris. In one embodiment described
herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab3
following expression of the full-length polynucleotides in a suitable host. In another
embodiment described, anti-NGF antibodies such as Ab3 or Fab fragments thereof may be
produced via expression of Ab3 polynucleotides in mammalian cells such as CHO, NSO or HEK
293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not
limited to, Pichia pastoris.
Antibody Ab4
Further described is the use of polynucleotides set forth below to produce
antibody Ab4 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA without appreciably inhibiting the association of NGF with p75, in methods of
treating pain in an individual comprising administering to said individual antibody Ab4
polypeptides. Further described are polynucleotides encoding antibody polypeptides for
treatment or prevention of pain and pain associated conditions having binding specificity to
NGF. In one embodiment described, polynucleotides described comprise, or alternatively
consist of, the following polynucleotide sequence encoding the variable light chain polypeptide
sequence of SEQ ID NO: 31:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC
ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGC
GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAA
ACGT (SEQ ID NO: 231).
In one embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 32:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC
ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGC
GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 232).
In another embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain
polypeptide sequence of SEQ ID NO: 33:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCTATGTAATGAT
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGAATCACTTGGAGTG
CTGGTACATACTACGCGAGCAGTGCGAAAGGCCGATTCACCATCTCCAGAGACAAT
TCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCTGTG
TATTACTGTGCTGGAGGTGGTGGTAGTATCTATGATATTTGGGGCCAAGGGACCCTC
GTCACCGTCTCGAGC (SEQ ID NO: 233).
In one embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 34:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCTATGTAATGAT
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGAATCACTTGGAGTG
CTGGTACATACTACGCGAGCAGTGCGAAAGGCCGATTCACCATCTCCAGAGACAAT
TCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCTGTG
TATTACTGTGCTGGAGGTGGTGGTAGTATCTATGATATTTGGGGCCAAGGGACCCTC
GTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC
TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTT
CCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACA
CCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACA
TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC
CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTA
CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT
ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC
AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT
GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ
ID NO: 234).
In a further embodiment described, polynucleotides encoding antibody fragments
for treatment or prevention of pain and pain associated conditions having binding specificity to
NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ
ID NO: 235; SEQ ID NO: 236; and SEQ ID NO: 237 which correspond to polynucleotides
encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light
chain variable sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32.
In a further embodiment described, polynucleotides encoding antibody fragments
h for treatment or prevention of pain and pain associated conditions having binding specificity to
NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ
ID NO: 238; SEQ ID NO: 239; and SEQ ID NO: 240 which correspond to polynucleotides
encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the
heavy chain variable sequence of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO:
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments described herein. In one embodiment
described, polynucleotides encoding antibody fragments for treatment or prevention of pain and
pain associated conditions having binding specificity to NGF comprise, or alternatively consist
of, one, two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 231 encoding the light chain variable sequence of
SEQ ID NO: 31; the polynucleotide SEQ ID NO: 232 encoding the light chain sequence of SEQ
ID NO: 32; the polynucleotide SEQ ID NO: 233 encoding the heavy chain variable sequence of
SEQ ID NO: 33; the polynucleotide SEQ ID NO: 234 encoding the heavy chain sequence of
SEQ ID NO: 34; polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 235; SEQ ID NO: 236; and SEQ ID NO: 237) of the light chain variable sequence of SEQ
ID NO: 31 or the light chain sequence of SEQ ID NO: 32; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 238; SEQ ID NO: 239; and SEQ ID NO:
240) of the heavy chain variable sequence of SEQ ID NO: 33 or the heavy chain sequence of
SEQ ID NO: 34.
In a preferred embodiment described, polynucleotides described comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments for
treatment or prevention of pain and pain associated conditions having binding specificity for
NGF. With respect to antibody Ab4, the polynucleotides encoding the full length Ab4 antibody
comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 232 encoding the light
chain sequence of SEQ ID NO: 32 and the polynucleotide SEQ ID NO: 234 encoding the heavy
chain sequence of SEQ ID NO: 34.
Another embodiment described contemplates these polynucleotides incorporated
into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in
fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable
Pichia species include, but are not limited to, Pichia pastoris. In one embodiment described
herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab4
following expression of the full-length polynucleotides in a suitable host. In another
embodiment described, anti-NGF antibodies for treatment or prevention of pain and pain
associated conditions such as Ab4 or Fab fragments thereof may be produced via expression of
Ab4 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect,
plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia)
and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab5
Further described is the use of polynucleotides set forth below to produce
antibody Ab5 polypeptides for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF, which inhibit the association of NGF with TrkA and the
association of NGF with p75, in methods of treating pain in an individual comprising
administering to said individual antibody Ab5 polypeptides. Further described are
polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain
associated conditions having binding specificity to NGF. In one embodiment described,
polynucleotides described comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 41:
GCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGA
GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTTACAGCAATTTAGCCTG
GTATCAGCAGAGACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCCACTCT
GGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTC
TCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCTCTTACTACTGTCAACAGGGTT
TTACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC
GT (SEQ ID NO: 241).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 42:
GCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGA
GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTTACAGCAATTTAGCCTG
GTATCAGCAGAGACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCCACTCT
GGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTC
TCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCTCTTACTACTGTCAACAGGGTT
TTACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC
GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT
CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA
AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA
GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 242).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 43:
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAACTATGCAGTGGGCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTGGTCGTAATG
GTAACACATGGTACGCGAGCTGGGCAAGAGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGGATCTGAAAATCACCAGTCCGACAAGCGAGGACACGGCCACATATTT
CTGTGCCAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCTGGGGCCC
AGGCACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 243).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 44:
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAACTATGCAGTGGGCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTGGTCGTAATG
GTAACACATGGTACGCGAGCTGGGCAAGAGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGGATCTGAAAATCACCAGTCCGACAAGCGAGGACACGGCCACATATTT
CTGTGCCAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCTGGGGCCC
AGGCACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCT
GGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT
CACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAA
AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC
ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG
TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGC
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA
TCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT
ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC
TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
TGA (SEQ ID NO: 244).
In a further optional embodiment described, polynucleotides encoding for
treatment or prevention of pain and pain associated conditions fragments having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 245; SEQ ID NO: 246; and SEQ ID NO: 247 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 41 or the light chain sequence of
SEQ ID NO: 42.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 248; SEQ ID NO: 249; and SEQ ID NO: 250 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 43 or the heavy chain sequence of
SEQ ID NO: 44.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 241 encoding the light chain variable sequence of SEQ ID NO: 41; the
polynucleotide SEQ ID NO: 242 encoding the light chain sequence of SEQ ID NO: 42; the
polynucleotide SEQ ID NO: 243 encoding the heavy chain variable sequence of SEQ ID NO: 43;
the polynucleotide SEQ ID NO: 244 encoding the heavy chain sequence of SEQ ID NO: 44;
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 245; SEQ ID
NO: 246; and SEQ ID NO: 247) of the light chain variable sequence of SEQ ID NO: 41 or the
light chain sequence of SEQ ID NO: 42; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 248; SEQ ID NO: 249; and SEQ ID NO: 250) of the heavy
chain variable sequence of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab5, the
polynucleotides encoding the full length Ab5 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 242 encoding the light chain sequence of SEQ ID NO: 42 and the
polynucleotide SEQ ID NO: 244 encoding the heavy chain sequence of SEQ ID NO: 44.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab5 following expression of the full-length polynucleotides in a suitable host.
In another optional embodiment described, anti-NGF antibodies such as Ab5 or Fab fragments
thereof may be produced via expression of Ab5 polynucleotides in mammalian cells such as
CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab6
Further optionally described is the use of polynucleotides set forth below to
produce antibody Ab6 polypeptides having binding specificity to NGF, which inhibits the
association of NGF with TrkA and the association of NGF with p75, in methods of treating pain
in an individual comprising administering to said individual antibody Ab6 polypeptides. Further
described are polynucleotides encoding for treatment or prevention of pain and pain associated
conditions polypeptides having binding specificity to NGF. In one embodiment described,
polynucleotides described comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 51:
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG
TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG
GAATCTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTACACTCT
CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAACAGGGTTT
TACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC
GT (SEQ ID NO: 251).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 52:
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG
TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG
GAATCTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTACACTCT
CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAACAGGGTTT
TACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC
GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT
CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA
AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA
GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 252).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 53:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAATCATTGGTCGTA
ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC
AATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCT
GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCT
GGGGCCCAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 253).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 54:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAATCATTGGTCGTA
ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC
AATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCT
GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCT
GGGGCCCAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCT
TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC
TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC
AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA
CGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG
TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA
CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC
AGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT
GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA
AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC
AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC
GGGTAAATGA (SEQ ID NO: 254).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 51 or the light chain sequence of
SEQ ID NO: 52.
In a further optional embodiment described, polynucleotides encoding for
treatment or prevention of pain and pain associated conditions fragments having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of
SEQ ID NO: 54.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 251 encoding the light chain variable sequence of SEQ ID NO: 51; the
polynucleotide SEQ ID NO: 252 encoding the light chain sequence of SEQ ID NO: 52; the
polynucleotide SEQ ID NO: 253 encoding the heavy chain variable sequence of SEQ ID NO: 53;
the polynucleotide SEQ ID NO: 254 encoding the heavy chain sequence of SEQ ID NO: 54;
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 255; SEQ ID
NO: 256; and SEQ ID NO: 257) of the light chain variable sequence of SEQ ID NO: 51 or the
light chain sequence of SEQ ID NO: 52; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260) of the heavy
chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab6, the
polynucleotides encoding the full length Ab6 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 252 encoding the light chain sequence of SEQ ID NO: 52 and the
polynucleotide SEQ ID NO: 254 encoding the heavy chain sequence of SEQ ID NO: 54.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab6 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab6 or Fab fragments thereof
may be produced via expression of Ab6 polynucleotides in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab7
Further described is the use of polynucleotides set forth below to produce
antibody Ab7 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA and the association of NGF with p75. in methods of treating pain in an
individual comprising administering to said individual antibody Ab7 polypeptides. Further
described are polynucleotides encoding antibody polypeptides for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF. In one embodiment
described, polynucleotides described comprise, or alternatively consist of, the following
polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO:
GCCGATGTTGTGATGACCCAGACTCCAGCCTCCGTGTCTCAACCTGTG
GGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGACATTTATAACTTATTGGC
CTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCATCCAC
TCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACA
CTCTCACCATCAGCGGCCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAAACA
ATTATCTTGTTACTACTTATGGTGTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCA
AACGT (SEQ ID NO: 261).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 62:
GCCGATGTTGTGATGACCCAGACTCCAGCCTCCGTGTCTCAACCTGTG
GGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGACATTTATAACTTATTGGC
CTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCATCCAC
TCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACA
CTCTCACCATCAGCGGCCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAAACA
ATTATCTTGTTACTACTTATGGTGTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCA
AACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA
AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA
AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC
ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAG
CAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC
TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 262).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 63:
CAGGAGCAGCTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGAC
ACCCCTGACACTCACCTGTACAGTCTCTGGATTCTCCCTCAGTAGCTATGCAATGAT
CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTGATACTG
ATACTAGCGCATACTACGCGAGCTGGGTGAAAGGCCGATTCACCATCTCCAGAACC
TCGACCACGGTGGATCTCAAAATCACTAGTCCGACAACCGAGGACACGGCCACCTA
TTTCTGTGCCAGATCTTATGCTGCTTATGGTGGTTATCCTGCTACTTTTGATCCCTGG
GGCCCAGGCACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 263).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 64:
CAGGAGCAGCTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGAC
ACCCCTGACACTCACCTGTACAGTCTCTGGATTCTCCCTCAGTAGCTATGCAATGAT
CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTGATACTG
ATACTAGCGCATACTACGCGAGCTGGGTGAAAGGCCGATTCACCATCTCCAGAACC
TCGACCACGGTGGATCTCAAAATCACTAGTCCGACAACCGAGGACACGGCCACCTA
TTTCTGTGCCAGATCTTATGCTGCTTATGGTGGTTATCCTGCTACTTTTGATCCCTGG
GGCCCAGGCACCCTGGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTC
CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG
GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC
CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT
GAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTG
ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG
GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC
CCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG
CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG
TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAATGA (SEQ ID NO: 264).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 265; SEQ ID NO: 266; and SEQ ID NO: 267 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 61 or the light chain sequence of
SEQ ID NO: 62.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 268; SEQ ID NO: 269; and SEQ ID NO: 270 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 63 or the heavy chain sequence of
SEQ ID NO: 64.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 261 encoding the light chain variable sequence of SEQ ID NO: 61; the
polynucleotide SEQ ID NO: 262 encoding the light chain sequence of SEQ ID NO: 62; the
polynucleotide SEQ ID NO: 263 encoding the heavy chain variable sequence of SEQ ID NO: 63;
the polynucleotide SEQ ID NO: 264 encoding the heavy chain sequence of SEQ ID NO: 64;
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 265; SEQ ID
NO: 266; and SEQ ID NO: 267) of the light chain variable sequence of SEQ ID NO: 61 or the
light chain sequence of SEQ ID NO: 62; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 268; SEQ ID NO: 269; and SEQ ID NO: 270) of the heavy
chain variable sequence of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab7, the
polynucleotides encoding the full length Ab7 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 262 encoding the light chain sequence of SEQ ID NO: 62 and the
polynucleotide SEQ ID NO: 264 encoding the heavy chain sequence of SEQ ID NO: 64.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab7 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab7 or Fab fragments thereof
may be produced via expression of Ab7 polynucleotides in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab8
Further described is the use of polynucleotides set forth below to produce
antibody Ab8 polypeptides having binding specificity to NGF, which inhibits the association of
NGF with TrkA and the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab8 polypeptides. Further
described are polynucleotides encoding antibody polypeptides for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF. In one embodiment
described, polynucleotides described comprise, or alternatively consist of, the following
polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTGAGGACATTTACAACTTATTGGCCTGG
TATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATTCTGCATCCACTCTG
GCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTC
ACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAACAACTAT
CTTGTTACTACTTATGGTGTTGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGT
(SEQ ID NO: 271).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 72:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTGAGGACATTTACAACTTATTGGCCTGG
TATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATTCTGCATCCACTCTG
GCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTC
ACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAACAACTAT
CTTGTTACTACTTATGGTGTTGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGT
ACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA
CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA
GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGC
TCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 272).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 73:
CAGGTACAGCTGGTGGAGTCTGGTGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCACCTTCAGTAGCTATGCAATGATC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTGATACTGA
TACTAGCGCATACTACGCAAGCAGTGTGAAAGGCCGATTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTACCTGCAAATGTCTAGCCTGAGAGCCGAGGACACGGCT
GTGTATTACTGTGCTAGATCTTATGCTGCTTATGGTGGTTATCCTGCTACTTTTGATC
CCTGGGGCCAAGGTACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 273).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 74:
CAGGTACAGCTGGTGGAGTCTGGTGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCACCTTCAGTAGCTATGCAATGATC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTGATACTGA
TACTAGCGCATACTACGCAAGCAGTGTGAAAGGCCGATTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTACCTGCAAATGTCTAGCCTGAGAGCCGAGGACACGGCT
GTGTATTACTGTGCTAGATCTTATGCTGCTTATGGTGGTTATCCTGCTACTTTTGATC
CCTGGGGCCAAGGTACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGG
TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC
CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC
CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC
AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAC
CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC
AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC
CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG
TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG
GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC
TCCGGGTAAATGA (SEQ ID NO: 274).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 275; SEQ ID NO: 276; and SEQ ID NO: 277 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 71 or the light chain sequence of
SEQ ID NO: 72.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 278; SEQ ID NO: 279; and SEQ ID NO: 280 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 73 or the heavy chain sequence of
SEQ ID NO: 74.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 271 encoding the light chain variable sequence of SEQ ID NO: 71; the
polynucleotide SEQ ID NO: 272 encoding the light chain sequence of SEQ ID NO: 72; the
polynucleotide SEQ ID NO: 273 encoding the heavy chain variable sequence of SEQ ID NO: 73;
the polynucleotide SEQ ID NO: 274 encoding the heavy chain sequence of SEQ ID NO: 74;
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 275; SEQ ID
NO: 276; and SEQ ID NO: 277) of the light chain variable sequence of SEQ ID NO: 71 or the
light chain sequence of SEQ ID NO: 72; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 278; SEQ ID NO: 279; and SEQ ID NO: 280) of the heavy
chain variable sequence of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab8, the
polynucleotides encoding the full length Ab8 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 272 encoding the light chain sequence of SEQ ID NO: 72 and the
polynucleotide SEQ ID NO: 274 encoding the heavy chain sequence of SEQ ID NO: 74.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab8 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab8 or Fab fragments thereof
may be produced via expression of Ab8 polynucleotides in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab9
Further described is the use of polynucleotides set forth below to produce
antibody Ab9 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA and the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab9 polypeptides. Further
described are polynucleotides encoding antibody polypeptides for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF. In one embodiment
described, polynucleotides described comprise, or alternatively consist of, the following
polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO:
GCCTATGATATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGA
GGCACAGTCACCATCAAGTGCCAGGCCAGTGAGAACATTGGTAGCTACTTAGCCTG
GTATCAGCAGAAACCAGGGCAGCCTCCCGAACTCCTGATCTACAGGGCGTCCACTCT
GGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCT
CACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGGTTA
TAATAGTGAGAATCTTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC
GT (SEQ ID NO: 281).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 82:
GCCTATGATATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGA
GGCACAGTCACCATCAAGTGCCAGGCCAGTGAGAACATTGGTAGCTACTTAGCCTG
GTATCAGCAGAAACCAGGGCAGCCTCCCGAACTCCTGATCTACAGGGCGTCCACTCT
GGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCT
CACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGGTTA
TAATAGTGAGAATCTTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC
GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT
CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA
AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA
GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 282).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 83:
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTATGTATTCAATGGGCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATGGATTAGTTATGGTG
GTACTGCATATTACGCGAGCTGGGCGAAGGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGGAGCTGAAGATCACCAGTCCGACAATCGAGGACACGGCCACCTATTT
CTGTGCCAGAGAGACTCCTGTTAATTATTATTTGGACATTTGGGGCCAGGGGACCCT
CGTCACCGTCTCGAGC (SEQ ID NO: 283).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 84:
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTATGTATTCAATGGGCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATGGATTAGTTATGGTG
GTACTGCATATTACGCGAGCTGGGCGAAGGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGGAGCTGAAGATCACCAGTCCGACAATCGAGGACACGGCCACCTATTT
CTGTGCCAGAGAGACTCCTGTTAATTATTATTTGGACATTTGGGGCCAGGGGACCCT
CGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC
CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACT
TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC
ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCC
AGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACAC
ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC
CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTA
CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT
ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC
AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT
GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ
ID NO: 284).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 285; SEQ ID NO: 286; and SEQ ID NO: 287 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 81 or the light chain sequence of
SEQ ID NO: 82.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 288; SEQ ID NO: 289; and SEQ ID NO: 290 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 83 or the heavy chain sequence of
SEQ ID NO: 84.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one optional embodiment described,
polynucleotides encoding antibody fragments for treatment or prevention of pain and pain
associated conditions having binding specificity to NGF comprise, or alternatively consist of,
one, two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 281 encoding the light chain variable sequence of
SEQ ID NO: 81; the polynucleotide SEQ ID NO: 282 encoding the light chain sequence of SEQ
ID NO: 82; the polynucleotide SEQ ID NO: 283 encoding the heavy chain variable sequence of
SEQ ID NO: 83; the polynucleotide SEQ ID NO: 284 encoding the heavy chain sequence of
SEQ ID NO: 84; polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 285; SEQ ID NO: 286; and SEQ ID NO: 287) of the light chain variable sequence of SEQ
ID NO: 81 or the light chain sequence of SEQ ID NO: 82; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 288; SEQ ID NO: 289; and SEQ ID NO:
290) of the heavy chain variable sequence of SEQ ID NO: 83 or the heavy chain sequence of
SEQ ID NO: 84.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab9, the
polynucleotides encoding the full length Ab9 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 282 encoding the light chain sequence of SEQ ID NO: 82 and the
polynucleotide SEQ ID NO: 284 encoding the heavy chain sequence of SEQ ID NO: 84.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab9 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab9 or Fab fragments thereof
may be produced via expression of Ab9 polynucleotides in mammalian cells such as CHO, NSO
or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab10
Further described is the use of polynucleotides set forth below to produce
antibody Ab10 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA and the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab10 polypeptides. Further
described are polynucleotides encoding antibody polypeptides having binding specificity to
NGF. In one embodiment described, polynucleotides described comprise, or alternatively
consist of, the following polynucleotide sequence encoding the variable light chain polypeptide
sequence of SEQ ID NO: 91:
GCCTATGATATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGCCAGGCCAGTGAGAACATTGGTAGCTACTTAGCCTGGT
ATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAGGGCTTCCACTCTGG
CATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA
CCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAACAGGGTTACA
ATAGTGAGAATCTTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGT
(SEQ ID NO: 291).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 92:
GCCTATGATATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGCCAGGCCAGTGAGAACATTGGTAGCTACTTAGCCTGGT
ATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAGGGCTTCCACTCTGG
CATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA
CCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAACAGGGTTACA
ATAGTGAGAATCTTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGT
ACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA
CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA
GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGC
TCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 292).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 93:
CAGGTACAGCTGGTGGAGTCTGGTGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCACCTTCAGTATGTATTCAATGGGC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATGGATTAGTTATGG
TGGTACTGCATACTACGCTAGCAGCGCTAAGGGCCGATTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTACCTGCAAATGTCTAGCCTGAGAGCCGAGGACACGGCT
GTGTATTACTGTGCTAGAGAGACTCCTGTTAATTACTACTTGGACATTTGGGGCCAA
GGTACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 293).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 94:
CAGGTACAGCTGGTGGAGTCTGGTGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCACCTTCAGTATGTATTCAATGGGC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATGGATTAGTTATGG
TGGTACTGCATACTACGCTAGCAGCGCTAAGGGCCGATTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTACCTGCAAATGTCTAGCCTGAGAGCCGAGGACACGGCT
GTGTATTACTGTGCTAGAGAGACTCCTGTTAATTACTACTTGGACATTTGGGGCCAA
GGTACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTG
GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAA
GGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG
GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG
TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAA
ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC
CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA
TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC
CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA
CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC
ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT
GA (SEQ ID NO: 294).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 295; SEQ ID NO: 296; and SEQ ID NO: 297 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 91 or the light chain sequence of
SEQ ID NO: 92.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 298; SEQ ID NO: 299; and SEQ ID NO: 300 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 93 or the heavy chain sequence of
SEQ ID NO: 94.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 291 encoding the light chain variable sequence of SEQ ID NO: 91; the
polynucleotide SEQ ID NO: 292 encoding the light chain sequence of SEQ ID NO: 92; the
polynucleotide SEQ ID NO: 293 encoding the heavy chain variable sequence of SEQ ID NO: 93;
the polynucleotide SEQ ID NO: 294 encoding the heavy chain sequence of SEQ ID NO: 94;
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 295; SEQ ID
NO: 296; and SEQ ID NO: 297) of the light chain variable sequence of SEQ ID NO: 91 or the
light chain sequence of SEQ ID NO: 92; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 298; SEQ ID NO: 299; and SEQ ID NO: 300) of the heavy
chain variable sequence of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab10, the
polynucleotides encoding the full length Ab10 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 292 encoding the light chain sequence of SEQ ID NO: 92 and the
polynucleotide SEQ ID NO: 294 encoding the heavy chain sequence of SEQ ID NO: 94.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab10 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab10 or Fab fragments thereof
may be produced via expression of Ab10 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab11
Further described is the use of polynucleotides set forth below to produce
antibody Ab11 polypeptides having binding specificity to NGF, which inhibits the association of
NGF with TrkA and the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab11 polypeptides. Further
described are polynucleotides encoding antibody polypeptides having binding specificity to
NGF. In one embodiment described, polynucleotides described comprise, or alternatively
consist of, the following polynucleotide sequence encoding the variable light chain polypeptide
sequence of SEQ ID NO: 101:
GCATTCGAATTGACCCAGACTCCATCCTCCGTGGAGGCAGCTGTGGGA
GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAACATTGTTACCAATTTAGCCTGG
TATCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCACTCTG
GCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTC
ACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTATTTCTGTCAGAGCTATGAT
GGTTTTAATAGTGCTGGGTTCGGCGGAGGGACCGAGGTGGTGGTCAAACGT (SEQ
ID NO: 301).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 102:
GCATTCGAATTGACCCAGACTCCATCCTCCGTGGAGGCAGCTGTGGGA
GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAACATTGTTACCAATTTAGCCTGG
TATCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCACTCTG
GCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTC
ACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTATTTCTGTCAGAGCTATGAT
GGTTTTAATAGTGCTGGGTTCGGCGGAGGGACCGAGGTGGTGGTCAAACGTACGGT
AGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAC
TGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTG
GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGG
ACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGAC
TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCC
CGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 302).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 103:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTGGCTACGACATGAGCTG
GGTCCGCCAGGCTCCAGGAAAGGGGCTGGAATACATCGGACTCATTAGTTATGATG
GTAACACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT
CTGTGCCAGAAGTCTTTATGCTGGTCCTAATGCTGGTATCGGACCGTTTAACATCTG
GGGCCAGGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 303).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 104:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTGGCTACGACATGAGCTG
GGTCCGCCAGGCTCCAGGAAAGGGGCTGGAATACATCGGACTCATTAGTTATGATG
GTAACACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT
CTGTGCCAGAAGTCTTTATGCTGGTCCTAATGCTGGTATCGGACCGTTTAACATCTG
GGGCCAGGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTT
CCCCCTGGCAcCCTCCTCCaAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT
GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA
CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA
GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAAC
GTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTG
TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG
AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC
AGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT
GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA
AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC
AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC
GGGTAAATGA (SEQ ID NO: 304).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 305; SEQ ID NO: 306; and SEQ ID NO: 307 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 101 or the light chain sequence of
SEQ ID NO: 102.
In a further optional embodiment described, polynucleotides encoding for
treatment or prevention of pain and pain associated conditions fragments having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 308; SEQ ID NO: 309; and SEQ ID NO: 310 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 103 or the heavy chain sequence
of SEQ ID NO: 104.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 301 encoding the light chain variable sequence of SEQ ID NO: 101; the
polynucleotide SEQ ID NO: 302 encoding the light chain sequence of SEQ ID NO: 102; the
polynucleotide SEQ ID NO: 303 encoding the heavy chain variable sequence of SEQ ID NO:
103; the polynucleotide SEQ ID NO: 304 encoding the heavy chain sequence of SEQ ID NO:
104; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 305;
SEQ ID NO: 306; and SEQ ID NO: 307) of the light chain variable sequence of SEQ ID NO:
101 or the light chain sequence of SEQ ID NO: 102; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 308; SEQ ID NO: 309; and SEQ ID NO:
310) of the heavy chain variable sequence of SEQ ID NO: 103 or the heavy chain sequence of
SEQ ID NO: 104.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab11, the
polynucleotides encoding the full length Ab11 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 302 encoding the light chain sequence of SEQ ID NO: 102 and the
polynucleotide SEQ ID NO: 304 encoding the heavy chain sequence of SEQ ID NO: 104.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab11 following expression of the full-length polynucleotides in a suitable host.
In another optional embodiment described, anti-NGF antibodies such as Ab11 or Fab fragments
thereof may be produced via expression of Ab11 polynucleotides in mammalian cells such as
CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab12
Further described is the use of polynucleotides set forth below to produce
antibody Ab12 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA and the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab12 polypeptides. Further
described are polynucleotides encoding antibody polypeptides for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF. In one embodiment
described, polynucleotides described comprise, or alternatively consist of, the following
polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO:
111:
GCATTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAACATTGTTACCAACTTAGCCTGG
TATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATGGTGCATCCACTCTG
GCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTC
ACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAGAGCTATGAT
GGTTTCAATAGTGCTGGTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGT (SEQ
ID NO: 311).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 112:
GCATTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAACATTGTTACCAACTTAGCCTGG
TATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATGGTGCATCCACTCTG
GCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTC
ACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAGAGCTATGAT
GGTTTCAATAGTGCTGGTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGTACGGT
AGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAC
TGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTG
GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGG
ACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGAC
TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCC
CGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 312).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 113:
CAGGTACAGCTGGTGGAGTCTGGTGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCTCCCTCAGTGGCTACGACATGAG
CTGGGTCCGTCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGGACTCATTAGTTATG
ATGGTAACACATACTACGCGACCTCCGCGAAAGGCCGATTCACCATCTCCAGAGAC
AATTCCAAGAACACGCTGTACCTGCAAATGTCTAGCCTGAGAGCCGAGGACACGGC
TGTGTATTACTGTGCTAGAAGTCTTTATGCTGGTCCTAATGCTGGTATCGGACCGTTT
AACATCTGGGGCCAAGGTACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 313).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 114:
CAGGTACAGCTGGTGGAGTCTGGTGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCTCCCTCAGTGGCTACGACATGAG
CTGGGTCCGTCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGGACTCATTAGTTATG
ATGGTAACACATACTACGCGACCTCCGCGAAAGGCCGATTCACCATCTCCAGAGAC
AATTCCAAGAACACGCTGTACCTGCAAATGTCTAGCCTGAGAGCCGAGGACACGGC
TGTGTATTACTGTGCTAGAAGTCTTTATGCTGGTCCTAATGCTGGTATCGGACCGTTT
AACATCTGGGGCCAAGGTACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCT
GGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG
GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT
ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA
TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCC
AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA
GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGG
AGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGC
CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA
GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT
CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC
TGTCTCCGGGTAAATGA (SEQ ID NO: 314).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 315; SEQ ID NO: 316; and SEQ ID NO: 317 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 111 or the light chain sequence of
SEQ ID NO: 112.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 318; SEQ ID NO: 319; and SEQ ID NO: 320 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 113 or the heavy chain sequence
of SEQ ID NO: 114.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 311 encoding the light chain variable sequence of SEQ ID NO: 111; the
polynucleotide SEQ ID NO: 312 encoding the light chain sequence of SEQ ID NO: 112; the
polynucleotide SEQ ID NO: 313 encoding the heavy chain variable sequence of SEQ ID NO:
113; the polynucleotide SEQ ID NO: 314 encoding the heavy chain sequence of SEQ ID NO:
114; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 315;
SEQ ID NO: 316; and SEQ ID NO: 317) of the light chain variable sequence of SEQ ID NO:
111 or the light chain sequence of SEQ ID NO: 112; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 318; SEQ ID NO: 319; and SEQ ID NO:
320) of the heavy chain variable sequence of SEQ ID NO: 113 or the heavy chain sequence of
SEQ ID NO: 114.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab12, the
polynucleotides encoding the full length Ab12 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 312 encoding the light chain sequence of SEQ ID NO: 112 and the
polynucleotide SEQ ID NO: 314 encoding the heavy chain sequence of SEQ ID NO: 114.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab12 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab12 or Fab fragments thereof
may be produced via expression of Ab12 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab13
Further described is the use of polynucleotides set forth below to produce
antibody Ab13 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA and the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab13 polypeptides. Further
described are polynucleotides encoding antibody polypeptides having binding specificity to
NGF. In one embodiment described, polynucleotides described comprise, or alternatively
consist of, the following polynucleotide sequence encoding the variable light chain polypeptide
sequence of SEQ ID NO: 121:
GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGA
GGCACAGTCAGCATCAGTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCC
ACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCGGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCGACGTGCAGTGTGACGCTGCTGCCACTTACTACTGTGCAGGC
GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGT (SEQ ID NO: 321).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 122:
GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGA
GGCACAGTCAGCATCAGTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCC
ACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCGGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCGACGTGCAGTGTGACGCTGCTGCCACTTACTACTGTGCAGGC
GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA (SEQ ID NO: 322).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 123:
CAGTCGGTGGAGGCGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACCTACTGGATGAGCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGACATTTATTTTAGTA
ATGAAGAAACAAACTACGCGAGCTGGGCGAAAGGCCGATTTACCATCTCCAAAACC
TCGACCACGGTGGATCTGAATGTCATCAGTCCGACAACCGAGGACACGGCCACCTA
TTTCTGTGCCAGAGGTTCTCCTGATGTTGATATTGGTATAGATATGTGGGGCCCGGG
CACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 323).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 124:
CAGTCGGTGGAGGCGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACCTACTGGATGAGCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGACATTTATTTTAGTA
ATGAAGAAACAAACTACGCGAGCTGGGCGAAAGGCCGATTTACCATCTCCAAAACC
TCGACCACGGTGGATCTGAATGTCATCAGTCCGACAACCGAGGACACGGCCACCTA
TTTCTGTGCCAGAGGTTCTCCTGATGTTGATATTGGTATAGATATGTGGGGCCCGGG
CACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGC
ACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG
ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC
GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG
GTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC
AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAAC
TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCT
CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAG
CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC
CGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA
TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATG
A (SEQ ID NO: 324).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 325; SEQ ID NO: 326; and SEQ ID NO: 327 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 121 or the light chain sequence of
SEQ ID NO: 122.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 328; SEQ ID NO: 329; and SEQ ID NO: 330 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 123 or the heavy chain sequence
of SEQ ID NO: 124.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 321 encoding the light chain variable sequence of SEQ ID NO: 121; the
polynucleotide SEQ ID NO: 322 encoding the light chain sequence of SEQ ID NO: 122; the
polynucleotide SEQ ID NO: 323 encoding the heavy chain variable sequence of SEQ ID NO:
123; the polynucleotide SEQ ID NO: 324 encoding the heavy chain sequence of SEQ ID NO:
124; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 325;
SEQ ID NO: 326; and SEQ ID NO: 327) of the light chain variable sequence of SEQ ID NO:
121 or the light chain sequence of SEQ ID NO: 122; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 328; SEQ ID NO: 329; and SEQ ID NO:
330) of the heavy chain variable sequence of SEQ ID NO: 123 or the heavy chain sequence of
SEQ ID NO: 124.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab13, the
polynucleotides encoding the full length Ab13 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 322 encoding the light chain sequence of SEQ ID NO: 122 and the
polynucleotide SEQ ID NO: 324 encoding the heavy chain sequence of SEQ ID NO: 124.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab13 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab13 or Fab fragments thereof
may be produced via expression of Ab13 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab14
Further described is the use of polynucleotides set forth below to produce
antibody Ab14 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA and the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab14 polypeptides. Further
described are polynucleotides encoding antibody polypeptides having binding specificity to
NGF. In one embodiment described, polynucleotides described comprise, or alternatively
consist of, the following polynucleotide sequence encoding the variable light chain polypeptide
sequence of SEQ ID NO: 131:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC
ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGC
GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAA
ACGT (SEQ ID NO: 331).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 132:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC
ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGC
GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 332).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 133:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTACCTACTGGATGAG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGACATTTACTTTA
GTAATGAAGAAACAAACTACGCGAGCAGCGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACAC
TGCTGTGTATTACTGTGCTAGAGGTTCTCCTGATGTTGATATTGGTATAGATATGTGG
GGCCCAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 333).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 134:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTACCTACTGGATGAG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGACATTTACTTTA
GTAATGAAGAAACAAACTACGCGAGCAGCGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACAC
TGCTGTGTATTACTGTGCTAGAGGTTCTCCTGATGTTGATATTGGTATAGATATGTGG
GGCCCAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTC
CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG
GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC
CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT
GAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTG
ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG
GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC
CCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG
CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG
TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAATGA (SEQ ID NO: 334).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 335; SEQ ID NO: 336; and SEQ ID NO: 337 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 131 or the light chain sequence of
SEQ ID NO: 132.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 338; SEQ ID NO: 339; and SEQ ID NO: 340 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 133 or the heavy chain sequence
of SEQ ID NO: 134.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 331 encoding the light chain variable sequence of SEQ ID NO: 131; the
polynucleotide SEQ ID NO: 332 encoding the light chain sequence of SEQ ID NO: 132; the
polynucleotide SEQ ID NO: 333 encoding the heavy chain variable sequence of SEQ ID NO:
133; the polynucleotide SEQ ID NO: 334 encoding the heavy chain sequence of SEQ ID NO:
134; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 335;
SEQ ID NO: 336; and SEQ ID NO: 337) of the light chain variable sequence of SEQ ID NO:
131 or the light chain sequence of SEQ ID NO: 132; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 338; SEQ ID NO: 339; and SEQ ID NO:
340) of the heavy chain variable sequence of SEQ ID NO: 133 or the heavy chain sequence of
SEQ ID NO: 134.
In a preferred optional embodiment described, polynucleotides of the invention
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab14, the
polynucleotides encoding the full length Ab14 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 332 encoding the light chain sequence of SEQ ID NO: 132 and the
polynucleotide SEQ ID NO: 334 encoding the heavy chain sequence of SEQ ID NO: 134.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab14 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab14 or Fab fragments thereof
may be produced via expression of Ab14 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab15
Further described is the use of polynucleotides set forth below to produce
antibody Ab15 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA without appreciably inhibiting the association of NGF with p75, in methods of
treating pain in an individual comprising administering to said individual antibody Ab15
polypeptides. Further described are polynucleotides encoding antibody polypeptides for
treatment or prevention of pain and pain associated conditions having binding specificity to
NGF. In one embodiment described, polynucleotides described comprise, or alternatively
consist of, the following polynucleotide sequence encoding the variable light chain polypeptide
sequence of SEQ ID NO: 141:
GCAGCCGTGCTGACCCAGACACCATCGCCCGTGTCTGCAGCTGTGGGA
GACACAGTCACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCC
AATCTGCCATCTGGGGTCCCATCACGGTTCAGCGGCAGTGGATCTGGGACACAGTTC
ACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGC
GATTATGATGATGATACTGATAATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGT (SEQ ID NO: 341).
In one embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 142:
GCAGCCGTGCTGACCCAGACACCATCGCCCGTGTCTGCAGCTGTGGGA
GACACAGTCACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCC
AATCTGCCATCTGGGGTCCCATCACGGTTCAGCGGCAGTGGATCTGGGACACAGTTC
ACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGC
GATTATGATGATGATACTGATAATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 342).
In another embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain
polypeptide sequence of SEQ ID NO: 143:
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCTATGCAATGATCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTTGGAGTGGTG
GCACCTACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACC
ACGGTGGATCTGCAAATCACCAGTCCGACAACCGAGGACGCGGCCACCTATTTCTGT
GCCGCAGGTGGTGGTAGTATTTATGATGTTTGGGGCCCGGGCACCCTGGTCACCGTC
TCGAGC (SEQ ID NO: 343).
In one embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 144:
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCTATGCAATGATCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTTGGAGTGGTG
GCACCTACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACC
ACGGTGGATCTGCAAATCACCAGTCCGACAACCGAGGACGCGGCCACCTATTTCTGT
GCCGCAGGTGGTGGTAGTATTTATGATGTTTGGGGCCCGGGCACCCTGGTCACCGTC
TCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC
ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC
GGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA
GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACC
GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGT
GAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTC
AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA
GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG
GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACC
AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT
GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAG
GTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA
CTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO: 344).
In a further embodiment described, polynucleotides encoding antibody fragments
for treatment or prevention of pain and pain associated conditions having binding specificity to
NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ
ID NO: 345; SEQ ID NO: 346; and SEQ ID NO: 347 which correspond to polynucleotides
encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light
chain variable sequence of SEQ ID NO: 141 or the light chain sequence of SEQ ID NO: 142.
In a further embodiment described, polynucleotides encoding antibody fragments
for treatment or prevention of pain and pain associated conditions having binding specificity to
NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ
ID NO: 348; SEQ ID NO: 349; and SEQ ID NO: 350 which correspond to polynucleotides
encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the
heavy chain variable sequence of SEQ ID NO: 143 or the heavy chain sequence of SEQ ID NO:
144.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 341 encoding the light chain variable sequence of SEQ ID NO: 141; the
polynucleotide SEQ ID NO: 342 encoding the light chain sequence of SEQ ID NO: 142; the
polynucleotide SEQ ID NO: 343 encoding the heavy chain variable sequence of SEQ ID NO:
143; the polynucleotide SEQ ID NO: 344 encoding the heavy chain sequence of SEQ ID NO:
144; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 345;
SEQ ID NO: 346; and SEQ ID NO: 347) of the light chain variable sequence of SEQ ID NO:
141 or the light chain sequence of SEQ ID NO: 142; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 348; SEQ ID NO: 349; and SEQ ID NO:
350) of the heavy chain variable sequence of SEQ ID NO: 143 or the heavy chain sequence of
SEQ ID NO: 144.
In a preferred embodiment described, polynucleotides described comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments
having binding specificity for NGF. With respect to antibody Ab15, the polynucleotides
encoding the full length Ab15 antibody comprise, or alternatively consist of, the polynucleotide
SEQ ID NO: 342 encoding the light chain sequence of SEQ ID NO: 142 and the polynucleotide
SEQ ID NO: 344 encoding the heavy chain sequence of SEQ ID NO: 144.
Another embodiment described contemplates these polynucleotides incorporated
into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in
fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable
Pichia species include, but are not limited to, Pichia pastoris. In one embodiment described
herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab15
following expression of the full-length polynucleotides in a suitable host. In another
embodiment described, anti-NGF antibodies such as Ab15 or Fab fragments thereof may be
produced via expression of Ab15 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab16
Further described is the use of polynucleotides set forth below to produce
antibody Ab16 polypeptides for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF, which inhibit the association of NGF with TrkA without
appreciably inhibiting the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab16 polypeptides. Further
described are polynucleotides encoding antibody polypeptides for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF. In one embodiment
described, polynucleotides described comprise, or alternatively consist of, the following
polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO:
151:
GCCCTGGTGATGACCCAGACTCCATCCTCCACGTCTGAACCAGTGGGA
GGCACAGTCACCATCAATTGCCAGGCTAGTCAGAATATTGGTAACGACCTATCCTGG
TATCAGCAGAAACCAGGGCAGCCTCCCGAGCTCCTAATCTATTCTACATCCAAACTG
GCAACTGGGGTCCCAAAGCGGTTCAGTGGCAGCAGATCTGGGACACAGTTCACTCT
CACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCTAGGTGTTTA
TAGTTATATTAGTGATGATGGTAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGT (SEQ ID NO: 351).
In one embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 152:
GCCCTGGTGATGACCCAGACTCCATCCTCCACGTCTGAACCAGTGGGA
GGCACAGTCACCATCAATTGCCAGGCTAGTCAGAATATTGGTAACGACCTATCCTGG
TATCAGCAGAAACCAGGGCAGCCTCCCGAGCTCCTAATCTATTCTACATCCAAACTG
GCAACTGGGGTCCCAAAGCGGTTCAGTGGCAGCAGATCTGGGACACAGTTCACTCT
CACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCTAGGTGTTTA
TAGTTATATTAGTGATGATGGTAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 352).
In another embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain
polypeptide sequence of SEQ ID NO: 153:
CAGTCGGTGGAGGAGTTCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAATAACTATGCAATGACCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGGATCATTGGTAGTATTG
GTACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCTTCATCTCCAAAACCTCGA
CCACTGTGGATCTGAAAATCATTAGTCCGACAACCGAGGACACGGCCACCTATTTCT
GTGCCAGAGATGCTGGCGTTACTGTTGATGGTTATGGCTACTACTTTAACATCTGGG
GCCCAGGCACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 353).
In one embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 154:
CAGTCGGTGGAGGAGTTCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAATAACTATGCAATGACCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGGATCATTGGTAGTATTG
GTACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCTTCATCTCCAAAACCTCGA
CCACTGTGGATCTGAAAATCATTAGTCCGACAACCGAGGACACGGCCACCTATTTCT
GTGCCAGAGATGCTGGCGTTACTGTTGATGGTTATGGCTACTACTTTAACATCTGGG
GCCCAGGCACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCC
CCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG
TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC
AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC
AGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG
AATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAG
TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG
TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG
TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT
ACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC
CCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC
AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG
TAAATGA (SEQ ID NO: 354).
In a further embodiment described, polynucleotides encoding antibody fragments
for treatment or prevention of pain and pain associated conditions having binding specificity to
NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ
ID NO: 355; SEQ ID NO: 356; and SEQ ID NO: 357 which correspond to polynucleotides
encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light
chain variable sequence of SEQ ID NO: 151 or the light chain sequence of SEQ ID NO: 152.
In a further embodiment described, polynucleotides encoding antibody fragments
for treatment or prevention of pain and pain associated conditions having binding specificity to
NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ
ID NO: 358; SEQ ID NO: 359; and SEQ ID NO: 360 which correspond to polynucleotides
encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the
heavy chain variable sequence of SEQ ID NO: 153 or the heavy chain sequence of SEQ ID NO:
154.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 351 encoding the light chain variable sequence of SEQ ID NO: 151; the
polynucleotide SEQ ID NO: 352 encoding the light chain sequence of SEQ ID NO: 152; the
polynucleotide SEQ ID NO: 353 encoding the heavy chain variable sequence of SEQ ID NO:
153; the polynucleotide SEQ ID NO: 354 encoding the heavy chain sequence of SEQ ID NO:
154; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 355;
SEQ ID NO: 356; and SEQ ID NO: 357) of the light chain variable sequence of SEQ ID NO:
151 or the light chain sequence of SEQ ID NO: 152; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 358; SEQ ID NO: 359; and SEQ ID NO:
360) of the heavy chain variable sequence of SEQ ID NO: 153 or the heavy chain sequence of
SEQ ID NO: 154.
In a preferred embodiment described, polynucleotides described comprise, or
alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments
having binding specificity for NGF. With respect to antibody Ab16, the polynucleotides
encoding the full length Ab16 antibody comprise, or alternatively consist of, the polynucleotide
SEQ ID NO: 352 encoding the light chain sequence of SEQ ID NO: 152 and the polynucleotide
SEQ ID NO: 354 encoding the heavy chain sequence of SEQ ID NO: 154.
Another embodiment described contemplates these polynucleotides incorporated
into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in
fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable
Pichia species include, but are not limited to, Pichia pastoris. In one embodiment described
herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab16
following expression of the full-length polynucleotides in a suitable host. In another
embodiment described, anti-NGF antibodies such as Ab16 or Fab fragments thereof may be
produced via expression of Ab16 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example
diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab17
Further described is the use of polynucleotides set forth below to produce
antibody Ab17 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA without appreciably inhibiting the association of NGF with p75, in methods of
treating pain in an individual comprising administering to said individual antibody Ab17
polypeptides. Further described are polynucleotides encoding antibody polypeptides for
treatment or prevention of pain and pain associated conditions having binding specificity to
NGF. In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the variable light chain
polypeptide sequence of SEQ ID NO: 161:
GCCATCGAAATGACCCAGACTCCATTCTCCGTGTCTGCAGCTGTGGGA
GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGACCATTAGCAACTACTTAGCCTG
GTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCAATCT
GGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCT
CACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGGTTA
TACTATCAGTAATGTTGATAACAATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGT (SEQ ID NO: 361).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 162:
GCCATCGAAATGACCCAGACTCCATTCTCCGTGTCTGCAGCTGTGGGA
GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGACCATTAGCAACTACTTAGCCTG
GTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCAATCT
GGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCT
CACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGGTTA
TACTATCAGTAATGTTGATAACAATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 362).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 163:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGGGATC
CCTGACACTCACCTGCGCAGCCTCTGGATTCTCCCTCACTGGCTACAACTTGGTCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATTCATTAGTTATGGTG
ATACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGACTCTGACGATCACCGATCTGCAACCTTCAGACACGGGCACCTATTTC
TGTGCCAGAGAGACTGCTAATACTTATGATTATGGCATCTGGGGCCCAGGCACCCTC
GTCACCGTCTCGAGC (SEQ ID NO: 363).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 164:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGGGATC
CCTGACACTCACCTGCGCAGCCTCTGGATTCTCCCTCACTGGCTACAACTTGGTCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATTCATTAGTTATGGTG
ATACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGACTCTGACGATCACCGATCTGCAACCTTCAGACACGGGCACCTATTTC
TGTGCCAGAGAGACTGCTAATACTTATGATTATGGCATCTGGGGCCCAGGCACCCTC
GTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC
TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTT
CCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACA
CCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACA
TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC
CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTA
CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT
ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC
AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT
GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ
ID NO: 364).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 365; SEQ ID NO: 366; and SEQ ID NO: 367 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 161 or the light chain sequence of
SEQ ID NO: 162.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 368; SEQ ID NO: 369; and SEQ ID NO: 370 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 163 or the heavy chain sequence
of SEQ ID NO: 164.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 361 encoding the light chain variable sequence of SEQ ID NO: 161; the
polynucleotide SEQ ID NO: 362 encoding the light chain sequence of SEQ ID NO: 162; the
polynucleotide SEQ ID NO: 363 encoding the heavy chain variable sequence of SEQ ID NO:
163; the polynucleotide SEQ ID NO: 364 encoding the heavy chain sequence of SEQ ID NO:
164; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 365;
SEQ ID NO: 366; and SEQ ID NO: 367) of the light chain variable sequence of SEQ ID NO:
161 or the light chain sequence of SEQ ID NO: 162; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 368; SEQ ID NO: 369; and SEQ ID NO:
370) of the heavy chain variable sequence of SEQ ID NO: 163 or the heavy chain sequence of
SEQ ID NO: 164.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab17, the
polynucleotides encoding the full length Ab17 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 362 encoding the light chain sequence of SEQ ID NO: 162 and the
polynucleotide SEQ ID NO: 364 encoding the heavy chain sequence of SEQ ID NO: 164.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one optional
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab17 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab17 or Fab fragments thereof
may be produced via expression of Ab17 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab18
Further described is the use of polynucleotides set forth below to produce
antibody Ab18 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA and the association of NGF with p75, in methods of treating pain in an
individual comprising administering to said individual antibody Ab18 polypeptides. Further
described are polynucleotides encoding antibody polypeptides for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF. In one optional
embodiment described, polynucleotides described comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence of
SEQ ID NO: 171:
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGTCAGGCTAGTCAGACCATTAGCAACTACTTAGCCTGG
TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAATCTG
GAATCTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGAACAGAATTCACTCTC
ACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGTCAACAGGGTTAT
ACTATCAGTAATGTTGATAACAATGTTTTCGGCGGAGGAACCAAGGTGGAAATCAA
ACGT (SEQ ID NO: 371).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 172:
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGTCAGGCTAGTCAGACCATTAGCAACTACTTAGCCTGG
TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAATCTG
GAATCTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGAACAGAATTCACTCTC
ACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGTCAACAGGGTTAT
ACTATCAGTAATGTTGATAACAATGTTTTCGGCGGAGGAACCAAGGTGGAAATCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 372).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 173:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTGGCTACAACTTGGT
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGATTCATTAGTTATG
GTGATACCACATACTACGCTAGCTCTGCTAAAGGCCGATTCACCATCTCCAGAGACA
ATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCTG
TGTATTACTGTGCTAGAGAGACTGCTAATACTTATGATTATGGCATCTGGGGCCAAG
GGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 373).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 174:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTGGCTACAACTTGGT
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGATTCATTAGTTATG
GTGATACCACATACTACGCTAGCTCTGCTAAAGGCCGATTCACCATCTCCAGAGACA
ATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCTG
TGTATTACTGTGCTAGAGAGACTGCTAATACTTATGATTATGGCATCTGGGGCCAAG
GGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG
CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG
GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT
GGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAA
CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC
TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCA
GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA
AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATC
CCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCT
ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC
ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA
TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATG
A (SEQ ID NO: 374).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 375; SEQ ID NO: 376; and SEQ ID NO: 377 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 171 or the light chain sequence of
SEQ ID NO: 172.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 378; SEQ ID NO: 379; and SEQ ID NO: 380 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 173 or the heavy chain sequence
of SEQ ID NO: 174.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one optional embodiment described,
polynucleotides encoding antibody fragments for treatment or prevention of pain and pain
associated conditions having binding specificity to NGF comprise, or alternatively consist of,
one, two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 371 encoding the light chain variable sequence of
SEQ ID NO: 171; the polynucleotide SEQ ID NO: 372 encoding the light chain sequence of
SEQ ID NO: 172; the polynucleotide SEQ ID NO: 373 encoding the heavy chain variable
sequence of SEQ ID NO: 173; the polynucleotide SEQ ID NO: 374 encoding the heavy chain
sequence of SEQ ID NO: 174; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 375; SEQ ID NO: 376; and SEQ ID NO: 377) of the light chain variable
sequence of SEQ ID NO: 171 or the light chain sequence of SEQ ID NO: 172; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 378; SEQ ID
NO: 379; and SEQ ID NO: 380) of the heavy chain variable sequence of SEQ ID NO: 173 or the
heavy chain sequence of SEQ ID NO: 174.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity for NGF. With respect to antibody Ab18, the polynucleotides encoding the full length
Ab18 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 372
encoding the light chain sequence of SEQ ID NO: 172 and the polynucleotide SEQ ID NO: 374
encoding the heavy chain sequence of SEQ ID NO: 174.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab18 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab18 or Fab fragments thereof
may be produced via expression of Ab18 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab19
Further described is the use of polynucleotides set forth below to produce
antibody Ab19 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA and the association of NGF with p75. in methods of treating pain in an
individual comprising administering to said individual antibody Ab19 polypeptides. Further
described are polynucleotides encoding antibody polypeptides for treatment or prevention of
pain and pain associated conditions having binding specificity to NGF. In one embodiment
described, polynucleotides described comprise, or alternatively consist of, the following
polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO:
181:
GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGA
GGCACAGTCAGCATCAGTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTATTTA
TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCTTCC
ACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCGACGTGCAGTGTGACGCTGCTGCCACTTACTACTGTGCAGGC
GGTTATAGTAGTAGTAGTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGT (SEQ ID NO: 381).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 182:
GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGA
GGCACAGTCAGCATCAGTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTATTTA
TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCTTCC
ACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCGACGTGCAGTGTGACGCTGCTGCCACTTACTACTGTGCAGGC
GGTTATAGTAGTAGTAGTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 382).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 183:
CAGTCGGTGGAGGCGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCC
CTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACCTACTGGATGTCCTGG
GTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGACATTTATTTTAGTAA
TGAGGAAACAAACTACGCGACCTGGGCGAAAGGCCGATTTACCATCTCCAAAACCT
CGACCACGGTGGATCTGAATGTCATCAGTCCGACAACCGAGGACACGGCCACCTAT
TTCTGTGCAAGAGGTTCTCCTGATGTTGAGATTGCTATAGATATGTGGGGCCAGGGC
ACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 383).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 184:
CAGTCGGTGGAGGCGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCC
CTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACCTACTGGATGTCCTGG
GTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGACATTTATTTTAGTAA
TGAGGAAACAAACTACGCGACCTGGGCGAAAGGCCGATTTACCATCTCCAAAACCT
CGACCACGGTGGATCTGAATGTCATCAGTCCGACAACCGAGGACACGGCCACCTAT
TTCTGTGCAAGAGGTTCTCCTGATGTTGAGATTGCTATAGATATGTGGGGCCAGGGC
ACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCA
CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACA
AGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT
CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC
TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA
CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT
CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCC
GGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATG
A (SEQ ID NO: 384).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 385; SEQ ID NO: 386; and SEQ ID NO: 387 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 181 or the light chain sequence of
SEQ ID NO: 182.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 388; SEQ ID NO: 389; and SEQ ID NO: 390 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 183 or the heavy chain sequence
of SEQ ID NO: 184.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 381 encoding the light chain variable sequence of SEQ ID NO: 181; the
polynucleotide SEQ ID NO: 382 encoding the light chain sequence of SEQ ID NO: 182; the
polynucleotide SEQ ID NO: 383 encoding the heavy chain variable sequence of SEQ ID NO:
183; the polynucleotide SEQ ID NO: 384 encoding the heavy chain sequence of SEQ ID NO:
184; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 385;
SEQ ID NO: 386; and SEQ ID NO: 387) of the light chain variable sequence of SEQ ID NO:
181 or the light chain sequence of SEQ ID NO: 182; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 388; SEQ ID NO: 389; and SEQ ID NO:
390) of the heavy chain variable sequence of SEQ ID NO: 183 or the heavy chain sequence of
SEQ ID NO: 184.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab19, the
polynucleotides encoding the full length Ab19 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 382 encoding the light chain sequence of SEQ ID NO: 182 and the
polynucleotide SEQ ID NO: 384 encoding the heavy chain sequence of SEQ ID NO: 184.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one optional
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab19 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab19 or Fab fragments thereof
may be produced via expression of Ab19 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab20
Further described is the use of polynucleotides set forth below to produce
antibody Ab20 polypeptides for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF, which inhibits the association of NGF with TrkA and the
association of NGF with p75, in methods of treating pain in an individual comprising
administering to said individual antibody Ab20 polypeptides. Further described are
polynucleotides encoding antibody polypeptides having binding specificity to NGF. In one
optional embodiment described, polynucleotides described comprise, or alternatively consist of,
the following polynucleotide sequence encoding the variable light chain polypeptide sequence of
SEQ ID NO: 191:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC
ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGC
GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAA
ACGT (SEQ ID NO: 391).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 192:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTACTTA
TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC
ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGC
GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAA
ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 392).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 193:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTACCTACTGGATGAG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGACATTTACTTTA
GTAATGAAGAAACAAACTACGCGACCAGCGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACAC
TGCTGTGTATTACTGTGCTAGAGGTTCTCCTGATGTTGAGATTGCTATAGATATGTGG
GGCCAAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 393).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 194:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTACCTACTGGATGAG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGACATTTACTTTA
GTAATGAAGAAACAAACTACGCGACCAGCGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACAC
TGCTGTGTATTACTGTGCTAGAGGTTCTCCTGATGTTGAGATTGCTATAGATATGTGG
GGCCAAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTC
CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG
GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC
CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT
GAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTG
ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG
GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC
CCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG
CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG
TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAATGA (SEQ ID NO: 394).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 395; SEQ ID NO: 396; and SEQ ID NO: 397 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 191 or the light chain sequence of
SEQ ID NO: 192.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 398; SEQ ID NO: 399; and SEQ ID NO: 400 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 193 or the heavy chain sequence
of SEQ ID NO: 194.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 391 encoding the light chain variable sequence of SEQ ID NO: 191; the
polynucleotide SEQ ID NO: 392 encoding the light chain sequence of SEQ ID NO: 192; the
polynucleotide SEQ ID NO: 393 encoding the heavy chain variable sequence of SEQ ID NO:
193; the polynucleotide SEQ ID NO: 394 encoding the heavy chain sequence of SEQ ID NO:
194; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 395;
SEQ ID NO: 396; and SEQ ID NO: 397) of the light chain variable sequence of SEQ ID NO:
191 or the light chain sequence of SEQ ID NO: 192; and polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 398; SEQ ID NO: 399; and SEQ ID NO:
400) of the heavy chain variable sequence of SEQ ID NO: 193 or the heavy chain sequence of
SEQ ID NO: 194.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab20, the
polynucleotides encoding the full length Ab20 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 392 encoding the light chain sequence of SEQ ID NO: 192 and the
polynucleotide SEQ ID NO: 394 encoding the heavy chain sequence of SEQ ID NO: 194.
Another optional embodiment described contemplates these polynucleotides
incorporated into an expression vector for expression in mammalian cells such as CHO, NSO,
HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast
Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one
embodiment described herein (infra), Fab fragments may be produced by enzymatic digestion
(e.g., papain) of Ab20 following expression of the full-length polynucleotides in a suitable host.
In another embodiment described, anti-NGF antibodies such as Ab20 or Fab fragments thereof
may be produced via expression of Ab20 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species
include, but are not limited to, Pichia pastoris.
Antibody Ab21
Further described is the use of polynucleotides set forth below to produce
antibody Ab21 polypeptides having binding specificity to NGF, which inhibit the association of
NGF with TrkA and the association of NGF with p75 in methods of treating pain in an individual
comprising administering to said individual antibody Ab21 polypeptides. Further described are
polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain
associated conditions having binding specificity to NGF. In one embodiment described,
polynucleotides described comprise, or alternatively consist of, the following polynucleotide
sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 51:
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG
TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG
GAATCTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTACACTCT
CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAACAGGGTTT
TACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC
GT (SEQ ID NO: 251).
In one embodiment described, polynucleotides described optionally comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 401:
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG
TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG
GAATCTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTACACTCT
CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAACAGGGTTT
TACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC
GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT
CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA
AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA
GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 403).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the variable heavy
chain polypeptide sequence of SEQ ID NO: 53:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAATCATTGGTCGTA
ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC
AATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCT
GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCT
GGGGCCCAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 253).
In one optional embodiment described, polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 402:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAATCATTGGTCGTA
ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC
AATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCT
GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTACTACGTCTTTAACATCT
GGGGCCCAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCT
TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC
TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC
AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA
CGTGAATCACAAGCCCAGCAACACCAAGGTGGACGCGAGAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG
TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA
CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC
AGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT
GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA
AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC
AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC
GGGTAAATGA (SEQ ID NO: 404).
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 51 or the light chain sequence of
SEQ ID NO: 401.
In a further optional embodiment described, polynucleotides encoding antibody
fragments for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide
sequences of SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260 which correspond to
polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of
SEQ ID NO: 402.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 251 encoding the light chain variable sequence of SEQ ID NO: 51; the
polynucleotide SEQ ID NO: 403 encoding the light chain sequence of SEQ ID NO: 401; the
polynucleotide SEQ ID NO: 253 encoding the heavy chain variable sequence of SEQ ID NO: 53;
the polynucleotide SEQ ID NO: 404 encoding the heavy chain sequence of SEQ ID NO: 402;
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 255; SEQ ID
NO: 256; and SEQ ID NO: 257) of the light chain variable sequence of SEQ ID NO: 51 or the
light chain sequence of SEQ ID NO: 401; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260) of the heavy
chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 402.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody Ab21, the
polynucleotides encoding the full length Ab21 antibody comprise, or alternatively consist of, the
polynucleotide SEQ ID NO: 403 encoding the light chain sequence of SEQ ID NO: 401 and the
polynucleotide SEQ ID NO: 404 encoding the heavy chain sequence of SEQ ID NO: 402.
Another optional embodiment described contemplates these polynucleotides incorporated into an
expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal,
insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia
species include, but are not limited to, Pichia pastoris. In one optional embodiment described
herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab21
following expression of the full-length polynucleotides in a suitable host. In another
embodiment described, anti-NGF antibodies such as Ab21 or Fab fragments thereof may be
produced via expression of Ab21 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not
limited to, Pichia pastoris.
Antibody fragment Fab2
Further described is the use of polynucleotides set forth below to produce
antibody fragment Fab2 polypeptides that inhibit the association of NGF with TrkA and p75 for
treatment or prevention of pain and pain associated conditions having binding specificity to NGF
in methods of treating pain in an individual comprising administering to said individual antibody
Ab1 polypeptides . Further described are polynucleotides encoding antibody fragment
polypeptides for treatment or prevention of pain and pain associated conditions having binding
specificity to NGF. In one embodiment described, Fab polynucleotides described comprise, or
alternatively consist of, the following polynucleotide sequence encoding the light chain
polypeptide sequence of SEQ ID NO: 407:
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG
TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG
GAATCTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTACACTCT
CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAACAGGGTTT
TACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC
GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT
CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA
AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA
GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 409).
In another optional embodiment described, polynucleotides described comprise,
or alternatively consist of, the following polynucleotide sequence encoding the heavy chain
polypeptide sequence of SEQ ID NO: 408:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG
CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAATCATTGGTCGTA
ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC
AATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCT
GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTACTACGTCTTTAACATCT
GGGGCCCAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCT
TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC
TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC
AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA
CGTGAATCACAAGCCCAGCAACACCAAGGTGGACGCGAGAGTTGAGCCCAAATCTT
GTGACAAAACTCACTAG (SEQ ID NO: 410).
In a further optional embodiment described, polynucleotides encoding Fab
antibody fragments having binding specificity to NGF comprise one or more of the
polynucleotide sequences of SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257 which
correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or
hypervariable regions) of the light chain variable sequence of SEQ ID NO: 51 or the light chain
sequence of SEQ ID NO: 409.
In a further optional embodiment described, polynucleotides encoding Fab
antibody fragments for treatment or prevention of pain and pain associated conditions having
binding specificity to NGF comprise one or more of the polynucleotide sequences of SEQ ID
NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260 which correspond to polynucleotides encoding
the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain
variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 410.
Also described are polynucleotide sequences including one or more of the
polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and
pain associated conditions described herein. In one embodiment described, polynucleotides
encoding antibody fragments for treatment or prevention of pain and pain associated conditions
having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more,
including all of the following polynucleotides encoding antibody fragments: the polynucleotide
SEQ ID NO: 251 encoding the light chain variable sequence of SEQ ID NO: 51; the
polynucleotide SEQ ID NO: 409 encoding the light chain sequence of SEQ ID NO: 407; the
polynucleotide SEQ ID NO: 253 encoding the heavy chain variable sequence of SEQ ID NO: 53;
the polynucleotide SEQ ID NO: 410 encoding the heavy chain sequence of SEQ ID NO: 408;
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 255; SEQ ID
NO: 256; and SEQ ID NO: 257) of the light chain variable sequence of SEQ ID NO: 51 or the
light chain sequence of SEQ ID NO: 407; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260) of the heavy
chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 408.
In a preferred optional embodiment described, polynucleotides described
comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding)
fragments having binding specificity for NGF. With respect to antibody fragment Fab2, the
polynucleotides encoding the Fab fragment include the polynucleotide SEQ ID NO: 409
encoding the light chain sequence of SEQ ID NO: 407 and the polynucleotide SEQ ID NO: 410
encoding the heavy chain sequence of SEQ ID NO: 408.
Another embodiment described contemplates these polynucleotides incorporated
into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in
fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable
Pichia species include, but are not limited to, Pichia pastoris. In one embodiment described
herein (infra), Fab fragments may be produced via expression of Fab2 polynucleotides in
mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial
systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast
strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
In one embodiment, described is an isolated polynucleotide comprising a
polynucleotide encoding an anti-NGF V antibody amino acid sequence selected from SEQ ID
NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, or
402, or encoding a variant thereof wherein at least one framework residue (FR residue) has been
substituted with an amino acid present at the corresponding position in a rabbit anti-NGF
antibody V polypeptide or a conservative amino acid substitution.
In another optional embodiment, described is an isolated polynucleotide
comprising the polynucleotide sequence encoding an anti-NGF V antibody amino acid sequence
of 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, or 401,
or encoding a variant thereof wherein at least one framework residue (FR residue) has been
substituted with an amino acid present at the corresponding position in a rabbit anti-NGF
antibody V polypeptide or a conservative amino acid substitution.
In yet another optional embodiment, described are one or more heterologous
polynucleotides comprising a sequence encoding the polypeptides contained in SEQ ID NO:1
and SEQ ID NO:3; SEQ ID NO:11 and SEQ ID NO:13; SEQ ID NO:21 and SEQ ID NO:23;
SEQ ID NO:31 and SEQ ID NO:33; SEQ ID NO:411 and SEQ ID NO:43; SEQ ID NO:51 and
SEQ ID NO:53, SEQ ID NO:61 and SEQ ID NO:63; SEQ ID NO:71 and SEQ ID NO:73; SEQ
ID NO:81 and SEQ ID NO:83; SEQ ID NO:91 and SEQ ID NO:93; SEQ ID NO:101 and SEQ
ID NO:103; SEQ ID NO:111 and SEQ ID NO:113; SEQ ID NO:121 and SEQ ID NO:123; SEQ
ID NO:131 and SEQ ID NO:133; SEQ ID NO:141 and SEQ ID NO:143; SEQ ID NO:151 and
SEQ ID NO:153; SEQ ID NO:161 and SEQ ID NO:163; SEQ ID NO:171 and SEQ ID NO:173;
SEQ ID NO:181 and SEQ ID NO:183; SEQ ID NO:191 and SEQ ID NO:193; or SEQ ID
NO:401 and SEQ ID NO:403.
In another embodiment, described is an isolated polynucleotide that expresses a
polypeptide containing at least one CDR polypeptide derived from an anti-NGF antibody
wherein said expressed polypeptide alone specifically binds NGF or specifically binds NGF
when expressed in association with another polynucleotide sequence that expresses a polypeptide
containing at least one CDR polypeptide derived from an anti-NGF antibody for treatment or
prevention of pain and pain associated conditions wherein said at least one CDR is selected from
those contained in the V or V polypeptides of SEQ ID NO: 1, 3, 11, 13, 21, 23, 31, 33, 41, 43,
51, 53, 61, 63, 71, 73, 81, 83, 91, 93, 101, 103, 111, 113, 121, 123, 131, 133, 141, 143, 151, 153,
161, 163, 171, 173, 181, 183, 191, 193, 401 or SEQ ID NO:403.
Host cells and vectors comprising said polynucleotides are also described.
Further described are vectors comprising the polynucleotide sequences encoding
the variable heavy and light chain polypeptide sequences, as well as the individual
complementarity-determining regions (CDRs, or hypervariable regions), as set forth herein, as
well as host cells comprising said vector sequences. In one embodiment described, the host cell
is a yeast cell. In another embodiment described, the yeast host cell belongs to the genus Pichia.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in
the art with a complete disclosure and description of how to make and use the subject invention,
and are not intended to limit the scope of what is regarded as the invention. Efforts have been
made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature,
concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless
otherwise indicated, parts are parts by weight, molecular weight is average molecular weight,
temperature is in degrees centigrade; and pressure is at or near atmospheric.
EXAMPLE 1
This example describes culture methods that improved the purity of recombinant
antibodies produced from cultured P. pastoris cells. When a bolus of ethanol was added during
culturing, the resulting antibodies exhibited a great decrease in the concentration of an undesired
product-associated variant.
Methods
To generate the inoculum, diploid P. pastoris was grown using a medium
composed of the following nutrients (percentages are given as w/v): yeast extract 3%, anhydrous
dextrose 2%, YNB 1.34%, Biotin 0.004% and 100 mM potassium phosphate (pH 6.0). The
inoculum medium for runs L355, L357, L358, L359 and L360 was composed of the following
nutrients (percentages are given as w/v): yeast extract 3%, glycerol 2%, YNB 1.34%, Biotin
0.004%, 200 mM potassium phosphate (pH 6.0) The inoculum was grown for approximately 24
hours to 29 hours in a shaking incubator at 30ºC and 300 rpm. A 10% inoculum was then added
to Labfors 2.5L working volume vessels containing 1 L sterile growth medium. The growth
medium was composed of the following nutrients: potassium sulfate 18.2 g/L, ammonium
phosphate monobasic 36.4 g/L, potassium phosphate dibasic 12.8 g/L, magnesium sulfate
heptahydrate 3.72 g/L, sodium citrate dihydrate 10 g/L, glycerol 40 g/L, yeast extract 30 g/L,
PTM1 trace metals 4.35 mL/L, and antifoam 204 1.67 mL/L. The PTM1 trace metal solution
was comprised of the following components: cupric sulfate pentahydrate 6 g/L, sodium iodide
0.08 g/L, manganese sulfate hydrate 3 g/L, sodium molybdate dihyrate 0.2 g/L, boric acid 0.02
g/L, cobalt chloride 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65 g/L, biotin 0.2
g/L, and sulfuric acid 5 mL/L. The yeast strain was engineered to express Ab-A antibody from
four integrated genomic copies of the heavy chain coding sequence (SEQ ID NO: 441) and 3
copies of the light chain coding sequence (SEQ ID NO: 440). The heavy chain gene copies were
integrated into the pGAP locus (3 copies) and HIS4 TT locus (1 copy) while the 3 light chain
gene copies were integrated into the pGAP locus. The antibody chain gene copies were each
under the control of the GAP promoter. The bioreactor process control parameters were set as
follows: Agitation 1000 rpm, airflow 1.35 standard liter per minute, temperature 28ºC and pH
was controlled (at 6) using ammonium hydroxide. No oxygen supplementation was provided.
Following addition of the inoculum, fermentation cultures were grown for
approximately 12 to 16 hours (the “growth phase”). The growth phase ended when the initial
glycerol in the medium was consumed, as which was detected by a dissolved oxygen (“DO”)
spike (a sudden increase in the dissolved oxygen concentration). The cultures were then starved
for approximately three hours after the dissolved oxygen spike (“starvation phase”) for run L306.
For other runs, the ethanol bolus was added immediately after DO spike. A bolus of ethanol was
then added to the reactor to give a final concentration of 1% ethanol (w/v). Control cultures
were treated identically, except that the bolus addition of ethanol was omitted. The fermentation
cultures were allowed to equilibrate for 15 to 30 minutes (“equilibration phase”). After the
equilibration phase, feed was added at a constant rate of 30 g/L/hr for 40 minutes (“transition
phase”). For the remainder of the culture (“production phase”) the ethanol concentration was
detected using an ethanol sensing probe (Raven Biotech) which was used to control the feed rate,
with the feed rate being set at 15 g/L/hr when the ethanol concentration was below the set point,
or 7.5 g/L/hr when the ethanol concentration was above the set point. In instances in which the
high feed rate of 15 g/L/hr was not high enough to maintain ethanol at set point (which occurred
in the L315 fermentation run), the high feed rate was set to 22.5 g/L/hr while the low feed rate
was set to 15 g/L/hr. The same set point was maintained whether or not an ethanol bolus had
been added to the culture (production of ethanol by the yeast caused the set point to be reached
without the bolus addition of ethanol). The feed was composed of the following components:
yeast extract 50 g/L, anhydrous dextrose 500 g/L, magnesium sulfate heptahydrate 3 g/L, PTM1
trace metals 12 mL/L, and sodium citrate dihydrate 0.5g/L The total fermentation time was
typically 85 hours to 97 hours in these experiments, though longer and shorter times can also be
used.
After the production phase, fermentation cultures had PEI (polyethyleneimine)
and EDTA (ethylenediaminetetraacetic acid) added to 0.05% w/v and 3 mM final concentrations
respectively. The cultures were then spun in a centrifuge and antibodies were purified from the
culture supernatant by Protein A affinity. Briefly, approximately 20 mL of 0.2m clarified
supernatants from harvested fermentation broth were diluted with the same volume of
equilibration buffer (20 mM Histidine pH6). From this diluted broth, 20 mL were then loaded
onto a pre-equilibrated 1 mL HiTrap MabSelect Sure column (GE, Piscataway, NJ). The column
was subsequently washed using 40 column volumes of equilibration buffer. The antibody bound
onto the column was eluted using a step gradient into 100% elution buffer (100 mM Citric Acid
pH 3.0). One mL fractions were collected and immediately neutralized using 100mL of 2M Tris
buffer pH 8.0. Protein containing fractions were determined by measuring absorbance at 280nM
and protein-containing fractions were pooled.
Protein A purified antibodies were analyzed for purity by SDS-PAGE. For non-
reduced samples, SDS-PAGE was carried out using precast polyacrylamide gels (NuPAGE®
Bis-Tris Gels) containing a 4%-12% polyacrylamide gradient, using NuPAGE® MES SDS
running buffer and NuPAGE® LDS Sample Buffer (all from Invitrogen, Carlsbad, Ca.) in accord
with the manufacturer’s instructions. Proteins were then visualized by Coomassie blue staining.
Reduced samples were processed in the same manner except that samples were reduced prior to
loading using the NuPAGE® Sample Reducing Agent (Invitrogen, Carlsbad, Ca.) in accord with
the manufacturer’s instructions.
Results
To determine the effect of a bolus addition of ethanol during culture on antibody
purity, the antibody Ab-A was produced from yeast cultures with or without the addition of a
bolus of ethanol to a final concentration of 1% (10 g/L) at the end of the growth phase and prior
to the production phase. The production phase was continued for 97 hours (, 87 hours
(, or 86 hours (. The antibody produced by each culture was then harvested from
the culture media, purified by Protein A affinity chromatography.
SDS-PAGE was used to detect the relative abundance of the full antibody, the
“half antibody” or H1L1 complex (containing one heavy and one light chain) and the H2L1
complex (containing two heavy chains and one light chain). The abundance of the H1L1 and
H2L1 complexes was greatly decreased in the cultures that were produced with the bolus
addition of ethanol. This improvement was reproduced in three experiments shown in ,
compare lanes 2 and 3 (with bolus) to 5 (no bolus); , compare lane 2 (with bolus) to 3
(no bolus); , compare lanes 2 and 4 (with bolus) to lanes 5-7 (no bolus). Under reducing
conditions, the H1L1, H2L1, and full antibody species were each separated into individual heavy
and light chains, confirming the identity of the 75 kDa band as consisting of one light and one
heavy chain joined by a disulfide-linkage (FIGS. 1B and 2B; lane order is the same as FIGS. 1A
and 1B, respectively).
The decrease in abundance of the H1L1 species was then quantified using ImageJ
to plot the gel band density along the length of the non-reduced gels (FIGS. 1C-E and 2C-D,
respectively corresponding to , lanes 2, 3, and 5, and , lanes 2 and 3, and , corresponding to lanes 2 and 4-6 of ). The area under the H1L1 peak was quantified
and results are tabulated in FIGS. 1F, 2E, and 3B. Based on these measurements, the addition of
an ethanol bolus prior to antibody production decreased the relative abundance of the 75 kDa
band by about 90% in , by about 85% in , and by about 87% in .
In summary, these results demonstrate that the concentration of the H1L1 species
was greatly decreased by the bolus addition of ethanol to the culture, resulting in decreased
production of the H1L1 species between about 85% to 90%.
EXAMPLE 2
This example extends the results obtained in Example 1 by demonstrating that the
same methods produced a similar improvement in antibody purity when used during production
of two additional antibodies.
Methods
Antibodies Ab-B and Ab-C were recombinantly produced using the methods
described in Example 1. Antibody Ab-C was expressed from a yeast strain engineered contain
four copies of the heavy chain coding sequence (SEQ ID NO: 439) and three copies of the light
chain coding sequence (SEQ ID NO: 438). Samples were taken from the reactors and culture
supernatant containing antibodies was collected after a total fermentation time of 67 hours (T67)
or 87 hours (T87) for the Ab-B antibody and for 86 hours (T86) for the Ab-C antibody, purified
by Protein A affinity, and analyzed by SDS-PAGE as described in Example 1.
Results
Antibodies Ab-B ( and Ab-C ( were produced with or without a
bolus addition of ethanol to a final concentration of 1% at the end of the growth phase and prior
to the production phase. For the antibodies produced without the bolus addition of ethanol, the
H1L1 or half antibody species, and the H2L1 species were each observed as a prominent band
(, lines 6 and 7; , lanes 6 and 7; , lanes 5 and 6). The intensity of these
bands were greatly decreased for the culture produced with a bolus addition of ethanol (,
lanes 2-3; , lanes 2-3; , lane 3). Under reducing conditions, the H1L1 and H2L1
bands were separated into individual heavy and light chains, confirming the identity of these
species as consisting full length heavy and light chains (FIGS. 4B, 4D, and 5B; lane order is the
same as in FIGS. 4A, 4D, and 5A, respectively).
The decrease in abundance of the H1L1 species was then quantified using ImageJ
to plot the gel band density along the length of the non-reduced gels. FIGS. 4E and 4F tabulate
the area contained in the H1L1 peaks shown in FIGS. 4A (T67) and 4C (T87), respectively,
demonstrating that the bolus addition of ethanol produced about a 73% reduction in the relative
abundance of H1L1 complexes at the earlier time point shown and about a 34% average
reduction in the relative abundance of H1L1 complexes at the later time point shown in .
Similarly, tabulates the area contained in the H1L1 peaks shown in FIGS. 5A,
demonstrating about a 61% average reduction in the relative abundance of H1L1 complexes by
the bolus addition of ethanol.
In summary, these results demonstrate that the concentration of the H1L1 and
H2L1 species were decreased by between about 61% and 73% by the bolus addition of ethanol to
the culture for two additional antibodies having binding specificity for different targets.
EXAMPLE 3
This example further describes the improved purity of recombinant antibodies
produced from cultured P. pastoris cells through addition of a bolus of ethanol during culturing.
In addition to greatly decreasing the abundance of the H1L1 and H2L1 species, this example
further demonstrates a decrease in the concentration of other product-associated variants.
Methods
Recombinant antibodies Ab-A, Ab-B, and Ab-C were prepared and purified from
P. pastoris cultures as described in Examples 1 and 2. Antibodies were produced either with or
without a bolus addition of ethanol to a final concentration of 1% (w/v) at the end of the growth
phase and prior to the production phase. To analyze the purity of protein A purified antibody
preparations, size exclusion high-performance liquid chromatography (SE-HPLC) was used.
Briefly, an Agilent (Santa Clara, CA) 1200 Series HPLC with UV detection instrument was
used. For sample separation, a TSKgel 3000SW 7.8x300 mm column connected with a
TSKgel Guard SW 6x40 mm from Tosoh Bioscience (King of Prussia, PA) was used. A
solution of 100 mM sodium phosphate, 200 mM sodium chloride pH 6.5 was used as mobile
phase with a flow rate of 0.5 mL/min in isocratic mode and absorbance at UV 215nm was
monitored. Before injection of samples the column was equilibrated until a stable baseline was
achieved. Samples were diluted to a concentration of 1 mg/mL using mobile phase and a 30 mL
volume was injected. To monitor column performance, BioRad (Hercules, CA) gel filtration
standards were used.
Results
The Ab-A antibody preparations described in Example 1 and additional
preparations produced using the same methods were expressed in yeast, purified by protein A
affinity, and then analyzed for purity using size exclusion chromatography (SEC). Under the
condition used, the half antibody (H1L1) species co-elutes with the full antibody, which is
thought to be due to non-covalent association between pairs of half antibodies. However, this
method allows purity to be assessed with respect to other product-associated variants, such
complexes having aberrant stoichiometry, fragments, glycosylated forms, and aggregates.
SE-HPLC data are shown for Ab-A samples produced without (FIGS. 6A, 6C,
and 6E) or with (FIGS. 6B, 6D, and 6F) a bolus addition of ethanol. A product-associated
variant consisting of an aggregate of two full antibodies (containing four heavy and four light
chains) was detected (arrow), and the abundance thereof was reduced on average in the samples
prepared with the bolus addition. shows quantification of the purity of Ab-A by
determining the percentage of the antibody preparation contained in the main peak (containing
the full antibody). The bolus addition of ethanol increased the average percentage contained in
the main peak from 80.3% up to 90.6%.
Similar analysis was performed for the Ab-B and Ab-C antibody preparations
described in Example 2, quantified in FIGS. 8 and 9, respectively. Overall purity of the Ab-B
antibody was improved, with the average fraction in the main peak increasing from 76% to 79%
at T67 and from 60% to 73% at T87. For the Ab-C antibody, there was little detectable
difference in antibody purity assessed by this method, apparently due to the high initial purity of
the Ab-C antibody even without the bolus addition.
In summary, these results demonstrate that a bolus addition of ethanol to the
culture can decrease the concentration of other product-associated variants in addition to the
half-antibody species.
EXAMPLE 4
This example describes the further confirmation of the identity of the 75 kDa
product associated variant as a half antibody species containing only one antibody heavy chain
and only one antibody light chain. This hypothesis was based on several observations. First, the
75 kDa band was present in protein A purified samples (see Example 1) indicating that it
contained at least the protein A binding portion of an antibody heavy chain. Second, the 75 kDa
band was prominent in non-reduced samples analyzed by SDS-PAGE (see, e.g., , lanes
2-3), but under reducing conditions the same samples did not contain any bands of comparable
intensity (other than the expected light and heavy chains), indicating that the 75 kDa band does
not include any components other than antibody heavy and light chains (or other species having
the same electrophoretic mobility). Third, disappearance of the 75 kDa band from the reduced
samples also indicates that its constituents are linked by at least one disulfide bond. Fourth, SEC
analysis had demonstrated co-elution of the 75 kDa species with the full antibody, strongly
suggesting that the 75 kDa species can non-covalently self-associate to form a full antibody (or
another complex of the same apparent hydrodynamic radius). Finally, the apparent molecular
weight of about 75 kDa (determined by reference to electrophoresis standards), taken together
with the observation (from denaturing gels) that this complex was only made up of full-length
antibody chains, was consistent with complex containing only one heavy chain and only one
light chain, but was inconsistent with other complexes.
Methods
Mass spectrometry was used to detect the relative abundance of heavy chains
lacking the inter-heavy chain disulfide bonds (normally found at amino acids 220 and 223) in
different samples. Two-hundred and fifty micrograms of each sample was added into an
Eppendorf tube. An appropriate amount (~450 μL) of denaturing buffer (6 M Guanidine-HCl, 1
mM EDTA, 0.25 M Tris, pH 7.5) was added to the tube to obtain a final volume of 500 μL and a
sample concentration of 0.5 mg/mL. Twelve and a half microliters of 2 M Iodoacetamide was
added into each sample to alkylate any free cysteine. The samples were vortexed then incubated
at room temperature, in the dark, for 30 ± 5 minutes. The samples were then desalted using NAP-
columns pre-equilibrated with digestion buffer (0.1 M Tris-HCl, pH 7.5). Each sample solution
was added to separate (pre-equilibrated) columns and allowed to enter the column bed. One
milliliter of digestion buffer was added to each column and the eluent was collected into
Eppendorf tubes. The samples were divided into equal aliquots containing approximately 50 μg
of material (five 200 μL aliquots). The alkylated and desalted aliquots were stored at -20 °C until
needed. One aliquot of each sample (alkylated, and desalted) was used for each digestion.
Trypsin solution at 0.5 mg/mL was added to each sample aliquot at a 1:25 w:w ratio of
trypsin:protein (4 μL). All trypsin tubes were incubated at 37 ± 2 ºC for 4 hours. After
incubation, the enzymatic digestion was quenched by adding 1 μL of Trifluoroacetic acid to each
tube. The samples were then divided into two equal portions, reduced and non-reduced. Half the
samples were reduced in the presence of 1 M DTT for 1 hour at 37 ± 2 ºC. The contents of both
reduced and nonreduced samples were transferred to HPLC vials and placed in the autosampler
for analysis.
MS and MS/MS data was collected on a Micromass Q-TOF Ultima mass
spectrometer using electrospray ionization (ESI) in positive ion mode. Data was acquired from
m/z 200-1950 in MS mode. Prior to analysis, the mass spectrometer was calibrated using a 5th
order fit on fragment ions of [Glu ]-Fibrinopeptide covering a range from m/z 175 to 1285. The
injections volumes were adjusted to achieve an on-column load of approximately 20 pmoles of
protein.
Results
Based on the observations discussed above, Applicants hypothesized that the 75
kDa band was a “half antibody” species containing one heavy antibody chain and one light
antibody chain covalently linked to one another through disulfide bonds, and that pairs of half
antibody complexes could non-covalently associate to form a complex having the same
stoichiometry as a full antibody (two heavy chains and two light chains) but lacking the disulfide
linkages between the two heavy chains (“unlinked heavy chains”). Based thereon, it was
predicted the relative abundance of the 75 kDa band would correlate with the relative proportion
of unlinked heavy chains, which was determined using mass spectrometry analysis of trypsin
digested antibody samples.
The peptide fragments of interest for this study were the T17 trypsin fragment of
the heavy chain (T17H) which is composed of amino acids 217-242, respectively. Amino acids
220 and 223 are responsible for disulfide bonding between antibody heavy chains. Typically,
free cysteine analysis can be conducted by determining a ratio of alkylated cysteine residues to
non-alkylated residues in reduced samples after a tryptic digestion. However, alkylated species
were not present in either lot of material. It was hypothesized the two cysteine residues of the
peptide fragment of interest, T17H, were bonding to each other, or the cysteines were protected
by the antibody’s quaternary structure. In either case, the cysteine residues would not be
accessible for alkylation. Instead, the analytical approach was to utilize the ratio of non-reduced
species to reduced species in both lots to calculate a percent difference. It was observed that the
non-reduced samples had a 2 Da decrease in molecular weight, indicative of disulfide bonding.
This behavior was exploited to calculate the percent free T17H. The theoretical mass of the non-
reduced T17H species is 2727.41 Da (disulfide bonding between Cys220 & Cys223) and
2729.41 Da for reduced peptide.
The extracted ion chromatograms of reduced and non-reduced samples were
analyzed for representative charge states. ratio of counts between the non-reduced to reduced
samples calculates the free T17H species to be 2.3% in the antibody produced with a bolus
addition of ethanol, and 26.1% in the sample produced without a bolus addition of ethanol.
These results are presented in tabular form in Fig. 9.
Thus, the abundance of heavy chains lacking the inter-heavy chain disulfide bond
was greatly increased in the sample produced without the bolus addition of ethanol, further
confirming the identity of this species as containing one heavy chain and one light chain but
lacking an inter-heavy chain disulfide bond. Moreover, the detection of a species 2 Da lighter
than the expected mass indicated that heavy chain in the H1L1 species may contain an extra
disulfide bond to itself which may interfere with the formation of the normal inter-heavy chain
disulfide bond.
EXAMPLE 5
This example demonstrates a correlation between cell viability and antibody
purity. The addition of an ethanol bolus generally improved cell viability and antibody purity for
Ab-A and the Ab-B antibody. Further, the Ab-C antibody, which already exhibited high purity
even without the bolus addition, also exhibited higher culture viability. Taken together, these
results suggest that the improvement in antibody purity resulting from a bolus addition of ethanol
is at least partially attributable to increased culture viability.
Methods
Culture viability was determined using a Cellometer (Nexcelom). Culture samples
were diluted with PBS so that the final cell count was within 1 x 10 to 5 x 10 cells/mL. One
half of the sample was then treated to heat conditions of 75°C for 10 minutes as positive control
for propidium iodide (PI) staining. The untreated sample and treated sample were then mixed
with PI (20 uL of sample plus 20 uL of PI). The sample was then placed in a slide cassette and
viability was determined by counting the number of non-fluorescing cells then dividing by the
total number of cells. Cells that are dead have taken up the propidium iodide and are fluorescing
so the positive control heat killed sample should show less than 1% cells alive.
Results
Cell viability was determined for antibody-producing cultures grown with or
without a bolus addition of ethanol as described in Examples 1 and 2. As described above,
purity of the Ab-A and Ab-B antibodies were greatly improved by a bolus addition of ethanol to
the yeast culture (see Examples 1-2 and FIGS. 1-4). The addition of an ethanol bolus improved
cell viability for these cultures as well. For the Ab-A antibody, viability improved from 91.9%
to 97.2% on average (), while for the Ab-B antibody, viability improved from 84.8% to
95.1% on average (). Due to the already high purity of the Ab-C antibody produced even
without the bolus addition of ethanol, the improvements in the purity of this antibody resulting
from the bolus addition of ethanol were more modest (see Example 3 and . Consistent
with the observation that high cell viability correlated with higher antibody purity, the Ab-C
antibody cultures exhibited high cell viability (95.8%% on average) in the absence of a bolus
addition of ethanol, which was little changed by the bolus addition of ethanol (96.8%).
Taken together, these results indicate that the improvement in antibody purity
resulting from the addition of an ethanol bolus may be in part caused by (or at least correlates
with) an improvement in cell viability.
EXAMPLE 6
This example demonstrates that a similar improvement in antibody purity can be
attained with varying ethanol bolus concentrations.
Methods
The Ab-A antibody was produced as in Example 1, except that the bolus addition
of ethanol was 5 g/L (0.5% w/v), 10 g/L (1% w/v), or 15 g/L (1.5% w/v). Antibodies samples
were purified from the culture media at 63 and 86 hours and purified by protein A affinity, then
the purity was analyzed by non-reduced SDS-PAGE as in Example 1.
Results
Antibody purity was similarly high irrespective of the bolus concentration added
(between 0.5% and 1.5% w/v), at both time points tested, 63 hours (A) and 86 hours
(FIG, 14B). Detected levels of the H1L1 and H2L1 species were similarly low in each culture.
These results indicate that the improvement in antibody purity can be attained
while varying the ethanol bolus concentration.
EXAMPLE 7
This example demonstrates that similar improvements in antibody purity can be
attained while varying the duration of the “starvation period” between the dissolved oxygen
spike and the addition of the ethanol bolus to the cultures.
Methods
The Ab-A antibody was produced as in Example 1, except that the duration of the
starvation period, the time between the dissolved oxygen spike (indicating exhaustion of the
carbon source in the culture) and the bolus addition of ethanol, was either 0 hours or 3 hours.
Antibodies samples were purified from the culture media and purified by protein A affinity, then
the purity was analyzed by non-reduced SDS-PAGE as in Example 1.
Results
Antibody purity was similarly high irrespective of the variation of the starvation
period between 0 and 3 hours (A, compare lanes 5 (0 hours starvation) and 6 (3 hours
starvation)). Detected levels of the H1L1 and H2L1 species were similarly low in each culture.
These results indicate that the improvement in antibody purity can be attained
with a varying the duration of the starvation period.
EXAMPLE 8
This example tests the effect on antibody purity from varying the duration of the
“equilibration period” between the addition of the ethanol bolus and commencement of adding
the feed to the cultures.
Methods
The Ab-B antibody was produced as in Example 2, except that the duration of the
equilibration period, the time between addition of the ethanol bolus and the commencement of
feeding the culture, was 0, 30, or 60 minutes. Additionally, the yeast strain from which the Ab-B
antibody was produced contained three copies of the light chain gene instead of four. Antibodies
samples were purified from the culture media and purified by protein A affinity, then the purity
was analyzed by non-reduced SDS-PAGE as in Example 1. Viability was also assessed using
the methods described in Example 5.
Results
Antibody purity was similarly high for an equilibration period of 0 or 30 minutes
(A, lanes 7 and 8 (0 minutes equilibration time) and lane 3 (30 minutes equilibration
time). However, detected levels of the H1L1 and H2L1 species were increased in the culture
with the 60 minute equilibration period (A, lanes 5 and 6).
Viability was also assessed for each culture at the 23 hour and 85 hour time
points. For the 60 minute equilibration period, viability was between 75% and 80% at 23 hours,
while at the same time point viability was approximately 88-90% for the 0 and 30 minute
equilibration periods (B). Subsequently, at 85 hours, viability had improved but
remained somewhat reduced for the 60 minute equilibration period relative to the 0 and 30
minute equilibration periods (C).
These results indicate that the improvement in antibody purity can be attained
while varying the equilibration period at least between 0 and 30 minutes, while some loss of
viability and purity may occur for an equilibration period of 60 minutes or longer (though purity
may still be improved relative to a control culture without a bolus addition of ethanol).
The above description of various illustrated embodiments of the invention is not
intended to be exhaustive or to limit the invention to the precise form disclosed. While specific
embodiments of, and examples for, the invention are described herein for illustrative purposes,
various equivalent modifications are possible within the scope of the invention, as those skilled
in the relevant art will recognize. The teachings provided herein of the invention can be applied
to other purposes, other than the examples described above.
The invention may be practiced in ways other than those particularly described in
the foregoing description and examples. Numerous modifications and variations of the invention
are possible in light of the above teachings and, therefore, are within the scope of the appended
claims.
These and other changes can be made to the invention in light of the above
detailed description. In general, in the following claims, the terms used should not be construed
to limit the invention to the specific embodiments disclosed in the specification and the claims.
Accordingly, the invention is not limited by the disclosure, but instead the scope of the invention
is to be determined entirely by the following claims.
Certain teachings related to methods for obtaining a clonal population of antigen-
specific B cells were disclosed in U.S. Provisional patent application no. 60/801,412, filed May
19, 2006, the disclosure of which is herein incorporated by reference in its entirety.
Certain teachings related to humanization of rabbit-derived monoclonal antibodies
and preferred sequence modifications to maintain antigen binding affinity were disclosed in
International Application No. , corresponding to International Publication
No. WO/2008/144757, entitled “Novel Rabbit Antibody Humanization Methods and Humanized
Rabbit Antibodies”, filed May 21, 2008, the disclosure of which is herein incorporated by
reference in its entirety.
Certain teachings related to producing antibodies or fragments thereof using
mating competent yeast and corresponding methods were disclosed in U.S. Patent application no.
11/429,053, filed May 8, 2006, (U.S. Patent Application Publication No. US2006/0270045), the
disclosure of which is herein incorporated by reference in its entirety.
The entire disclosure of each document cited herein (including patents, patent
applications, journal articles, abstracts, manuals, books, or other disclosures), including each
document cited in the Background, Summary, Detailed Description, and Examples, is hereby
incorporated by reference herein in its entirety.
Claims (75)
- WHAT WE CLAIM IS: 1) A method of producing a full length antibody, comprising: a. providing a growth culture comprising Pichia pastoris cells comprising genes that provide for the expression of said full length antibody and growing said Pichia yeast in said growth culture for about 12-16 hours (“growth phase”); b. adding ethanol to said culture after said growth phase in a single bolus addition (“bolus”) which bolus results in a concentration of ethanol in said culture of between 0.5% and 1.5% w/v, and c. culturing said culture to produce said secreted full length antibody, wherein said culturing comprises adding a feed comprising a fermentable carbon source to said culture; and further wherein said antibody does not comprise any of: (i) an anti-human NGF antibody comprising the variable light (V ) chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 5, 6, and 7 and the variable heavy (V ) chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 8, 9, and 10; (ii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 15, 16, and 17 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 18, 19, and 20; (iii) an anti-human NGF antibody comprising the VL chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 25, 26, and 27 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 28, 29, and 30; (iv) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 35, 36, and 37 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 38, 39, and 40; (v) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 45, 46, and 47 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 48, 49, and 50; (vi) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 55, 56, and 57 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 58, 59, and 60; (vii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 65, 66, and 67 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 68, 69, and 70; (viii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 75, 76, and 77 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 78, 79, and 80; (ix) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 85, 86, and 87 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 88, 89, and 90; (x) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 95, 96, and 97 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 98, 99, and 100; (xi) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 105, 106, and 107 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 108, 109, and 110; (xii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 115, 116, and 117 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 118, 119, and 120; (xiii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 125, 126, and 127 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 128, 129, and 130; (xiv) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 135, 136, and 137 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 138, 139, and 140; (xv) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 145, 146, and 147 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 148, 149, and 150; (xvi) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 155, 156, and 157 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 158, 159, and 160; (xvii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 165, 166, and 167 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 168, 169, and 170; (xviii) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 175, 176, and 177 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 178, 179, and 180; (xix) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 185, 186, and 187 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 188, 189, and 190; and (xx) an anti-human NGF antibody comprising the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 195, 196, and 197 and the V chain CDR1, CDR2, and CDR3 polypeptides of SEQ ID NO: 198, 199, and 200.
- 2) The method of claim 1, wherein the ethanol bolus enhances the formation of stable disulfide bonds relative to the same method effected in the absence of the bolus of ethanol.
- 3) The method of claim 1, wherein said antibody contains one or more polypeptides comprising at least one disulfide bond.
- 4) The method of any one of claims 1 to 3, which decreases the relative abundance of one or more product-associated variants relative to the same method effected in the absence of the bolus of ethanol.
- 5) The method of any one of claims 1 to 3, which decreases the relative abundance of product-associated variants having a higher or lower apparent molecular weight than said antibody as detected by size exclusion chromatography or gel electrophoresis relative to the same method effected in the absence of the bolus of ethanol.
- 6) The method of any one of claims 1 to 3, which decreases the relative abundance of antibodies having aberrant stoichiometry relative to the same method effected in the absence of the bolus of ethanol.
- 7) The method of any one of claims 1 to 3, which decreases the relative abundance of antibodies having aberrant disulfide bonds relative to the same method effected in the absence of the bolus of ethanol.
- 8) The method of any one of claims 1 to 3, which decreases the relative abundance of antibodies having reduced cysteines relative to the same method effected in the absence of the bolus of ethanol.
- 9) The method of any one of claims 1 to 3, which decreases the relative abundance of antibodies having aberrant glycosylation relative to the same method effected in the absence of the bolus of ethanol.
- 10) The method of claim 3, which modulates the formation or stability of inter-heavy chain disulfide bonds.
- 11) The method of claim 3 or 10, which modulates the formation or stability of disulfide bonds linking the light and heavy chains.
- 12) The method of any one of claims 3, 10, or 11, which decreases the relative abundance of one or more product-associated variants relative to the same method effected in the absence of the bolus of ethanol.
- 13) The method of claim 12 wherein said product-associated variants comprise one or more of the H1L1, H2L1, and H4L4 product-associated variants.
- 14) The method of any one of claims 3 or 10-13, which increases the purity of said antibody relative to said method effected in the absence of said bolus of ethanol.
- 15) The method of any one of the foregoing claims, wherein step (c) comprises providing oxygen to said cells.
- 16) The method of claim 15, wherein said providing oxygen comprises agitating said culture.
- 17) The method of claim 15 or 16, wherein said providing oxygen comprises contacting said culture with a gas mixture comprising oxygen.
- 18) The method of any one of the foregoing claims, wherein said fermentable carbon source comprises glucose or galactose.
- 19) The method of claim 18, wherein said fermentable carbon source comprises glucose.
- 20) The method of claim 18, wherein said fermentable carbon source comprises one or more of glucose, citrate, sorbitol, xylose, trehalose, arabinose, galactose, fructose, melibiose, lactose, maltose, rhamnose, ribose, mannose, mannitol, and raffinose.
- 21) The method of any one of the foregoing claims, wherein the culture of step (a) is produced by adding a carbon source to said culture, and culturing said culture until the carbon source is depleted.
- 22) The method of claim 21, wherein said carbon source comprises one or more of: glucose, ethanol, citrate, sorbitol, xylose, trehalose, arabinose, galactose, fructose, melibiose, lactose, maltose, rhamnose, ribose, mannose, mannitol, and raffinose.
- 23) The method of claim 21 or 22, wherein the depletion of the carbon source is determined by detecting a decrease in the metabolic activity of said Pichia pastoris cells.
- 24) The method of claim 23, wherein said decrease in the metabolic activity of said Pichia pastori cells is identified by detecting a decrease in the consumption of oxygen by said Pichia pastoris cells, by detecting an increase in pH in the culture, by detecting stabilization of the wet cell mass, or by detecting an increase in the concentration of ammonia in the culture.
- 25) The method of claim 24, wherein said decrease in the consumption of oxygen by said Pichia pastoris cells is identified by detecting an increase in the concentration of dissolved oxygen in said culture.
- 26) The method of any one of the foregoing claims, wherein the genes that provide for expression of said antibody are integrated into one or more genomic loci.
- 27) The method of claim 26, wherein at least one of said genomic loci are selected from the group consisting of the pGAP locus, 3’ AOX TT locus; PpURA5; OCH1; AOX1; HIS4; GAP; pGAP; 3’ AOX TT; ARG; and the HIS4 TT locus.
- 28) The method of any one of the foregoing claims, wherein at least one of the genes encoding said subunits of the antibody are expressed under control of an inducible or constitutive promoter.
- 29) The method of claim 28, wherein said inducible promoter is selected from the group consisting of the AOX1, CUP1, tetracycline inducible, thiamine inducible, and FLD1 promoters.
- 30) The method of any one of the foregoing claims, wherein at least one of the genes encoding said subunits of the antibody are expressed under control of a promoter selected from the group consisting of: the CUP1, AOX1, ICL1, glyceraldehydephosphate dehydrogenase (GAP), FLD1, ADH1, alcohol dehydrogenase II, GAL4, PHO3, PHO5, and Pyk promoters, tetracycline inducible promoters, thiamine inducible promoters, chimeric promoters derived therefrom, yeast promoters, mammalian promoters, insect promoters, plant promoters, reptile promoters, amphibian promoters, viral promoters, and avian promoters.
- 31) The method of any one of the foregoing claims, wherein said Pichia pastoris cell is a diploid, tetraploid cell, or polyploid.
- 32) The method of any one of the foregoing claims, further comprising purifying said antibody from said Pichia pastoris cells or from the culture medium.
- 33) The method of claim 32, wherein said antibody is purified from an intracellular component, cytoplasm, nucleoplasm, or a membrane of said Pichia pastoris cells.
- 34) The method of claim 32, wherein said Pichia pastoris cells secrete said antibody into the culture medium.
- 35) The method of claim 34, wherein said antibody is purified from said culture medium.
- 36) The method of any one of the foregoing claims, wherein said antibody comprises a monospecific or bispecific antibody.
- 37) The method of any one of the foregoing claims, wherein said antibody comprises a human antibody or a humanized antibody.
- 38) The method of claim 37, wherein said humanized antibody is of mouse, rat, rabbit, goat, sheep, or cow origin.
- 39) The method of claim 37, wherein said humanized antibody is of rabbit origin.
- 40) The method of any one of the foregoing claims, wherein said antibody comprises a monovalent, bivalent, or multivalent antibody.
- 41) The method of any one of the foregoing claims, wherein said antibody is purified from said culture by protein A and/or protein G affinity.
- 42) The method of any one of the foregoing claims, wherein at least one of the genes that provide for expression of a subunit of said antibody is optimized for expression in said Pichia pastoris cell.
- 43) The method of any one of the foregoing claims, wherein the purity of said antibody is assessed by measuring the fraction of the antibody produced by said eukaryotic cell that is contained in antibody complexes having the expected apparent hydrodynamic radius, is contained in antibody complexes having the expected molecular weight, and / or specifically binds a target of said antibody.
- 44) The method of any one of the foregoing claims, wherein the yield of said antibody is assessed by determining the amount of antibody produced by said Pichia pastoris cell discounting any product-associated variants that are abnormally glycosylated, contained in antibody complexes other than complexes having the expected apparent hydrodynamic radius, contained in antibody complexes having the expected molecular weight, and / or that fail to specifically bind to the target of said antibody.
- 45) The method of claim 32 or 33, wherein the molecular weight of said antibody is determined by non-reducing SDS-PAGE.
- 46) The method of any one of the foregoing claims, wherein said method further comprises purifying said antibody.
- 47) The method of any one of the foregoing claims, wherein said culture cell produces a supernatant antibody titer of at least 100 mg / L, at least 150 mg / L, at least 200 mg / L, at least 250 mg / L, at least 300 mg / L, between 100 and 300 mg / L, between 100 and 500 mg / L, between 100 and 1000 mg / L, at least 1000 mg / L, at least 1250 mg/liter, at least 1500 mg/liter, at least about 1750 mg/liter, at least about 2000 mg/liter, at least about 10000 mg/liter, or more.
- 48) The method of any one of the foregoing claims, wherein one or more subunits of said antibody are expressed from more than one gene copy.
- 49) The method of any one of the foregoing claims, wherein said antibody is expressed from between 1-10 copies of a gene encoding the light chain of said antibody and from 1-10 copies of a gene encoding the heavy chain of said antibody.
- 50) The method of any one of the foregoing claims, wherein genes that provide for expression of said antibody are integrated into genome of said cells.
- 51) The method of any one of the foregoing claims, wherein genes that provide for expression of said antibody are contained on an extrachromosomal element, plasmid, or artificial chromosome.
- 52) The method of claim 50 or 51, wherein said cells comprise more copies of the gene that provide for the expression of the light chain of said antibody than copies of the gene that provide for expression of the heavy chain of said antibody.
- 53) The method of claim 50 or 51, wherein the respective number of copies of the gene encoding the heavy chain of said antibody and the number of copies of the gene encoding the light chain of said antibody in said cells are: 2 and 2, 2 and 3, 3 and 3, 3 and 4, 3 and 5, 4 and 3, 4 and 4, 4 and 5, 4 and 6, 5 and 4, 5 and 5, 5 and 6, or 5 and 7.
- 54) The method of claim 50 or 51, wherein the respective number of copies of the gene encoding the heavy chain of said antibody and the number of copies of the gene encoding the light chain of said antibody in said cells are: 2 and 1, 3 and 1, 4 and 1, 5 and 1, 6 and 1, 7 and 1, 8 and 1, 9 and 1, 10 and 1, 1 and 2, 2 and 2, 3 and 2, 4 and 2, 5 and 2, 6 and 2, 7 and 2, 8 and 2, 9 and 2, 10 and 2, 1 and 3, 2 and 3, 3 and 3, 4 and 3, 5 and 3, 6 and 3, 7 and 3, 8 and 3, 9 and 3, 10 and 3, 1 and 4, 2 and 4, 3 and 4, 4 and 4, 5 and 4, 6 and 4, 7 and 4, 8 and 4, 9 and 4, 10 and 4, 1 and 5, 2 and 5, 3 and 5, 4 and 5, 5 and 5, 6 and 5, 7 and 5, 8 and 5, 9 and 5, 10 and 5, 1 and 6, 2 and 6, 3 and 6, 4 and 6, 5 and 6, 6 and 6, 7 and 6, 8 and 6, 9 and 6, 10 and 6, 1 and 7, 2 and 7, 3 and 7, 4 and 7, 5 and 7, 6 and 7, 7 and 7, 8 and 7, 9 and 7, 10 and 7, 1 and 8, 2 and 8, 3 and 8, 4 and 8, 5 and 8, 6 and 8, 7 and 8, 8 and 8, 9 and 8, 10 and 8, 1 and 9, 2 and 9, 3 and 9, 4 and 9, 5 and 9, 6 and 9, 7 and 9, 8 and 9, 9 and 9, 10 and 9, 1 and 10, 2 and 10, 3 and 10, 4 and 10, 5 and 10, 6 and 10, 7 and 10, 8 and 10, 9 and 10, 10 and 10.
- 55) The method of any one of the foregoing claims, wherein the culture of step (c) is grown in a production medium.
- 56) The method of claim 55, wherein said production medium is a minimal medium.
- 57) The method of claim 55, wherein said minimal medium lacks selective agents.
- 58) The method of claim 55, wherein said minimal medium lacks pre-formed amino acids or other complex biomolecules.
- 59) The method of claim 55, wherein the production medium is a complex medium.
- 60) The method of claim 59, wherein the complex medium comprises one or more of yeast extract, soy peptones, and other plant peptones.
- 61) The method of any one of the foregoing claims, wherein the culture of step (c) is grown to a high cell density.
- 62) The method of claim 61, wherein said high cell density is at least 50 g/L.
- 63) The method of claim 61, wherein said high cell density is at least 100 g/L.
- 64) The method of claim 61, wherein said high cell density is at least 300 g/L.
- 65) The method of claim 61, wherein said high cell density is at least 400 g/L.
- 66) The method of claim 61, wherein said high cell density is at least 500 g/L.
- 67) The method of claim 61, wherein said high cell density is at least 750 g/L.
- 68) The method of any one of the foregoing claims, wherein the Pichia pastoris cells are cultured for at least 20 doublings and maintain high levels of expression of said antibody after said at least 20 doublings.
- 69) The method of any one of the foregoing claims, wherein the Pichia pastoris cells of step (c) are cultured for at least 50 doublings and maintain high levels of expression of said antibody after said at least 50 doublings.
- 70) The method of any one of the foregoing claims, wherein the Pichia pastoris cells of step (c) are cultured for at least 100 doublings and maintain high levels of expression of said antibody after said at least 100 doublings.
- 71) The method of any one of the foregoing claims, wherein at least one subunit of said antibody comprises a secretion signal.
- 72) The method or composition of claim 71, wherein the secretion signal comprises one or more polypeptides selected from the group consisting of: SEQ ID NOS: 414 to 437 or any combination thereof.
- 73) Full-length antibodies when produced by a method of any one of claims 1 to 72.
- 74) A method as claimed in any one of claims 1 to 72, of producing full-length antibodies, substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.
- 75) Full-length antibodies as claimed in claim 73, substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ718187A NZ718187B2 (en) | 2011-05-20 | 2012-05-08 | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as pichia pastoris |
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161488660P | 2011-05-20 | 2011-05-20 | |
US61/488,660 | 2011-05-20 | ||
US201161496873P | 2011-06-14 | 2011-06-14 | |
US201161496860P | 2011-06-14 | 2011-06-14 | |
US61/496,873 | 2011-06-14 | ||
US61/496,860 | 2011-06-14 | ||
US201161525307P | 2011-08-19 | 2011-08-19 | |
US61/525,307 | 2011-08-19 | ||
PCT/US2012/036953 WO2012161956A2 (en) | 2011-05-20 | 2012-05-08 | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as pichia pastoris |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ618635A NZ618635A (en) | 2016-09-30 |
NZ618635B2 true NZ618635B2 (en) | 2017-01-05 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11447540B2 (en) | Pichia pastoris yeast cultures comprising reduced antibody-associated variants | |
US11225667B2 (en) | Multi-copy strategy for high-titer and high-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris | |
EP2744903B1 (en) | Multi-copy strategy for high-titer and high-purity production of multi-subunit proteins such as a antibodies in transformed microbes such as pichia pastoris | |
US11214610B2 (en) | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris | |
JP6491696B2 (en) | High-purity production of multi-subunit proteins such as antibodies in transformed microorganisms such as Pichia pastoris | |
US20190390239A1 (en) | Temperature shift for high yield expression of polypeptides in yeast and other transformed cells | |
AU2016273912A1 (en) | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris | |
NZ618635B2 (en) | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as pichia pastoris | |
NZ718187B2 (en) | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as pichia pastoris | |
AU2012259225A1 (en) | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris | |
NZ721102B2 (en) | Multi-copy strategy for high-titer and high-purity production of multi-subunit proteins such as antibodies in transformed microbes such as pichia pastoris | |
NZ621199B2 (en) | Multi-copy strategy for high-titer and high-purity production of multi-subunit proteins such as antibodies in transformed microbes such as pichia pastoris |