NZ617725B2 - Antibodies against g-csfr and uses thereof - Google Patents
Antibodies against g-csfr and uses thereof Download PDFInfo
- Publication number
- NZ617725B2 NZ617725B2 NZ617725A NZ61772512A NZ617725B2 NZ 617725 B2 NZ617725 B2 NZ 617725B2 NZ 617725 A NZ617725 A NZ 617725A NZ 61772512 A NZ61772512 A NZ 61772512A NZ 617725 B2 NZ617725 B2 NZ 617725B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- seq
- amino acid
- set forth
- sequence set
- acid sequence
- Prior art date
Links
- 102000004965 antibodies Human genes 0.000 title claims abstract description 432
- 108090001123 antibodies Proteins 0.000 title claims abstract description 432
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 434
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 434
- 230000027455 binding Effects 0.000 claims abstract description 209
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims abstract description 115
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims abstract description 115
- 210000004027 cells Anatomy 0.000 claims abstract description 112
- 241000282414 Homo sapiens Species 0.000 claims abstract description 99
- 101700074459 CA1B Proteins 0.000 claims abstract description 77
- 239000000203 mixture Substances 0.000 claims abstract description 38
- 230000011664 signaling Effects 0.000 claims abstract description 36
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 33
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 33
- 241000282567 Macaca fascicularis Species 0.000 claims abstract description 20
- 210000002798 bone marrow cell Anatomy 0.000 claims abstract description 9
- 210000003714 Granulocytes Anatomy 0.000 claims abstract description 6
- 230000012010 growth Effects 0.000 claims abstract description 6
- 230000001332 colony forming Effects 0.000 claims abstract description 3
- 235000018102 proteins Nutrition 0.000 claims description 424
- 150000001413 amino acids Chemical group 0.000 claims description 212
- 229920001184 polypeptide Polymers 0.000 claims description 147
- 235000001014 amino acid Nutrition 0.000 claims description 94
- 239000000427 antigen Substances 0.000 claims description 89
- 108091007172 antigens Proteins 0.000 claims description 89
- 102000038129 antigens Human genes 0.000 claims description 89
- 229960003767 Alanine Drugs 0.000 claims description 74
- 235000004279 alanine Nutrition 0.000 claims description 74
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-Methionine Natural products CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 67
- 102100006433 CSF3R Human genes 0.000 claims description 65
- 108010054017 Granulocyte Colony-Stimulating Factor Receptors Proteins 0.000 claims description 65
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 60
- 238000004166 bioassay Methods 0.000 claims description 39
- 229960004452 Methionine Drugs 0.000 claims description 38
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 38
- 235000006109 methionine Nutrition 0.000 claims description 38
- 229960003136 leucine Drugs 0.000 claims description 37
- 235000005772 leucine Nutrition 0.000 claims description 37
- 229960002885 Histidine Drugs 0.000 claims description 34
- 235000014304 histidine Nutrition 0.000 claims description 34
- 230000035693 Fab Effects 0.000 claims description 32
- 229960004441 Tyrosine Drugs 0.000 claims description 32
- 235000002374 tyrosine Nutrition 0.000 claims description 32
- 238000006467 substitution reaction Methods 0.000 claims description 29
- 230000001404 mediated Effects 0.000 claims description 27
- 229960001153 serine Drugs 0.000 claims description 27
- 235000004400 serine Nutrition 0.000 claims description 27
- MTCFGRXMJLQNBG-UWTATZPHSA-N D-serine Chemical compound OC[C@@H](N)C(O)=O MTCFGRXMJLQNBG-UWTATZPHSA-N 0.000 claims description 26
- 229940021015 I.V. solution additive Amino Acids Drugs 0.000 claims description 26
- 150000001875 compounds Chemical class 0.000 claims description 26
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 25
- 229960005190 Phenylalanine Drugs 0.000 claims description 25
- 235000008729 phenylalanine Nutrition 0.000 claims description 25
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 24
- 229960002743 Glutamine Drugs 0.000 claims description 24
- 210000000440 Neutrophils Anatomy 0.000 claims description 24
- 235000004554 glutamine Nutrition 0.000 claims description 24
- 101700027814 CDR3 Proteins 0.000 claims description 23
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 23
- 102100005175 CSF1R Human genes 0.000 claims description 22
- 101700063802 CSF1R Proteins 0.000 claims description 22
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 22
- 239000004472 Lysine Substances 0.000 claims description 22
- 229960003646 lysine Drugs 0.000 claims description 22
- 235000018977 lysine Nutrition 0.000 claims description 22
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 21
- 229960002449 Glycine Drugs 0.000 claims description 19
- 239000004471 Glycine Substances 0.000 claims description 19
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 18
- 230000002829 reduced Effects 0.000 claims description 18
- 239000004475 Arginine Substances 0.000 claims description 17
- 208000004235 Neutropenia Diseases 0.000 claims description 17
- 229960002429 Proline Drugs 0.000 claims description 17
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims description 17
- 229960003121 arginine Drugs 0.000 claims description 17
- 235000009697 arginine Nutrition 0.000 claims description 17
- 230000035755 proliferation Effects 0.000 claims description 17
- 235000013930 proline Nutrition 0.000 claims description 17
- 229960002989 Glutamic Acid Drugs 0.000 claims description 16
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 16
- 235000013922 glutamic acid Nutrition 0.000 claims description 16
- 239000004220 glutamic acid Substances 0.000 claims description 16
- 125000003372 histidine group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 claims description 16
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 16
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 15
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 15
- 239000004473 Threonine Substances 0.000 claims description 15
- 229960004799 Tryptophan Drugs 0.000 claims description 15
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 15
- 229960002898 threonine Drugs 0.000 claims description 15
- 235000008521 threonine Nutrition 0.000 claims description 15
- 206010003246 Arthritis Diseases 0.000 claims description 14
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical compound C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 claims description 14
- 229960000310 ISOLEUCINE Drugs 0.000 claims description 14
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 14
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 14
- 235000014705 isoleucine Nutrition 0.000 claims description 14
- 238000004519 manufacturing process Methods 0.000 claims description 14
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 12
- 229960004295 valine Drugs 0.000 claims description 12
- 239000004474 valine Substances 0.000 claims description 12
- 235000014393 valine Nutrition 0.000 claims description 12
- 125000000511 arginine group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 11
- 108091006028 chimera Proteins 0.000 claims description 11
- 108060001251 CD34 Proteins 0.000 claims description 10
- 102100016492 CD34 Human genes 0.000 claims description 10
- -1 arganine Chemical compound 0.000 claims description 10
- 201000011510 cancer Diseases 0.000 claims description 10
- 201000006417 multiple sclerosis Diseases 0.000 claims description 10
- 230000001939 inductive effect Effects 0.000 claims description 9
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 9
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 9
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 230000001264 neutralization Effects 0.000 claims description 8
- 229960005261 Aspartic Acid Drugs 0.000 claims description 7
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 7
- 235000003704 aspartic acid Nutrition 0.000 claims description 7
- 229960001230 Asparagine Drugs 0.000 claims description 6
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 claims description 6
- 206010061218 Inflammation Diseases 0.000 claims description 6
- 235000009582 asparagine Nutrition 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 6
- 230000004054 inflammatory process Effects 0.000 claims description 6
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 5
- 206010003816 Autoimmune disease Diseases 0.000 claims description 4
- 108010070144 Single-Chain Antibodies Proteins 0.000 claims description 4
- 102000005632 Single-Chain Antibodies Human genes 0.000 claims description 4
- 201000009596 autoimmune hypersensitivity disease Diseases 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 4
- 200000000018 inflammatory disease Diseases 0.000 claims description 4
- 125000001116 prolino group Chemical group [H]OC(=O)C1([H])N(*)C([H])([H])C([H])([H])C1([H])[H] 0.000 claims description 2
- 230000002685 pulmonary Effects 0.000 claims description 2
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 claims 2
- 102100000197 CD24 Human genes 0.000 abstract 1
- 108060001249 CD24 Proteins 0.000 abstract 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 165
- 230000035492 administration Effects 0.000 description 25
- 230000035772 mutation Effects 0.000 description 25
- 102000005962 receptors Human genes 0.000 description 25
- 108020003175 receptors Proteins 0.000 description 25
- 241000699666 Mus <mouse, genus> Species 0.000 description 20
- 108010045030 monoclonal antibodies Proteins 0.000 description 18
- 102000005614 monoclonal antibodies Human genes 0.000 description 18
- 238000000034 method Methods 0.000 description 17
- 230000004044 response Effects 0.000 description 17
- 108020001756 ligand binding domains Proteins 0.000 description 16
- 230000000875 corresponding Effects 0.000 description 14
- 230000002757 inflammatory Effects 0.000 description 14
- 239000003446 ligand Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 11
- 230000036809 Fabs Effects 0.000 description 10
- 230000036499 Half live Effects 0.000 description 10
- 238000005755 formation reaction Methods 0.000 description 10
- 238000002823 phage display Methods 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 9
- 238000010494 dissociation reaction Methods 0.000 description 9
- 230000005593 dissociations Effects 0.000 description 9
- 241000124008 Mammalia Species 0.000 description 8
- 125000000539 amino acid group Chemical group 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 230000001965 increased Effects 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- NFGXHKASABOEEW-UHFFFAOYSA-N (+)-methoprene Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 7
- 241000282412 Homo Species 0.000 description 7
- 230000001363 autoimmune Effects 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 7
- 230000002708 enhancing Effects 0.000 description 7
- 238000002955 isolation Methods 0.000 description 7
- 239000002609 media Substances 0.000 description 7
- 210000000130 stem cell Anatomy 0.000 description 7
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 7
- 210000004072 Lung Anatomy 0.000 description 6
- 108010021466 Mutant Proteins Proteins 0.000 description 6
- 102000008300 Mutant Proteins Human genes 0.000 description 6
- 241000700159 Rattus Species 0.000 description 6
- 108010033725 Recombinant Proteins Proteins 0.000 description 6
- 102000007312 Recombinant Proteins Human genes 0.000 description 6
- 206010039073 Rheumatoid arthritis Diseases 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 6
- 230000005591 charge neutralization Effects 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 230000001808 coupling Effects 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 6
- 238000005859 coupling reaction Methods 0.000 description 6
- 235000018417 cysteine Nutrition 0.000 description 6
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 6
- 230000036541 health Effects 0.000 description 6
- 230000001900 immune effect Effects 0.000 description 6
- 230000004968 inflammatory condition Effects 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 229960000060 monoclonal antibodies Drugs 0.000 description 6
- 238000006386 neutralization reaction Methods 0.000 description 6
- 229920002704 polyhistidine Polymers 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 210000004369 Blood Anatomy 0.000 description 5
- 102100006435 CSF3 Human genes 0.000 description 5
- 230000036947 Dissociation constant Effects 0.000 description 5
- 108010029961 Filgrastim Proteins 0.000 description 5
- 229960004177 Filgrastim Drugs 0.000 description 5
- 230000033115 angiogenesis Effects 0.000 description 5
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 239000006143 cell culture media Substances 0.000 description 5
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000006011 modification reaction Methods 0.000 description 5
- 238000010369 molecular cloning Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 230000001225 therapeutic Effects 0.000 description 5
- 210000003719 B-Lymphocytes Anatomy 0.000 description 4
- 241000283707 Capra Species 0.000 description 4
- 229960002433 Cysteine Drugs 0.000 description 4
- 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 4
- 210000003958 Hematopoietic Stem Cells Anatomy 0.000 description 4
- 108010058683 Immobilized Proteins Proteins 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- 210000002540 Macrophages Anatomy 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 108090000526 Papain Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 206010046851 Uveitis Diseases 0.000 description 4
- 150000001412 amines Chemical class 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000002860 competitive Effects 0.000 description 4
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 4
- 230000001809 detectable Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 201000002491 encephalomyelitis Diseases 0.000 description 4
- 230000001976 improved Effects 0.000 description 4
- 238000010348 incorporation Methods 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 210000004962 mammalian cells Anatomy 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000000159 protein binding assay Methods 0.000 description 4
- 230000001105 regulatory Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 101710027066 ALB Proteins 0.000 description 3
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000018358 Immunoglobulins Human genes 0.000 description 3
- 108060003951 Immunoglobulins Proteins 0.000 description 3
- 210000003734 Kidney Anatomy 0.000 description 3
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 3
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 3
- 206010027476 Metastasis Diseases 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 229940055729 Papain Drugs 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 210000002966 Serum Anatomy 0.000 description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 3
- 108010001801 Tumor Necrosis Factor-alpha Proteins 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 238000002784 cytotoxicity assay Methods 0.000 description 3
- 231100000263 cytotoxicity test Toxicity 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 230000001419 dependent Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000002552 dosage form Substances 0.000 description 3
- 229940079593 drugs Drugs 0.000 description 3
- 230000003511 endothelial Effects 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000007429 general method Methods 0.000 description 3
- 230000002068 genetic Effects 0.000 description 3
- 210000004602 germ cell Anatomy 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000011068 load Methods 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 229960000485 methotrexate Drugs 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- 235000019834 papain Nutrition 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 230000003389 potentiating Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000002285 radioactive Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- UCSJYZPVAKXKNQ-HZYVHMACSA-N 1-[(1S,2R,3R,4S,5R,6R)-3-carbamimidamido-6-{[(2R,3R,4R,5S)-3-{[(2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-(methylamino)oxan-2-yl]oxy}-4-formyl-4-hydroxy-5-methyloxolan-2-yl]oxy}-2,4,5-trihydroxycyclohexyl]guanidine Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- WLKSPGHQGFFKGE-UHFFFAOYSA-N 1-chloropropan-2-yl N-(3-chlorophenyl)carbamate Chemical compound ClCC(C)OC(=O)NC1=CC=CC(Cl)=C1 WLKSPGHQGFFKGE-UHFFFAOYSA-N 0.000 description 2
- ZTDCFXVUXBPHCE-UHFFFAOYSA-L 2-[bis(carboxylatomethyl)amino]acetate;hydron;nickel(3+) Chemical compound [H+].[Ni+3].[O-]C(=O)CN(CC([O-])=O)CC([O-])=O ZTDCFXVUXBPHCE-UHFFFAOYSA-L 0.000 description 2
- 101700033661 ACTB Proteins 0.000 description 2
- 102100011550 ACTB Human genes 0.000 description 2
- 101710032514 ACTI Proteins 0.000 description 2
- 102200014890 ATG4B C78A Human genes 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 230000037250 Clearance Effects 0.000 description 2
- 102000000503 Collagen Type II Human genes 0.000 description 2
- 108010041390 Collagen Type II Proteins 0.000 description 2
- 229920000453 Consensus sequence Polymers 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 2
- 229960004397 Cyclophosphamide Drugs 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 229940110715 ENZYMES FOR TREATMENT OF WOUNDS AND ULCERS Drugs 0.000 description 2
- 210000002889 Endothelial Cells Anatomy 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 229910052693 Europium Inorganic materials 0.000 description 2
- 102000005720 Glutathione Transferase family Human genes 0.000 description 2
- 108010070675 Glutathione Transferase family Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 210000004408 Hybridomas Anatomy 0.000 description 2
- 108010000345 Immobilized Antibodies Proteins 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 210000001616 Monocytes Anatomy 0.000 description 2
- 210000003007 Myelin Sheath Anatomy 0.000 description 2
- 102100015197 NAAA Human genes 0.000 description 2
- 101700053699 NAAA Proteins 0.000 description 2
- 210000001672 Ovary Anatomy 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 108091005771 Peptidases Proteins 0.000 description 2
- 102000035443 Peptidases Human genes 0.000 description 2
- 210000003819 Peripheral blood mononuclear cell Anatomy 0.000 description 2
- 208000007400 Relapsing-Remitting Multiple Sclerosis Diseases 0.000 description 2
- 101710043164 Segment-4 Proteins 0.000 description 2
- 210000001179 Synovial Fluid Anatomy 0.000 description 2
- 108091008153 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 210000001744 T-Lymphocytes Anatomy 0.000 description 2
- 229940104230 Thymidine Drugs 0.000 description 2
- 108060008683 Tumor Necrosis Factor Receptors Proteins 0.000 description 2
- 102000003298 Tumor Necrosis Factor Receptors Human genes 0.000 description 2
- 230000036462 Unbound Effects 0.000 description 2
- 101700038759 VP1 Proteins 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000003042 antagnostic Effects 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 230000000890 antigenic Effects 0.000 description 2
- 239000008365 aqueous carrier Substances 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 230000035512 clearance Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000003246 corticosteroid Substances 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000006471 dimerization reaction Methods 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N ethanolamine Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 101700012826 flaA Proteins 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037240 fusion proteins Human genes 0.000 description 2
- 101700005460 hemA Proteins 0.000 description 2
- 239000000185 hemagglutinin Substances 0.000 description 2
- 230000003308 immunostimulating Effects 0.000 description 2
- 230000001861 immunosuppresant Effects 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 230000000051 modifying Effects 0.000 description 2
- 230000000926 neurological Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000004091 panning Methods 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 230000036470 plasma concentration Effects 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000036678 protein binding Effects 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 2
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 210000001519 tissues Anatomy 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- AGNGYMCLFWQVGX-AGFFZDDWSA-N (E)-1-[(2S)-2-amino-2-carboxyethoxy]-2-diazonioethenolate Chemical compound OC(=O)[C@@H](N)CO\C([O-])=C\[N+]#N AGNGYMCLFWQVGX-AGFFZDDWSA-N 0.000 description 1
- RTGDFNSFWBGLEC-SYZQJQIISA-N 2-(morpholin-4-yl)ethyl (4E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-4-methylhex-4-enoate Chemical compound COC1=C(C)C=2COC(=O)C=2C(O)=C1C\C=C(/C)CCC(=O)OCCN1CCOCC1 RTGDFNSFWBGLEC-SYZQJQIISA-N 0.000 description 1
- 238000010600 3H thymidine incorporation assay Methods 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- 101700045663 ACP5 Proteins 0.000 description 1
- 101700007462 ADH1A Proteins 0.000 description 1
- 102100000356 ADH1A Human genes 0.000 description 1
- 101700080270 ADH2 Proteins 0.000 description 1
- 102100001249 ALB Human genes 0.000 description 1
- 108010061171 Abatacept Proteins 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010007562 Adalimumab Proteins 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 229960003896 Aminopterin Drugs 0.000 description 1
- 229940064005 Antibiotic throat preparations Drugs 0.000 description 1
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 description 1
- 229940042052 Antibiotics for systemic use Drugs 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 210000000628 Antibody-Producing Cells Anatomy 0.000 description 1
- 229940042786 Antitubercular Antibiotics Drugs 0.000 description 1
- 102000003916 Arrestin Human genes 0.000 description 1
- 108090000328 Arrestin Proteins 0.000 description 1
- 208000006673 Asthma Diseases 0.000 description 1
- 229950011321 Azaserine Drugs 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N BRL-49594 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 210000000601 Blood Cells Anatomy 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102100017562 C5AR1 Human genes 0.000 description 1
- 101700049499 C5AR1 Proteins 0.000 description 1
- 102100003268 CD14 Human genes 0.000 description 1
- 101700027514 CD14 Proteins 0.000 description 1
- 102100005310 CTLA4 Human genes 0.000 description 1
- 101700054183 CTLA4 Proteins 0.000 description 1
- 101700013560 CUP1 Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 210000004671 Cell-Free System Anatomy 0.000 description 1
- 231100000023 Cell-mediated cytotoxicity Toxicity 0.000 description 1
- 206010057250 Cell-mediated cytotoxicity Diseases 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 206010059150 Cerebrosclerosis Diseases 0.000 description 1
- 210000001612 Chondrocytes Anatomy 0.000 description 1
- 208000007118 Chronic Progressive Multiple Sclerosis Diseases 0.000 description 1
- 230000035700 Clearance Rate Effects 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108009000280 Complement Activation Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 101710038747 EEF1A1 Proteins 0.000 description 1
- 208000000999 Encephalomyelitis, Autoimmune, Experimental Diseases 0.000 description 1
- 210000001163 Endosomes Anatomy 0.000 description 1
- 229940088598 Enzyme Drugs 0.000 description 1
- 229950009760 Epratuzumab Drugs 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 241000160765 Erebia ligea Species 0.000 description 1
- 108010008165 Etanercept Proteins 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N Ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 210000002744 Extracellular Matrix Anatomy 0.000 description 1
- 102100014838 FCGRT Human genes 0.000 description 1
- 102100012694 GAL Human genes 0.000 description 1
- 101700002119 GAL Proteins 0.000 description 1
- 101700058973 GAL4 Proteins 0.000 description 1
- 101700014301 GALK1 Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N Glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- 229960003180 Glutathione Drugs 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 1
- 229940093922 Gynecological Antibiotics Drugs 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006822 Human Serum Albumin Proteins 0.000 description 1
- 108010064750 Humanized Monoclonal Antibodies Proteins 0.000 description 1
- 102000015434 Humanized Monoclonal Antibodies Human genes 0.000 description 1
- 229940072221 IMMUNOGLOBULINS Drugs 0.000 description 1
- 101700061808 IPP Proteins 0.000 description 1
- 108010053490 Infliximab Proteins 0.000 description 1
- 229920001137 Initiator element Polymers 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 210000000554 Iris Anatomy 0.000 description 1
- 208000003456 Juvenile Arthritis Diseases 0.000 description 1
- 206010059176 Juvenile idiopathic arthritis Diseases 0.000 description 1
- 102100013180 KDR Human genes 0.000 description 1
- 101710030888 KDR Proteins 0.000 description 1
- 210000000822 Killer Cells, Natural Anatomy 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 102100011539 LGALS4 Human genes 0.000 description 1
- 101710015850 LGALS4 Proteins 0.000 description 1
- 208000009856 Lung Disease Diseases 0.000 description 1
- 210000001165 Lymph Nodes Anatomy 0.000 description 1
- 108090000028 MMP12 Proteins 0.000 description 1
- 102100000165 MS4A1 Human genes 0.000 description 1
- 101710010909 MS4A1 Proteins 0.000 description 1
- 241001559185 Mammalian rubulavirus 5 Species 0.000 description 1
- 108090000143 Mouse Proteins Proteins 0.000 description 1
- 102000006386 Myelin Proteins Human genes 0.000 description 1
- 108010083674 Myelin Proteins Proteins 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 229920002274 Nalgene Polymers 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000003729 Neprilysin Human genes 0.000 description 1
- 208000008795 Neuromyelitis Optica Diseases 0.000 description 1
- 206010029379 Neutrophilia Diseases 0.000 description 1
- 229960002450 Ofatumumab Drugs 0.000 description 1
- LZMPYSIUWPEIRA-XFXZXTDPSA-N Ofatumumab Chemical compound N1=C2C=3COCCC=3N=CC2=N\C1=C1\NOC=C1 LZMPYSIUWPEIRA-XFXZXTDPSA-N 0.000 description 1
- 102100018954 PECAM1 Human genes 0.000 description 1
- 101710027269 PECAM1 Proteins 0.000 description 1
- 101700007624 PLB Proteins 0.000 description 1
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N PMSF Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 1
- 101700051812 PPA1 Proteins 0.000 description 1
- 210000002741 Palatine Tonsil Anatomy 0.000 description 1
- 229940049954 Penicillin Drugs 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010087702 Penicillinase Proteins 0.000 description 1
- 229950009506 Penicillinase Drugs 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 210000002381 Plasma Anatomy 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- OIGNJSKKLXVSLS-VWUMJDOOSA-N Prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N Prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 229940024999 Proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000018210 Recoverin family Human genes 0.000 description 1
- 108010076570 Recoverin family Proteins 0.000 description 1
- 102000004330 Rhodopsin Human genes 0.000 description 1
- 108090000820 Rhodopsin Proteins 0.000 description 1
- 108010001645 Rituximab Proteins 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 101710044706 SFA1 Proteins 0.000 description 1
- 101700006328 SGT1B Proteins 0.000 description 1
- 235000003534 Saccharomyces carlsbergensis Nutrition 0.000 description 1
- 229940081969 Saccharomyces cerevisiae Drugs 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 108020004688 Small Nuclear RNA Proteins 0.000 description 1
- 229940005581 Sodium Lactate Drugs 0.000 description 1
- NGSFWBMYFKHRBD-UHFFFAOYSA-M Sodium lactate Chemical compound [Na+].CC(O)C([O-])=O NGSFWBMYFKHRBD-UHFFFAOYSA-M 0.000 description 1
- 210000000952 Spleen Anatomy 0.000 description 1
- 229960005322 Streptomycin Drugs 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102100009912 TEAD1 Human genes 0.000 description 1
- 101700066199 TEAD1 Proteins 0.000 description 1
- 101710005270 TEF1 Proteins 0.000 description 1
- 101700026084 THY1 Proteins 0.000 description 1
- 108060008443 TPPP Proteins 0.000 description 1
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 description 1
- 206010047461 Viral infection Diseases 0.000 description 1
- 208000001756 Virus Disease Diseases 0.000 description 1
- 229960003697 abatacept Drugs 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating Effects 0.000 description 1
- 230000003213 activating Effects 0.000 description 1
- 230000001154 acute Effects 0.000 description 1
- 229960002964 adalimumab Drugs 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 101710025952 adhT Proteins 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000000240 adjuvant Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 238000003450 affinity purification method Methods 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229930002945 all-trans-retinaldehyde Natural products 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003110 anti-inflammatory Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000001580 bacterial Effects 0.000 description 1
- 239000012523 bacterial endotoxin Substances 0.000 description 1
- 108010051561 belimumab Proteins 0.000 description 1
- 229960003270 belimumab Drugs 0.000 description 1
- 229960000626 benzylpenicillin Drugs 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 102000024070 binding proteins Human genes 0.000 description 1
- 108091007650 binding proteins Proteins 0.000 description 1
- 230000003115 biocidal Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000002051 biphasic Effects 0.000 description 1
- 230000000903 blocking Effects 0.000 description 1
- 210000004271 bone marrow stromal cells Anatomy 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L cacl2 Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 101700018328 ccdB Proteins 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 230000001413 cellular Effects 0.000 description 1
- 230000014564 chemokine production Effects 0.000 description 1
- 230000001684 chronic Effects 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 229960005188 collagen Drugs 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- LCGLNKUTAGEVQW-UHFFFAOYSA-N dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 1
- 201000009910 diseases by infectious agent Diseases 0.000 description 1
- 150000002019 disulfides Chemical class 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 108010007604 epratuzumab Proteins 0.000 description 1
- 229960000403 etanercept Drugs 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 108010086781 golimumab Proteins 0.000 description 1
- 229960001743 golimumab Drugs 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth media Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 125000004435 hydrogen atoms Chemical group [H]* 0.000 description 1
- 230000002209 hydrophobic Effects 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 230000003100 immobilizing Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 229960000598 infliximab Drugs 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 229940079866 intestinal antibiotics Drugs 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 201000002215 juvenile rheumatoid arthritis Diseases 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000005567 liquid scintillation counting Methods 0.000 description 1
- 239000012516 mab select resin Substances 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229960004866 mycophenolate mofetil Drugs 0.000 description 1
- 230000010807 negative regulation of binding Effects 0.000 description 1
- 230000035407 negative regulation of cell proliferation Effects 0.000 description 1
- 108010068617 neonatal Fc receptor Proteins 0.000 description 1
- 210000002569 neurons Anatomy 0.000 description 1
- 230000003472 neutralizing Effects 0.000 description 1
- 239000002687 nonaqueous vehicle Substances 0.000 description 1
- 230000003000 nontoxic Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 108010052070 ofatumumab Proteins 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 229940005935 ophthalmologic Antibiotics Drugs 0.000 description 1
- 210000000056 organs Anatomy 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 230000000242 pagocytic Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000000546 pharmaceutic aid Substances 0.000 description 1
- 230000000275 pharmacokinetic Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 108091008117 polyclonal antibodies Proteins 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 229960005205 prednisolone Drugs 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000069 prophylaxis Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000002797 proteolythic Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 239000003638 reducing agent Substances 0.000 description 1
- 230000002207 retinal Effects 0.000 description 1
- 235000020945 retinal Nutrition 0.000 description 1
- 239000011604 retinal Substances 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000003248 secreting Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 125000003607 serino group Chemical group [H]OC(=O)C([H])(N([H])*)C([H])([H])O[H] 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000003584 silencer Effects 0.000 description 1
- 229920001255 small nuclear ribonucleic acid Polymers 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- 239000001540 sodium lactate Substances 0.000 description 1
- 235000011088 sodium lactate Nutrition 0.000 description 1
- 101710011036 stiI Proteins 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 230000002459 sustained Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 102000003995 transcription factors Human genes 0.000 description 1
- 108090000464 transcription factors Proteins 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000002103 transcriptional Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
- 210000004881 tumor cells Anatomy 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000017613 viral reproduction Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
Discloses proteins that bind to G-CSFR and inhibit growth of colony forming units - granulocytes (CFU-G) from CD24+ bone marrow cells grown in the presence of G-CSF with an IC50 of at least about 0.2 NM. Also discloses are proteins that bind to both human and cynomolgus monkey G-CSFR with a similar affinity and neutralise G-CSF signalling. Also discloses proteins that bind specifically to the same epitope in human hG-CSFR as that bound by antibody C1.2. Also disclosed are proteins that bind to specific mutants of a domain of G-CSFR and competitively inhibit binding of C1.2. Also disclosed are proteins having specified CDR regions for heavy and light chains that bind hG-CSFR and neutralise G-CSF signalling. Further disclosed are related nucleic acids, cells expressing those nucleic acids and compositions comprising the proteins. affinity and neutralise G-CSF signalling. Also discloses proteins that bind specifically to the same epitope in human hG-CSFR as that bound by antibody C1.2. Also disclosed are proteins that bind to specific mutants of a domain of G-CSFR and competitively inhibit binding of C1.2. Also disclosed are proteins having specified CDR regions for heavy and light chains that bind hG-CSFR and neutralise G-CSF signalling. Further disclosed are related nucleic acids, cells expressing those nucleic acids and compositions comprising the proteins.
Description
"ANTIBODIES AGAINST G-CSFR AND USES THEREOF"
RELATED APPLICATION
The present application claims priority from US Provisional Patent Application
No. 61/496,351 entitled “Antibodies against G-CSFR and uses thereof” filed on 13
June 2011, the entire contents of which are hereby incorporated by referenced.
SEQUENCE LISTING
The present application is filed together with a Sequence Listing in electronic
form. The entire contents of the Sequence Listing are hereby incorporated by
reference.
FIELD
The present disclosure relates to antibodies that bind to granulocyte-colony
stimulating factor receptor (G-CSFR) receptor and uses thereof, e.g., in therapy.
BACKGROUND
Granulocyte colony-stimulating factor (G-CSF) is a major regulator of
granulocyte production. G-CSF is produced by bone marrow stromal cells, endothelial
cells, macrophages, and fibroblasts, and production is induced by inflammatory stimuli.
G-CSF acts through the G-CSF receptor (G-CSFR), which is expressed on early
myeloid progenitors, mature neutrophils, monocytes/macrophages, T and B
lymphocytes and endothelial cells. Mice deficient in G-CSF or the G-CSFR exhibit
marked neutropenia, demonstrating the importance of G-CSF in steady-state
granulopoiesis. However, G-CSF appears to be dispensable for emergency
granulopoiesis, e.g., in response to an infection. G-CSF increases the production and
release of neutrophils, mobilizes hematopoietic stem and progenitor cell, and modulates
the differentiation, lifespan, and effector functions of mature neutrophils. G-CSF may
also exert effects on macrophages, including expansion of monocyte/macrophage
numbers, enhancement of phagocytic function, and regulation of inflammatory
cytokine and chemokine production. G-CSF has also been shown to mobilize
endothelial progenitor cells and induce or promote angiogenesis.
While G-CSF is used therapeutically, e.g., to treat neutropenia and/or mobilize
hematopoietic stem cells, it also has negative actions in some conditions, e.g.,
inflammatory conditions and/or cancer. For example, administration of G-CSF
exacerbates rheumatoid arthritis (RA), murine collagen-induced arthritis (CIA) and a
passive transfer model of CIA in rats. G-CSF has been found in the serum and synovial
fluid of RA patients. Furthermore, interleukin (IL)-1 and tumor necrosis factor α
(TNFα), which are found at increased levels in patients suffering from RA, induce the
production of G-CSF by human synovial fibroblasts and chondrocytes. Mice deficient
in G-CSF are resistant to the induction of acute and chronic inflammatory arthritis.
G-CSF has also been shown to play a role in multiple sclerosis (MS). For
example, G-CSF enhances adhesion of an auto-reactive T cell line model of MS to
extracellular matrix as effectively as interferon γ and TNFα, which are known to
exacerbate MS symptoms. Moreover, G-CSF deficient mice are resistant to
development of experimental autoimmune encephalomyelitis (EAE).
G-CSF and G-CSFR have also been tied to cancer, with studies showing that
this signaling pathway contributes to chemotherapy resistance, growth, survival,
invasiveness and metastasis of various cancers. Moreover, G-CSF has been shown to
induce to angiogenesis, a process important in the development of solid tumors.
It will be clear to the skilled person from the foregoing, that there is a need in
the art for reagents that reduce the signaling of G-CSF through the G-CSFR.
Exemplary agents will be suitable for use as therapeutics, e.g., to treat or prevent a G-
CSF-mediated condition.
SUMMARY
The present inventors have produced a class of proteins comprising antibody
binding sites (e.g., Fabs and antibodies) that bind to human G-CSFR (hG-CSFR) and
potently neutralize G-CSF signaling, e.g., prevent formation of granulocytes from
CD34 bone marrow cells and/or prevent cell proliferation in response to G-CSF and/or
reduce or prevent neutrophilia induced by administration of G-CSF. A class of proteins
identified by the inventors also cross-react cynomolgus monkey G-CSFR (cynoG-
CSFR), which facilitates pre-clinical studies with the proteins. A class of proteins
identified by the inventors bind to hG-CSFR with high affinity. A class of proteins
identified by the inventors are human antibodies, which are suitable for treatment of a
variety of conditions.
The present disclosure provides a protein comprising an antigen binding site of
an antibody, wherein the antigen binding site binds to hG-CSFR and neutralizes G-CSF
signaling, and wherein the protein inhibits growth of colony forming units –
granulocytes (CFU-G) from CD34 bone marrow cells grown in the presence of G-CSF
with an IC of at least about 0.2nM. For example, the IC is 0.1nM or less, for
50 50
example, 0.09nM or less, or 0.08nM or less, or 0.07nM or less, or 0.06nM or less
or0.05nM or less. In one example, the IC is 0.04nM or less. In another example, the
IC is 0.02nM or less. Methods for assessing IC of a protein in such an assay are
50 50
described herein. For example, the IC is determined in the presence of 10ng/ml of
hG-CSF.
In one example, the IC is determined by culturing CD34 bone marrow cells in
the presence of 10ng/ml stem cell factor and 10ng/ml hG-CSF. For example, the cells
are grown in semi-solid cell culture medium. In one example, the CFU-G are
enumerated after 14 days of culture.
The present disclosure additionally or alternatively provides a protein
comprising an antigen binding site of an antibody, wherein the antigen binding site
binds to both human and cynomolgus monkey G-CSFR with a similar affinity and
neutralizes G-CSF signaling. Such proteins are advantageous since they facilitate pre-
clinical studies in non-human mammals.
In one example, the affinity of the protein is determined using a biosensor, e.g.,
by surface plasmon resonance. For example, the ligand binding region or soluble hG-
CSFR or soluble cynoG-CSFR or hG-CSFR-Fc or cyno-G-CSFR-Fc is immobilized
and the affinity of the protein of the disclosure is determined.
The present disclosure additionally provides a provides a protein comprising an
antigen binding site of an antibody, wherein the antigen binding site binds specifically
to the same epitope in hG-CSFR as that bound by C1.2 (comprising a heavy chain
variable region (V ) comprising a sequence set forth in SEQ ID NO: 2 and a light chain
variable region (V ) comprising a sequence set forth in SEQ ID NO: 3) or C1.2G
(comprising a V comprising a sequence set forth in SEQ ID NO: 4 and a V
comprising a sequence set forth in SEQ ID NO: 5).
The present disclosure additionally or alternatively provides a protein
comprising an antigen binding site of an antibody, wherein (i) the protein competitively
inhibits binding of C1.2 (comprising a V comprising a sequence set forth in SEQ ID
NO: 2 and a V comprising a sequence set forth in SEQ ID NO: 3) or C1.2G
(comprising a V comprising a sequence set forth in SEQ ID NO: 4 and a V
comprising a sequence set forth in SEQ ID NO: 5) to hG-CSFR; (ii) the protein
neutralizes G-CSF signaling; and (iii) the level of binding of the protein to a
polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of:
(a) the arginine at position 287 of SEQ ID NO:1;
(b) the histidine at position 237 of SEQ ID NO:1;
(c) the methionine at position 198 of SEQ ID NO:1;
(d) the tyrosine at position 172 of SEQ ID NO:1;
(e) the leucine at position 171 of SEQ ID NO:1; or
(f) the leucine at position 111 of SEQ ID NO:1
is lower than the level of binding of the protein to a polypeptide of SEQ ID NO: 1.
The present disclosure additionally or alternatively provides a protein
comprising an antigen binding site of an antibody, wherein (i) the protein competitively
inhibits binding of C1.2 (comprising a V comprising a sequence set forth in SEQ ID
NO: 2 and a V comprising a sequence set forth in SEQ ID NO: 3) or C1.2G
(comprising a V comprising a sequence set forth in SEQ ID NO: 4 and a V
comprising a sequence set forth in SEQ ID NO: 5) to hG-CSFR; (ii) the protein
neutralizes G-CSF signaling; and (iii) preferentially binds to a polypeptide of SEQ ID
NO: 1 relative to its ability to bind to a polypeptide of SEQ ID NO: 1 in which an
alanine is substituted for any one of:
(a) the arginine at position 287 of SEQ ID NO:1;
(b) the histidine at position 237 of SEQ ID NO:1;
(c) the methionine at position 198 of SEQ ID NO:1;
(d) the tyrosine at position 172 of SEQ ID NO:1;
(e) the leucine at position 171 of SEQ ID NO:1; or
(f) the leucine at position 111 of SEQ ID NO:1.
The present disclosure additionally or alternatively provides a protein
comprising an antigen binding site of an antibody, wherein (i) the protein binds to hG-
CSFR; (ii) the protein neutralizes G-CSF signaling; and (iii) the level of binding of the
protein to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one
(a) the arginine at position 287 of SEQ ID NO:1
(b) the histidine at position 237 of SEQ ID NO:1;
(c) the methionine at position 198 of SEQ ID NO:1;
(d) the tyrosine at position 172 of SEQ ID NO:1;
(e) the leucine at position 171 of SEQ ID NO:1; or
(f) the leucine at position 111 of SEQ ID NO:1
is lower than the level of binding of the protein to a polypeptide of SEQ ID NO: 1.
The present disclosure additionally or alternatively provides a protein
comprising an antigen binding site of an antibody, wherein (i) the protein binds to hG-
CSFR; (ii) the protein neutralizes G-CSF signaling; and (iii) preferentially binds to a
polypeptide of SEQ ID NO: 1 relative to its ability to bind to a polypeptide of SEQ ID
NO: 1 in which an alanine is substituted for any one of:
(a) the arginine at position 287 of SEQ ID NO:1
(b) the histidine at position 237 of SEQ ID NO:1;
(c) the methionine at position 198 of SEQ ID NO:1;
(d) the tyrosine at position 172 of SEQ ID NO:1;
(e) the leucine at position 171 of SEQ ID NO:1; or
(f) the leucine at position 111 of SEQ ID NO:1.
In one example, the level of binding of the protein to the polypeptide comprising
the alanine substitution is reduced by at least about 10 fold or 20 fold or 50 fold or 100
fold or 150 fold or 200 fold compared to the binding of the protein to the polypeptide of
SEQ ID NO: 1. Preferably, the level of binding of the protein to the polypeptide
comprising the alanine substitution is reduced by at least about 50 fold. Preferably, the
level of binding of the protein to the polypeptide comprising the alanine substitution is
reduced by at least about 60 fold.
In one example, the antigen binding site of the protein does not detectably bind
to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the arginine at
position 287 of SEQ ID NO: 1.
In one example, the level of binding is assessed using a biosensor, e.g., by
surface plasmon resonance. For example, the protein is immobilized and the level of
binding to a polypeptide of SEQ ID NO: 1 or to a form of the polypeptide comprising
an alanine substitution is determined.
Additional forms of a polypeptide comprising the amino acids of SEQ ID NO: 1
with or without other substitutions bound or not significantly bound or not detectably
bound by a protein of the present disclosure are described herein and are to be taken to
apply mutatis mutandis to the present examples of the disclosure.
In one example, the antigen binding site cross-reacts with:
(i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the lysine
at position 167 of SEQ ID NO: 1; and/or
(ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the
histidine at position 168 of SEQ ID NO: 1.
In one example, the antigen binding site additionally cross-reacts with a
polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the leucine at
position 169 of SEQ ID NO: 1
In one example, the protein competitively inhibits the binding of C1.2
(comprising a V comprising a sequence set forth in SEQ ID NO: 2 and a V
comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a V
comprising a sequence set forth in SEQ ID NO: 4 and a V comprising a sequence set
forth in SEQ ID NO: 5) to one or more of:
(i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the lysine
at a position 167 of SEQ ID NO: 1; and/or
(ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the
histidine at position 168 of SEQ ID NO: 1.
In one example, a protein described herein according to any example binds to an
epitope comprising residues within one or two or three or four regions selected from
111-115, 170-176, 218-234 and/or 286-300 of SEQ ID NO: 1.
In one example, upon binding of a protein described herein according to any
example to a polypeptide of SEQ ID NO: 1 and cleavage using protelolytic enzymes
remains bound to one or two or three or four peptides comprising or consisting of
amino acids 111-115 of SEQ ID NO: 1 or amino acids 170-176 of SEQ ID NO: 1 or
amino acids 218-234 of SEQ ID NO: 1 or amino acids 286-300 of SEQ ID NO: 1.
In one example, the protein binds to a conformational epitope.
The present disclosure additionally or alternatively provides a protein that binds
to hG-CSFR and neutralizes G-CSF signaling, the protein comprising at least one of:
(i) a V comprising a complementarity determining region (CDR) 1 comprising a
sequence set forth in SEQ ID NO: 6, a CDR2 comprising a sequence set forth in SEQ
ID NO: 7 and a CDR3 comprising a sequence at least about 55% identity to the
sequence set forth in SEQ ID NO: 8;
(ii) a V comprising a sequence at least about 80%, such as 85% or 90% or 91% or
92% or 93% or 94% or 95% or 96% or 97% or 98% or 99% identical to a sequence set
forth in SEQ ID NO: 2 and/or 4;
(iii) a V comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 9, a
CDR2 comprising a sequence set forth in SEQ ID NO: 10 and a CDR3 comprising a
sequence at least about 33% identity to the sequence set forth in SEQ ID NO: 11; and
(iv) a V comprising a sequence at least about 80%, such as 85% or 90% or 91% or
92% or 93% or 94% or 95% or 96% or 97% or 98% or 99%identical to a sequence set
forth in SEQ ID NO: 3 and/or 5.
Such a protein can comprise any one or more of the functional activities
described herein, e.g., preferential binding to a polypeptide of SEQ ID NO: 1 relative to
the level of binding of a polypeptide of SEQ ID NO: 1 in which an alanine is
substituted for any one of:
(a) the arginine at position 287 of SEQ ID NO:1
(b) the histidine at position 237 of SEQ ID NO:1;
(c) the methionine at position 198 of SEQ ID NO:1;
(d) the tyrosine at position 172 of SEQ ID NO:1;
(e) the leucine at position 171 of SEQ ID NO:1; or
(f) the leucine at position 111 of SEQ ID NO:1.
In one example, the percentage identity at (ii) is at least about 95%.
In one example, the percentage identity at (iv) is at least about 94%.
In one example, differences between the recited sequence and the protein are
substitutions.
The skilled artisan will be capable of determining sites for mutations to a protein
of the disclosure, e.g., within a framework region of a variable region containing
protein. Moreover, the inventors have identified numerous sites in a V CDR3 and a
V CDR3 that can be mutated as well as numerous mutations that maintain activity of a
protein of the disclosure. For example a mutation, e.g., a substitution is within one or
more (e.g., 2 or 3 or 4) of the four C-terminal residues of HCDR3 and/or one or more
(e.g., 2 or 3 or 4 or 5 or 6) of the N-terminal or C-terminal residues of LCDR3. In one
example, the N-terminal five amino acids of V CDR3 are LGELG. In one example,
the three N-terminal amino acids of V CDR3 are QQS and/or the three C-terminal
amino acids of V CDR3 are PLT.
In one example, the V comprises a CDR3 comprising a sequence
LGELGX X X X , wherein:
1 2 3 4
X is selected from the group consisting of tryptophan, glutamine, methionine, serine,
phenylalanine, glutamic acid and histidine and/or is a neutral amino acid, such as
tryptophan, glutamine or methionine, for example, the amino acid is tryptophan;
X is an amino acid selected from the group consisting of phenylalanine, tyrosine,
methionine, serine, glycine and isoleucine, for example is phenylalanine, tyrosine,
methionine or serine, for example, the amino acid is phenylalanine;
X is an amino acid selected from the group consisting of aspartic acid, methionine,
glutamine, serine, leucine, valine, arginine and histidine, for example, is proline,
glutamic acid, alanine, leucine, phenylalanine or tyrosine, for example, the amino acid
is aspartic acid; and
X is any amino acid or an amino acid selected from the group consisting of proline,
glutamic acid, alanine, leucine, phenylalanine, tyrosine, threonine, asparagine, aspartic
acid, serine, glycine, arginine, and lysine, for example, the amino acid is proline.
In one example, the V comprises a CDR3 comprising a sequence
X X X X X X X X X , wherein:
1 2 3 4 5 6 7 8 9
X is an amino acid selected from the group consisting of glutamine, glutamic acid,
histidine, alanine and serine and/or is a hydrophilic amino acid, such as glutamine or
glutamic acid, for example, the amino acid is glutamine;
X is an amino acid selected from the group consisting of glutamine, valine,
phenylalanine, asparagine and glutamic acid, for example, the amino acid is glutamine;
X is an amino acid selected from the group consisting of serine and glycine, for
example, the amino acid is serine;
X is an amino acid selected from the group consisting of tryptophan, methionine,
phenylalanine, tyrosine, isoleucine and leucine, for example, the amino acid is
tryptophan or tyrosine;
X is any amino acid or an amino acid selected from the group consisting of glutamic
acid, methionine, glutamine, tryptophan, serine, valine, asparagine, glycine, alanine,
arganine, histidine, tyrosine, lysine and threonine, for example, the amino acid is
serine;
X is an amino acid selected from the group consisting of tyrosine, methionine,
isoleucine and threonine, for example, the amino acid is methionine, tyrosine or
threonine;
X is an amino acid selected from the group consisting of proline, alanine, histidine,
glycine and lysine, for example the amino acid is proline;
X is an amino acid selected from the group consisting of leucine, glutamine,
methionine, alanine, phenylalanine, isoleucine, lysine, histidine and glycine, for
example, the amino acid is leucine;
X is any amino acid or an amino acid selected from the group consisting of threonine,
phenylalanine, tyrosine, methionine, lysine, serine, histidine, proline, tryptophan,
isoleucine, glutamine, glycine and valine, for example, the amino acid is threonine.
The present disclosure additionally or alternatively provides a protein (e.g., an
antibody) that binds to hG-CSFR and neutralizes G-CSF signaling, the protein
comprising at least one variable region of an antibody selected from the group
consisting of:
(i) a V comprising an amino acid sequence set forth in SEQ ID NO: 2;
(ii) a V comprising an amino acid sequence set forth in SEQ ID NO: 3;
(iii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4;
(iv) a V comprising an amino acid sequence set forth in SEQ ID NO: 5;
(v) a V comprising an amino acid sequence set forth in SEQ ID NO: 14;
(vi) a V comprising an amino acid sequence set forth in SEQ ID NO: 15;
(vii) a V comprising an amino acid sequence set forth in SEQ ID NO: 16;
(viii) a V comprising an amino acid sequence set forth in SEQ ID NO: 17;
(ix) a V comprising an amino acid sequence set forth in SEQ ID NO: 18;
(x) a V comprising an amino acid sequence set forth in SEQ ID NO: 19;
(xi) a V comprising an amino acid sequence set forth in SEQ ID NO: 20;
(xii) a V comprising an amino acid sequence set forth in SEQ ID NO: 21;
(xiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 22;
(xiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 23;
(xv) a V comprising an amino acid sequence set forth in SEQ ID NO: 24;
(xvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 25;
(xvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 26;
(xviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 27;
(xix) a V comprising an amino acid sequence set forth in SEQ ID NO: 28;
(xx) a V comprising an amino acid sequence set forth in SEQ ID NO: 29;
(xxi) a V comprising an amino acid sequence set forth in SEQ ID NO: 30;
(xxii) a V comprising an amino acid sequence set forth in SEQ ID NO: 31;
(xxiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 32;
(xxiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 33;
(xxv) a V comprising an amino acid sequence set forth in SEQ ID NO: 34;
(xxvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 35;
(xxvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 36;
(xxviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 37;
(xxix) a V comprising an amino acid sequence set forth in SEQ ID NO: 38;
(xxx) a V comprising an amino acid sequence set forth in SEQ ID NO: 39;
(xxxi) a V comprising an amino acid sequence set forth in SEQ ID NO: 40;
(xxxii) a V comprising an amino acid sequence set forth in SEQ ID NO: 41;
(xxxiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 42;
(xxxiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 43;
(xxxv) a V comprising an amino acid sequence set forth in SEQ ID NO: 44;
(xxxvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 45;
(xxxvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 46;
(xxxviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 47;
(xxix) a V comprising an amino acid sequence set forth in SEQ ID NO: 48;
(xl) a V comprising an amino acid sequence set forth in SEQ ID NO: 49;
(xli) a V comprising an amino acid sequence set forth in SEQ ID NO: 50;
(xlii) a V comprising an amino acid sequence set forth in SEQ ID NO: 51;
(xliii) a V comprising an amino acid sequence set forth in SEQ ID NO: 52;
(xliv) a V comprising an amino acid sequence set forth in SEQ ID NO: 53;
(xlv) a V comprising an amino acid sequence set forth in SEQ ID NO: 54;
(xlvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 55;
(xlvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 56;
(xlviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 57;
(xlix) a V comprising an amino acid sequence set forth in SEQ ID NO: 58;
(l) a V comprising an amino acid sequence set forth in SEQ ID NO: 59;
(li) a V comprising an amino acid sequence set forth in SEQ ID NO: 60;
(lii) a V comprising an amino acid sequence set forth in SEQ ID NO: 61;
(liii) a V comprising an amino acid sequence set forth in SEQ ID NO: 62; and
(liv) a V comprising an amino acid sequence set forth in SEQ ID NO: 63.
In one example, a protein described herein comprises at least a V and a V ,
wherein the V and V bind to form a Fv comprising an antigen binding domain. The
skilled artisan will understand that the antigen binding domain comprises the binding
site of the antibody.
In one example, the V and the V are in a single polypeptide chain. For
example, the protein is:
(i) a single chain Fv fragment (scFv);
(ii) a dimeric scFv (di-scFv); or
(iii) at least one of (i) and/or (ii) linked to a constant region of an antibody, Fc or a
heavy chain constant domain (C ) 2 and/or C 3.
In one example, the V and V are in separate polypeptide chains.
For example, the protein is:
(i) a diabody;
(ii) a triabody;
(iii) a tetrabody;
(iv) a Fab;
(v) a F(ab’) ;
(vi) a Fv; or
(vii) one of (i) to (vi) linked to a constant region of an antibody, Fc or a heavy chain
constant domain (C ) 2 and/or C 3.
The foregoing proteins (described in the previous two lists) can also be referred
to as antigen binding domains of antibodies.
In one example, the protein is an antibody. In one example, the antibody is a
naked antibody.
In one example, a protein is chimeric, de-immunized, humanized, human or
primatized.
In one example, the protein or antibody is human.
The present disclosure additionally or alternatively provides an antibody that
binds to hG-CSFR and neutralizes G-CSF signaling, the antibody comprising:
(i) a V comprising an amino acid sequence set forth in SEQ ID NO: 2 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 3;
(ii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 5;
(iii) a V comprising an amino acid sequence set forth in SEQ ID NO: 15 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 14;
(iv) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 16;
(v) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 17;
(vi) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 18;
(vii) a V comprising an amino acid sequence set forth in SEQ ID NO: 20 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 19;
(viii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 21;
(ix) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 22;
(x) a V comprising an amino acid sequence set forth in SEQ ID NO: 24 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 23;
(ix) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 25;
(x) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 26;
(xi) a V comprising an amino acid sequence set forth in SEQ ID NO: 28 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 27;
(xii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 29;
(xiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 31 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 30;
(xiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 33 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 32;
(xv) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 34;
(xvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 36 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 35;
(xvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 38 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 37;
(xviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 40 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 39;
(xix) a V comprising an amino acid sequence set forth in SEQ ID NO: 42 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 41;
(xx) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 43;
(xxi) a V comprising an amino acid sequence set forth in SEQ ID NO: 45 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 44;
(xxii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 46;
(xxiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 47;
(xxiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 49 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 48;
(xxv) a V comprising an amino acid sequence set forth in SEQ ID NO: 51 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 50;
(xxvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 53 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 52;
(xxvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 54;
(xxviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 55 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 5;
(xxix) a V comprising an amino acid sequence set forth in SEQ ID NO: 57 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 56;
(xxx) a V comprising an amino acid sequence set forth in SEQ ID NO: 59 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 58;
(xxxi) a V comprising an amino acid sequence set forth in SEQ ID NO: 61 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 60;
(xxxii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 62;
(xxxiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 63; and
(xxxix) a V comprising three CDRs of a V set forth in any one or more of (i) to
(xxxiii) and a V comprising three CDRs of a V set forth in any one or more of (i) to
(xxxiii).
Sequences of exemplary V and V are described in Table 3, wherein the recited
V or V CDR3 sequence is substituted for the corresponding sequence in the V or V
H L H L
of C1.2 or C1.2G as described herein.
In one example, the present disclosure provides an antibody that binds to hG-
CSFR and neutralizes G-CSF signaling, the antibody comprising:
(i) a V comprising an amino acid sequence set forth in SEQ ID NO: 2 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 3; or
(ii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V
comprising an amino acid sequence set forth in SEQ ID NO: 5.
The present disclosure additionally or alternatively provides an antibody
comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:
64 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65.
In one example, the antibody binds to hG-CSFR and neutralizes G-CSF signaling.
The present disclosure additionally or alternatively provides an antibody
comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:
68 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65.
In one example, the antibody binds to hG-CSFR and neutralizes G-CSF signaling.
The present disclosure additionally or alternatively provides an antibody
comprising one heavy chain comprising an amino acid sequence set forth in SEQ ID
NO: 64 and one heavy chain comprising an amino acid sequence set forth in SEQ ID
NO: 68 and two light chains comprising an amino acid sequence set forth in SEQ ID
NO: 65. In one example, the antibody binds to hG-CSFR and neutralizes G-CSF
signaling.
Reference herein to a protein or antibody that “binds to” hG-CSFR provides
literal support for a protein or antibody that “binds specifically to” hG-CSFR.
In one example, a protein or antibody described herein does not significantly
bind to mouse G-CSFR and/or does not detectably bind to mouse G-CSFR.
In one example, a protein or antibody described herein according to any
example competitively inhibits binding of C1.2 and/or C1.2G to hG-CSFR or a cell
expressing same or SEQ ID NO: 1 or a soluble hG-CSFR (e.g., comprising amino acids
1-311 of SEQ ID NO: 1 fused to a Fc region of an antibody).
In one example, a protein or antibody described herein binds to a ligand binding
region of hG-CSFR and a ligand binding region of cynoG-CSFR with similar affinity.
In one example, the protein binds to soluble hG-CSFR and soluble cynoG-CSFR with
similar affinity. In one example, the protein binds to a polypeptide comprising SEQ ID
NO: 1 and to a polypeptide comprising SEQ ID NO: 67 with similar affinity. In one
example, the protein binds to hG-CSFR-Fc and cynoG-CSFR-Fc as described herein
with similar affinity. In one example, the affinity is at least about 2nM, for example, at
least about 1.5nM, such as at least about 1.2nM, 1.1nM or 1nM. In one example, the
0.5nM, such as, at least about 0.46nM or 0.45nM or 0.40nM or 0.39nM. In another
example, the affinity is at least about 0.1nM, such as at least about 0.09nM, for
example, at least about 0.08nM. In one example, the level of binding is assessed using
a biosensor, e.g., by surface plasmon resonance. For example, the ligand binding
region or soluble hG-CSFR or soluble cynoG-CSFR or hG-CSFR-Fc or cyno-G-CSFR-
Fc is immobilized and the level of binding to a protein of the disclosure is determined.
In another example, the protein of the disclosure is immobilized on, for
example, a biosensor and the level of binding of the ligand binding region or soluble
hG-CSFR or soluble cynoG-CSFR or hG-CSFR-Fc or cyno-G-CSFR-Fc is determined.
For example, the level of binding to the extracellular domain of hG-CSFR or cynoG-
CSFR is determined. . In accordance with this example, the affinity of the protein for
the extracellular domain of cynoG-CSFR is at least about 1nM, such as at least about
0.9nM, for example, at least about 0.75nM. For example, the affinity is at least about
0.7nM, such as at least about 0.6nM, for example, at least about 0.5nM. In one
example, the affinity is about 0.5nM. Alternatively, or additionally, the affinity of the
protein for the extracellular domain of hG-CSFR is at least about 7nM or 6nM or 5nM,
such as at least about 4nM, for example, at least about 3nM, e.g., at least about 2.5nM.
For example, the affinity is at least about 2.4 or 2.5nM.
The present disclosure also provides antigen binding domains or antigen binding
fragments of the foregoing antibodies.
In one example, a protein or antibody as described herein comprises a constant
region of an IgG4 antibody or a stabilized constant region of an IgG4 antibody. In one
example, the protein or antibody comprises an IgG4 constant region with a proline at
position 241 (according to the numbering system of Kabat (Kabat et al., Sequences of
Proteins of Immunological Interest Washington DC United States Department of
Health and Human Services, 1987 and/or 1991)).
The C-terminal lysine of the heavy chain constant region of a whole antibody of
the disclosure may be removed, for example, during production or purification of the
antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of
the antibody. Accordingly, whole antibodies may comprise antibody populations with
all C-terminal lysine residues removed, antibody populations with no C-terminal lysine
residues removed, and antibody populations having a mixture of antibodies with and
without the C-terminal lysine residue. In some examples, the antibody populations
may additionally comprise antibodies in which the C-terminal lysine residue is
removed in one of the heavy chain constant regions. Similarly, a composition of whole
antibodies may comprise the same or a similar mix of antibody populations with or
without the C-terminal lysine residue.
In one example, the stabilized constant region comprises a sequence from
position 119 to position 445 of SEQ ID NO: 64. In one example, the stabilized
constant region comprises a sequence from position 119 to position 444 of SEQ ID
NO: 68. In one example a protein or antibody as described herein or a composition of a
protein or antibody as described herein, comprises a heavy chain constant region,
including a stabilized heavy chain constant region, comprising a mixture of sequences
fully or partially with or without the C-terminal lysine residue.
In one example, an antibody of the disclosure comprises a V disclosed herein
linked or fused to an IgG4 constant region or stabilized IgG4 constant region (e.g., as
discussed above) and the V is linked to or fused to a kappa light chain constant region.
The present disclosure also provides a protein or antibody which inhibits G-
CSF-induced proliferation of a BaF3 cell expressing hG-CSFR with an IC of at least
about 6nM. For example, the IC is 5.9nM or less. In another example, the IC is
50 50
2nM or less or 1nM or less or 0.7nM or less or 0.6nM or less or 0.5nM or less. In one
example, the IC is determined by culturing BaF3 cells (e.g. about 2x10 cells) in the
presence of about 0.5ng/ml hG-CSF, e.g., for about 48 hours. In one example, the
proliferation of the BaF3 cells is determined by measuring 3-(4,5-Dimethylthiazol
yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.
The present disclosure also provides a protein or antibody which inhibits G-
CSF-induced proliferation of a BaF3 cell expressing hG-CSFR with an IC of at least
about 10μg/ml. For example, the IC is 5μg/ml or less. In another example, the IC is
50 50
3μg/ml or less or 2μg/ml or less or 1μg/ml or less. In one example, the IC is about
0.8μg/ml. In one example, the IC is determined by culturing BaF3 cells (e.g. about
1x10 cells) in the presence of about 10ng/ml hG-CSF, e.g., for about 48 hours. In one
example, the proliferation of the BaF3 cells is determined by measuring H-thymidine
incorporation.
In one example, a protein or antibody of the disclosure binds to a soluble hG-
CSFR comprising amino acid 1-311 of SEQ ID NO: 1 expressed as a fusion with an
antibody Fc region (hG-CSFR-Fc) with an affinity of at least about 1.5nM. For
example, the affinity is at least about 0.5nM or 0.4nM or 0.35nM or 0.33nM. In one
example, the affinity of the protein is determined using a biosensor, e.g., by surface
plasmon resonance. For example, the hG-CSFR-Fc is immobilized and the affinity of
the protein of the disclosure is determined.
In one example, a protein or antibody of the disclosure binds to hG-CSFR
expressed on the surface of a cell at an affinity of at least about 1nM, for example, at
least about 0.5nM, such as, at least 0.4nM, for example, at least 0.3nM, such as, at least
0.2nM.
In one example, a protein as described herein according to any example is
capable of reducing the number of neutrophils in circulation when or if administered to
a cynomolgus monkey. For example, the protein reduces the number of neutrophils in
circulation when or if administered to a cynomolgus monkey at a dose of between
0.05m/kg-30mg/kg, preferably between 0.1mg/kg-10mg/kg, e.g., administered at a dose
of 0.1mg/kg or 1mg/kg or 2mg/kg or 5 mg/kg or 10mg/kg. For example, the protein
reduces the number of neutrophils in circulation when or if administered following
administration of G-CSF or filgrastim or a PEGylated form thereof, e.g., when or if the
protein is administered about 12 hours after administration of G-CSF or filgrastim or a
PEGylated form thereof. In one example, the reduction is a 2 fold or 3 fold reduction.
In one example, the neutrophils are reduced about 10-24 hours, e.g., about 12 hours
following administration.
In one example, a protein or antibody as described herein is isolated and/or
recombinant.
In one example, a protein or antibody of the disclosure is conjugated to another
compound, for example, a detectable label or a compound that extends the half-life of
the protein or antibody, such as polyethylene glycol or an albumin binding protein.
The present disclosure also provides a nucleic acid encoding the protein or
antibody of the present disclosure.
In one example, such a nucleic acid is included in an expression construct in
which the nucleic acid is operably linked to a promoter. Such an expression construct
can be in a vector, e.g., a plasmid.
In examples of the disclosure directed to single polypeptide chain proteins, the
expression construct may comprise a promoter linked to a nucleic acid encoding that
polypeptide chain.
In examples directed to multiple polypeptide chains that form a protein, an
expression construct comprises a nucleic acid encoding a polypeptide comprising, e.g.,
a V operably linked to a promoter and a nucleic acid encoding a polypeptide
comprising, e.g., a V operably linked to a promoter.
In another example, the expression construct is a bicistronic expression
construct, e.g., comprising the following operably linked components in 5’ to 3’ order:
(i) a promoter
(ii) a nucleic acid encoding a first polypeptide;
(iii) an internal ribosome entry site; and
(iv) a nucleic acid encoding a second polypeptide,
wherein the first polypeptide comprises a V and the second polypeptide comprises a
V , or vice versa.
The present disclosure also contemplates separate expression constructs one of
which encodes a first polypeptide comprising a V and another of which encodes a
second polypeptide comprising a V . For example, the present disclosure also provides
a composition comprising:
(i) a first expression construct comprising a nucleic acid encoding a polypeptide
comprising a V operably linked to a promoter; and
(ii) a second expression construct comprising a nucleic acid encoding a polypeptide
comprising a V operably linked to a promoter.
The present disclosure also provides an isolated or recombinant cell expressing a
protein of the disclosure.
In one example, the cell comprises the expression construct of the disclosure or:
(i) a first expression construct comprising a nucleic acid encoding a polypeptide
comprising a V operably linked to a promoter; and
(ii) a second expression construct comprising a nucleic acid encoding a polypeptide
comprising a V operably linked to a promoter.
Examples of cells of the present disclosure include bacterial cells, yeast cells,
insect cells or mammalian cells.
The present disclosure additionally provides methods for producing a protein or
antibody of the disclosure. For example, such a method involves maintaining the
expression construct(s) of the disclosure under conditions sufficient for the protein to
be produced.
In one example, a method for producing a protein or antibody of the disclosure
comprises culturing the cell of the disclosure under conditions sufficient for the protein
or antibody to be produced and, optionally, secreted.
In one example, the method for producing a protein of the disclosure
additionally comprises isolating the protein or antibody and, optionally, formulating the
protein or antibody into a pharmaceutical composition.
The present disclosure additionally provides a composition comprising a protein
or antibody of the disclosure and a pharmaceutically acceptable carrier.
The present disclosure additionally provides a composition comprising:
(i) an antibody comprising a heavy chain comprising an amino acid sequence set
forth in SEQ ID NO: 64 and a light chain comprising an amino acid sequence set forth
in SEQ ID NO: 65; and
(ii) (a) an antibody comprising a heavy chain comprising an amino acid sequence
set forth in SEQ ID NO: 64 and a light chain comprising an amino acid
sequence set forth in SEQ ID NO: 65; and/or
(b) an antibody comprising one heavy chain comprising an amino acid
sequence set forth in SEQ ID NO: 64 and one heavy chain comprising an amino
acid sequence set forth in SEQ ID NO: 68 and two light chains comprising an
amino acid sequence set forth in SEQ ID NO: 65,
and, optionally, a pharmaceutically acceptable carrier.
The present disclosure also provides a method for treating or preventing a G-
CSF-mediated condition in a subject, the method comprising administering the protein,
antibody or composition of the disclosure. In this regard, a protein, antibody or
composition can be used to prevent a relapse of a condition, and this is considered
preventing the condition.
In one example, the G-CSF-mediated condition is an autoimmune disease, an
inflammatory disease or cancer. For example, the autoimmune disease or the
inflammatory disease is arthritis, multiple sclerosis, pulmonary inflammation or chronic
obstructive pulmonary disease.
In one example, the method comprises administering an amount of the protein or
antibody sufficient to reduce the number of neutrophils in a subject without inducing
neutropenia.
The present disclosure alternatively or additionally provides a method for
reducing the number of neutrophils in a subject without inducing neutropenia, the
method comprising administering a protein comprising an antigen binding site of an
antibody that binds (or specifically binds) to hG-CSFR to the subject. An exemplary
protein is an antibody or comprises an antigen binding domain thereof (e.g., a V
and/or a V ) or is an antigen binding fragment thereof. Exemplary proteins and
antibodies are described herein.
In one example, a method described herein comprises administering an amount
of the protein or antibody sufficient to reduce the number of neutrophils in a subject
without inducing moderate neutropenia.
In one example, a method described herein comprises administering an amount
of the protein or antibody sufficient to reduce the number of neutrophils in a subject
without inducing severe neutropenia.
In one example, a method described herein comprises administering between
about 0.05mg/kg and 30mg/kg of the protein or antibody. For example, the method
comprising administering between 0.1mg/kg and 10mg/kg or between 0.2mg/kg and
5mg/kg of the protein or antibody. In one example, the method comprises
administering about 0.5-2.0mg/kg of the protein or antibody.
The present disclosure also provides for use of a protein or antibody as
described herein in any example in medicine.
The present disclosure also provides for use of a protein or antibody as
described herein according to any example in the manufacture of a medicament to treat
a G-CSF-mediated condition.
The present disclosure also provides a method for localizing and/or detecting
and/or diagnosing and/or prognosing G-CSF-mediated condition associated with a cell
expressing G-CSFR, the method comprising detecting in vivo a protein or antibody as
described herein bound to the G-CSFR expressing cell, if present, wherein the protein
or antibody is conjugated to a detectable tag.
In one example, the method additionally comprises administering the protein to
the subject.
The present disclosure also provides a method for detecting G-CSFR or a cell
expressing same in a sample, the method comprising contacting the sample with a
protein or antibody as described herein according to any example such that a complex
forms and detecting the complex, wherein detection of the complex is indicative of G-
CSFR or a cell expressing same in the sample.
The present disclosure also provides a method for diagnosing or prognosing a
G-CSF-mediated condition, the method comprising performing a method as described
herein according to any example to detect G-CSFR or a cell expressing same, wherein
detection of the G-CSFR or cell expressing same is diagnostic or prognostic of the
condition.
The present disclosure also provides a kit comprising a protein or antibody as
described herein according to any example packaged with instructions for use in a
method as described herein.
KEY TO SEQUENCE LISTING
SEQ ID NO: 1 – amino acids 25-335 of Homo sapiens G-CSFR (hG-CSFR) with a C-
terminal polyhistidine tag
SEQ ID NO: 2 – V of C1.2
SEQ ID NO: 3 – V of C1.2
SEQ ID NO: 4 – V of C1.2G
SEQ ID NO: 5 – V of C1.2G
SEQ ID NO: 6 - HCDR1 of C1.2
SEQ ID NO: 7 - HCDR2 of C1.2
SEQ ID NO: 8 - HCDR3 of C1.2
SEQ ID NO: 9 - LCDR1 of C1.2
SEQ ID NO: 10 - LCDR2 of C1.2
SEQ ID NO: 11 - LCDR3 of C1.2
SEQ ID NO: 12 – consensus sequence of HCDR3 of C1.2
SEQ ID NO: 13 – consensus sequence of LCDR3 of C1.2
SEQ ID NO: 14 – V of antibody 987
SEQ ID NO: 15 – V of antibody 987
SEQ ID NO: 16 – V of antibody 95
SEQ ID NO: 17 – V of antibody 79
SEQ ID NO: 18 – V of antibody 83
SEQ ID NO: 19 – V of antibody 1003
SEQ ID NO: 20 – V of antibody 1003
SEQ ID NO: 21 – V of antibody 44
SEQ ID NO: 22 – V of antibody 97
SEQ ID NO: 23 – V of antibody 986
SEQ ID NO: 24 – V of antibody 986
SEQ ID NO: 25 – V of antibody 56
SEQ ID NO: 26 – V of antibody 77
SEQ ID NO: 27 – V of antibody 54
SEQ ID NO: 28 – V of antibody 54
SEQ ID NO: 29 – V of antibody 802
SEQ ID NO: 30 – V of antibody 967
SEQ ID NO: 31 – V of antibody 967
SEQ ID NO: 32 – V of antibody 989
SEQ ID NO: 33 – V of antibody 989
SEQ ID NO: 34 – V of antibody 63
SEQ ID NO: 35 – V of antibody 1002
SEQ ID NO: 36 – V of antibody 1002
SEQ ID NO: 37 – V of antibody 994
SEQ ID NO: 38 – V of antibody 994
SEQ ID NO: 39 - V of antibody 969
SEQ ID NO: 40 – V of antibody 969
SEQ ID NO: 41 – V of antibody 1000
SEQ ID NO: 42 – V of antibody 1000
SEQ ID NO: 43 – V of antibody 94
SEQ ID NO: 44 – V of antibody 975
SEQ ID NO: 45 – V of antibody 975
SEQ ID NO: 46 – V of antibody 75
SEQ ID NO: 47 – V of antibody 814
SEQ ID NO: 48 – V of antibody 973
SEQ ID NO: 49 – V of antibody 973
SEQ ID NO: 50 – V of antibody 977
SEQ ID NO: 51 – V of antibody 977
SEQ ID NO: 52 – V of antibody 984
SEQ ID NO: 53 – V of antibody 984
SEQ ID NO: 54 – V of antibody 61
SEQ ID NO: 55 – V of antibody 852
SEQ ID NO: 56 – V of antibody 996
SEQ ID NO: 57 – V of antibody 996
SEQ ID NO: 58 – V of antibody 43
SEQ ID NO: 59 – V of antibody 43
SEQ ID NO: 60 – V of antibody 999
SEQ ID NO: 61 – V of antibody 999
SEQ ID NO: 62 – V of antibody 870
SEQ ID NO: 63 – V of antibody 877
SEQ ID NO: 64 – Heavy chain of C1.2G with stabilized IgG4 constant region
SEQ ID NO: 65 – Light chain of C1.2G with kappa constant region
SEQ ID NO: 66 – sequence of exemplary h-GCSFR
SEQ ID NO: 67 – polypeptide comprising Ig and CRH domains of Macaca fascicularis
G-CSFR (cynoG-CSFR) with a C-terminal polyhistidine tag
SEQ ID NO: 68 - Heavy chain of C1.2G with stabilized IgG4 constant region and
lacking C-terminal lysine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graphical representation showing inhibition of G-CSF-mediated
proliferation of BaF3 cells by increasing concentrations of various anti-G-CSFR
antibodies. The relative IC values for each antibody were; 10.1μg/mL for mAb711,
37.4μg/ml for mAb 774, 0.8μg/mL for C1.2G and was not determinable for mAb 744.
Figure 2A is a graphical representation showing the relative binding of C1.2G
and mAb744 to a series of alanine point mutants of SEQ ID NO: 1 compared to their
binding to SEQ ID NO: 1 (positions of mutations are indicated with reference to SEQ
ID NO: 1). The fold decrease in K of the antibody for the mutant receptor compared
to SEQ ID NO: 1 is depicted.
Figure 2B is a graphical representation showing the relative binding of C1.2G,
mAb744 and mAb774 to a series of alanine point mutants of SEQ ID NO: 1 compared
to their binding to SEQ ID NO: 1 (positions of mutations are indicated with reference
to SEQ ID NO: 1). The fold decrease in K of the antibody for the mutant receptor
compared to SEQ ID NO: 1 is depicted.
Figure 3 is a graphical representation showing results of an assay in which
pegylated G-CSF was administered to cynomolgus monkeys and one day later C1.2
was administered. The number of neutrophils per μL blood was assessed.
DETAILED DESCRIPTION
General
Throughout this specification, unless specifically stated otherwise or the context
requires otherwise, reference to a single step, composition of matter, group of steps or
group of compositions of matter shall be taken to encompass one and a plurality (i.e.
one or more) of those steps, compositions of matter, groups of steps or groups of
compositions of matter.
Those skilled in the art will appreciate that the present disclosure is susceptible
to variations and modifications other than those specifically described. It is to be
understood that the disclosure includes all such variations and modifications. The
disclosure also includes all of the steps, features, compositions and compounds referred
to or indicated in this specification, individually or collectively, and any and all
combinations or any two or more of said steps or features.
The present disclosure is not to be limited in scope by the specific examples
described herein, which are intended for the purpose of exemplification only.
Functionally-equivalent products, compositions and methods are clearly within the
scope of the present disclosure.
Any example of the present disclosure herein shall be taken to apply mutatis
mutandis to any other example of the disclosure unless specifically stated otherwise.
Unless specifically defined otherwise, all technical and scientific terms used
herein shall be taken to have the same meaning as commonly understood by one of
ordinary skill in the art (for example, in cell culture, molecular genetics, immunology,
immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and
immunological techniques utilized in the present disclosure are standard procedures,
well known to those skilled in the art. Such techniques are described and explained
throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular
Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A
Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown
(editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL
Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical
Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors),
Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-
Interscience (1988, including all updates until present), Ed Harlow and David Lane
(editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988),
and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons
(including all updates until present).
The description and definitions of variable regions and parts thereof,
immunoglobulins, antibodies and fragments thereof herein may be further clarified by
the discussion in Kabat Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-
320, 1994, Chothia and Lesk J. Mol Biol. 196:901 -917, 1987, Chothia et al. Nature
342, 877-883, 1989 and/or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and
Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for
either meaning.
Throughout this specification the word “comprise”, or variations such as
“comprises” or “comprising”, will be understood to imply the inclusion of a stated
element, integer or step, or group of elements, integers or steps, but not the exclusion of
any other element, integer or step, or group of elements, integers or steps.
As used herein the term "derived from" shall be taken to indicate that a specified
integer may be obtained from a particular source albeit not necessarily directly from
that source.
Selected Definitions
For the purposes of nomenclature only and not limitation an exemplary
sequence of a human G-CSFR is set out in NCBI Reference Sequence: NP_000751.1
(and set out in SEQ ID NO: 66). The sequence of cynomolgus monkey G-CSFR can be
determined using sequences provided herein and/or in publically available databases
and/or determined using standard techniques (e.g., as described in Ausubel et al.,
(editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-
Interscience (1988, including all updates until present) or Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989))
Reference to human G-CSFR may be abbreviated to hG-CSFR and reference to
cynomolgus monkey G-CSFR may be abbreviated to cynoG-CSFR. Reference to
soluble G-CSFR refers to polypeptides comprising the ligand binding region of G-
CSFR. The Ig and CRH domains of the G-CSFR are involved in ligand binding and
receptor dimerization (Layton et al., J. Biol Chem., 272: 29735-29741, 1997 and
Fukunaga et al, EMBO J. 10: 2855-2865, 1991). Soluble forms of G-CSFR comprising
these portions of the receptor have been used in various studies of the receptor and
mutation of the free cysteines at positions 78, 163, and 228 of the receptor assists in
expression and isolation of the soluble receptor polypeptide (Mine et al., Biochem., 43:
2458-2464 2004) without affecting ligand binding. In the present studies soluble forms
of the receptor comprising amino acids 25-335 of hG-CSFR with mutations C78A,
C163S and C228S were used (e.g. SEQ ID NO:1) and the corresponding segment of
cynoG-CSFR with the cysteine mutations was used (e.g., SEQ ID NO 67) for studies
on the cynomolgus monkey receptor. Various point mutations of the soluble receptor
of SEQ ID NO:1 and SEQ ID NO: 67 have also been utilized. Reference to hG-CSFR-
Fc means the polypeptide of SEQ ID NO:1 wherein the C-terminal polyhistidine tag
has been replaced with an Fc sequence (e.g., a polypeptide comprising amino acids 1-
311 of SEQ ID NO: 1 fused to an Fc). cynoG-CSFR-Fc means the corresponding
segment of cynoG-CSFR with an Fc sequence attached to its C-terminus (e.g., a
polypeptide comprising amino acids 1-311 of SEQ ID NO: 67 fused to an Fc). The
inventors have shown that antibodies and proteins comprising antigen binding sites
thereof (e.g., Fab) bind to wild type hG-CSF polypeptides and to these mutant proteins
with highly similar affinity. Accordingly, studies using the mutant proteins are a model
of studies using hG-CSFR and/or cynoG-CSFR.
Reference herein to G-CSF includes native forms of G-CSF, mutant forms
thereof, e.g., filgrastim and pegylated forms of G-CSF or filgrastim. This term also
encompasses mutant forms of G-CSF retaining activity to bind to G-CSFR (e.g., hG-
CSFR) and induce signaling.
The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide
that by virtue of its origin or source of derivation is not associated with naturally-
associated components that accompany it in its native state; is substantially free of
other proteins from the same source. A protein may be rendered substantially free of
naturally associated components or substantially purified by isolation, using protein
purification techniques known in the art. By “substantially purified” is meant the
protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or
80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating
agents.
The term “recombinant” shall be understood to mean the product of artificial
genetic recombination. Accordingly, in the context of a recombinant protein
comprising an antibody antigen binding domain, this term does not encompass an
antibody naturally-occurring within a subject’s body that is the product of natural
recombination that occurs during B cell maturation. However, if such an antibody is
isolated, it is to be considered an isolated protein comprising an antibody antigen
binding domain. Similarly, if nucleic acid encoding the protein is isolated and
expressed using recombinant means, the resulting protein is a recombinant protein
comprising an antibody antigen binding domain. A recombinant protein also
encompasses a protein expressed by artificial recombinant means when it is within a
cell, tissue or subject, e.g., in which it is expressed.
The term “protein” shall be taken to include a single polypeptide chain, i.e., a
series of contiguous amino acids linked by peptide bonds or a series of polypeptide
chains covalently or non-covalently linked to one another (i.e., a polypeptide complex).
For example, the series of polypeptide chains can be covalently linked using a suitable
chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen
bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
The term “polypeptide” or “polypeptide chain” will be understood from the
foregoing paragraph to mean a series of contiguous amino acids linked by peptide
bonds.
As used herein, the term “antigen binding site” shall be taken to mean a
structure formed by a protein that is capable of binding or specifically binding to an
antigen. The antigen binding site need not be a series of contiguous amino acids, or
even amino acids in a single polypeptide chain. For example, in a Fv produced from
two different polypeptide chains the antigen binding site is made up of a series of
amino acids of a V and a V that interact with the antigen and that are generally,
however not always in the one or more of the CDRs in each variable region. In some
examples, an antigen binding site is a V or a V or a Fv.
The skilled artisan will be aware that an “antibody” is generally considered to be
a protein that comprises a variable region made up of a plurality of polypeptide chains,
e.g., a polypeptide comprising a V and a polypeptide comprising a V . An antibody
also generally comprises constant domains, some of which can be arranged into a
constant region, which includes a constant fragment or fragment crystallizable (Fc), in
the case of a heavy chain. A V and a V interact to form a Fv comprising an antigen
binding region that is capable of specifically binding to one or a few closely related
antigens. Generally, a light chain from mammals is either a κ light chain or a λ light
chain and a heavy chain from mammals is α, δ, ε, γ, or μ. Antibodies can be of any
type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG , IgG , IgG , IgG , IgA
1 2 3 4 1
and IgA ) or subclass. The term “antibody” also encompasses humanized antibodies,
primatized antibodies, human antibodies and chimeric antibodies.
The terms "full-length antibody," "intact antibody" or "whole antibody" are used
interchangeably to refer to an antibody in its substantially intact form, as opposed to an
antigen binding fragment of an antibody. Specifically, whole antibodies include those
with heavy and light chains including an Fc region. The constant domains may be wild-
type sequence constant domains (e.g., human wild-type sequence constant domains) or
amino acid sequence variants thereof.
As used herein, “variable region" refers to the portions of the light and/or heavy
chains of an antibody as defined herein that is capable of specifically binding to an
antigen and includes amino acid sequences of complementarity determining regions
(CDRs); i.e., CDRl, CDR2, and CDR3, and framework regions (FRs). Exemplary
variable regions comprise three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4)
together with three CDRs. In the case of a protein derived from an IgNAR, the protein
may lack a CDR2. V refers to the variable region of the heavy chain. V refers to the
variable region of the light chain.
As used herein, the term "complementarity determining regions” (syn. CDRs;
i.e., CDRl, CDR2, and CDR3) refers to the amino acid residues of an antibody variable
region the presence of which are necessary for antigen binding. Each variable region
typically has three CDR regions identified as CDRl, CDR2 and CDR3. The amino acid
positions assigned to CDRs and FRs can be defined according to Kabat Sequences of
Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987
and 1991 or other numbering systems in the performance of this disclosure, e.g., the
canonical numbering system of Chothia and Lesk J. Mol Biol. 196: 901-917, 1987;
Chothia et al. Nature 342, 877-883, 1989; and/or Al-Lazikani et al., J Mol Biol 273:
927-948, 1997; the IMGT numbering system of Lefranc et al., Devel. And Compar.
Immunol., 27: 55-77, 2003; or the AHO numbering system of Honnegher and Plükthun
J. Mol. Biol., 309: 657-670, 2001. For example, according to the numbering system of
Kabat, V framework regions (FRs) and CDRs are positioned as follows: residues 1-30
(FRl ), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and
103- 113 (FR4). According to the numbering system of Kabat, V FRs and CDRs are
positioned as follows: residues 1–23 (FRl), 24-34 (CDR1), 35-49 (FR2), 50-56
(CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4). The present disclosure is not
limited to FRs and CDRs as defined by the Kabat numbering system, but includes all
numbering systems, including those discussed above. In one example, reference herein
to a CDR (or a FR) is in respect of those regions according to the Kabat numbering
system.
"Framework regions" (FRs) are those variable region residues other than the
CDR residues.
As used herein, the term “Fv” shall be taken to mean any protein, whether
comprised of multiple polypeptides or a single polypeptide, in which a V and a V
associate and form a complex having an antigen binding site, i.e., capable of
specifically binding to an antigen. The V and the V which form the antigen binding
site can be in a single polypeptide chain or in different polypeptide chains.
Furthermore, an Fv of the disclosure (as well as any protein of the disclosure) may have
multiple antigen binding sites which may or may not bind the same antigen. This term
shall be understood to encompass fragments directly derived from an antibody as well
as proteins corresponding to such a fragment produced using recombinant means. In
some examples, the V is not linked to a heavy chain constant domain (C ) 1 and/or
the V is not linked to a light chain constant domain (C ). Exemplary Fv containing
polypeptides or proteins include a Fab fragment, a Fab’ fragment, a F(ab’) fragment, a
scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the
foregoing linked to a constant region or domain thereof, e.g., C 2 or C 3 domain, e.g.,
a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an
immunoglobulin, and can be produced by digestion of a whole antibody with the
enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a
heavy chain or can be produced using recombinant means. A "Fab' fragment" of an
antibody can be obtained by treating a whole antibody with pepsin, followed by
reduction, to yield a molecule consisting of an intact light chain and a portion of a
heavy chain comprising a V and a single constant domain. Two Fab' fragments are
obtained per antibody treated in this manner. A Fab’ fragment can also be produced by
recombinant means. A "F(ab')2 fragment” of an antibody consists of a dimer of two
Fab' fragments held together by two disulfide bonds, and is obtained by treating a
whole antibody molecule with the enzyme pepsin, without subsequent reduction. A
“Fab ” fragment is a recombinant fragment comprising two Fab fragments linked
using, for example a leucine zipper or a C 3 domain. A “single chain Fv” or “scFv” is a
recombinant molecule containing the variable region fragment (Fv) of an antibody in
which the variable region of the light chain and the variable region of the heavy chain
are covalently linked by a suitable, flexible polypeptide linker.
As used herein, the term “binds” in reference to the interaction of a protein or an
antigen binding site thereof with an antigen means that the interaction is dependent
upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on
the antigen. For example, an antibody recognizes and binds to a specific protein
structure rather than to proteins generally. If an antibody binds to epitope "A", the
presence of a molecule containing epitope “A” (or free, unlabeled “A”), in a reaction
containing labeled “A” and the protein, will reduce the amount of labeled “A” bound to
the antibody.
As used herein, the term “specifically binds” or “binds specifically” shall be
taken to mean that a protein of the disclosure reacts or associates more frequently, more
rapidly, with greater duration and/or with greater affinity with a particular antigen or
cell expressing same than it does with alternative antigens or cells. For example, a
protein binds to G-CSFR (e.g., hG-CSFR) with materially greater affinity (e.g., 20 fold
or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold) than it does to other
cytokine receptor or to antigens commonly recognized by polyreactive natural
antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens
naturally found in humans). In an example of the present disclosure, a protein that
“specifically binds” to one form of hG-CSFR or a polypeptide comprising a region
thereof (e.g., the ligand binding domain of hG-GCSFR) or a polypeptide comprising
amino acids 1-311 of SEQ ID NO: 1 with an affinity at least 20 fold or 40 fold or 60
fold or 80 fold or 100 fold or 150 fold or 200 fold greater than it does to a mutant form
of hG-CSFR or a polypeptide comprising a region thereof (e.g., a mutant form of the
ligand binding domain of h-GCSFR) or a mutant form of SEQ ID NO: 1 comprising an
alanine substituted for the native arginine at position 287. Additional exemplary
changes to SEQ ID NO: 1 and their effect on binding are described herein. Generally,
but not necessarily, reference to binding means specific binding, and each term shall be
understood to provide explicit support for the other term.
As used herein, the term “does not detectably bind” shall be understood to mean
that a protein, e.g., an antibody, binds to a candidate antigen at a level less than 10%, or
8% or 6% or 5% above background. The background can be the level of binding signal
detected in the absence of the protein and/or in the presence of a negative control
protein (e.g., an isotype control antibody) and/or the level of binding detected in the
presence of a negative control antigen. The level of binding is detected using biosensor
analysis (e.g. Biacore) in which the protein is immobilized and contacted with an
antigen.
As used herein, the term “does not significantly bind” shall be understood to
mean that the level of binding of a protein of the disclosure to a polypeptide is not
statistically significantly higher than background, e.g., the level of binding signal
detected in the absence of the protein and/or in the presence of a negative control
protein (e.g., an isotype control antibody) and/or the level of binding detected in the
presence of a negative control polypeptide. The level of binding is detected using
biosensor analysis (e.g. Biacore) in which the protein is immobilized and contacted
with an antigen.
As used herein, phrases referring to “reduced binding” or “binding being at a
lower level” in relation to an antigen will be understood to mean that an antibody binds
to an antigen (e.g., an alanine point mutant of SEQ ID NO:1 at any one of positions
287, 237, 198, 172, 171 or 111) with an affinity at least about 20 fold or 40 fold or 60
fold less than a control epitope or antigen (e.g. SEQ ID NO:1). For example, a protein
of the present disclosure can bind to a polypeptide of SEQ ID NO: 1 in which an
alanine is substituted for the histidine at position 237 at a level 20 fold or 40 fold or 60
fold less than it binds to a polypeptide of SEQ ID NO: 1. Preferably, the protein binds
at a level 20 fold less, more preferably 40 fold less, still more preferably 60 fold less.
A protein or antibody may be considered to “preferentially bind” to a
polypeptide if it binds that polypeptide with a dissociation constant (K ) that is less
than the protein’s or antibody's K for another polypeptide. In one example, a protein
or antibody is considered to preferentially bind to a polypeptide if it binds the
polypeptide with an affinity (i.e., K ) that is at least about 20 fold or 40 fold or 60 fold
or 80 fold or 100 fold or 120 fold or 140 fold or 160 fold more than the protein’s or
antibody's K for another polypeptide.
As used herein, the term “similar affinity” will be understood to mean that a
protein of the present disclosure binds to two antigens (e.g., the ligand binding domain
of G-CSFR from humans and from cynomolgus monkeys) with affinities that are
within about 5 fold or less of one another, e.g., within about 4, 3, 2, or 1 fold of one
another, such as, within about 0.5 fold of one another or the levels of binding are
substantially identical, e.g., when the affinity is assessed by immobilizing the two
antigens (e.g., the ligand binding domain of G-CSFR or extracellular domains from
humans and from cynomolgus monkeys) and contacting the immobilized proteins with
a protein of the disclosure.
For the purposes of clarification and as will be apparent to the skilled artisan
based on the exemplified subject matter herein, reference to “affinity” in this
specification is a reference to K of a protein or antibody.
For the purposes of clarification and as will be apparent to the skilled artisan
based on the description herein, reference to an “affinity of at least about” will be
understood to mean that the affinity (or K ) is equal to the recited value or higher (i.e.,
the value recited as the affinity is lower), i.e., an affinity of 2nM is greater than an
affinity of 3nM. Stated another way, this term could be “an affinity of X or less”,
wherein X is a value recited herein.
An “IC of at least about” will be understood to mean that the IC is equal to
50 50
the recited value or greater (i.e., the value recited as the IC is lower), i.e., an IC of
50 50
2nM is greater than an IC of 3nM. Stated another way, this term could be “an IC of
50 50
X or less”, wherein X is a value recited herein.
As used herein, the term “epitope” (syn. “antigenic determinant”) shall be
understood to mean a region of hG-CSFR to which a protein comprising an antigen
binding site of an antibody binds. This term is not necessarily limited to the specific
residues or structure to which the protein makes contact. For example, this term
includes the region spanning amino acids contacted by the protein and/or 5-10 or 2-5 or
1-3 amino acids outside of this region. In some examples, the epitope comprises a
series of discontinuous amino acids that are positioned close to one another when hG-
CSFR is folded, i.e., a “conformational epitope”. For example, a conformational
epitope comprises amino acids in one or more or two or more or all of the regions
corresponding to 111-115, 170-176, 218-234 and/or 286-300 of SEQ ID NO: 1. The
skilled artisan will also be aware that the term "epitope" is not limited to peptides or
polypeptides. For example, the term “epitope” includes chemically active surface
groupings of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl
side chains, and, in certain examples, may have specific three dimensional structural
characteristics, and/or specific charge characteristics.
The term “competitively inhibits” shall be understood to mean that a protein of
the disclosure (or an antigen binding site thereof) reduces or prevents binding of a
recited antibody or protein to G-CSFR, e.g., to hG-CSFR. This may be due to the
protein (or antigen binding site) and antibody binding to the same or an overlapping
epitope. It will be apparent from the foregoing that the protein need not completely
inhibit binding of the antibody, rather it need only reduce binding by a statistically
significant amount, for example, by at least about 10% or 20% or 30% or 40% or 50%
or 60% or 70% or 80% or 90% or 95%. Preferably, the protein reduces binding of the
antibody by at least about 30%, more preferably by at least about 50%, more
preferably, by at least about 70%, still more preferably by at least about 75%, even
more preferably, by at least about 80% or 85% and even more preferably, by at least
about 90%. Methods for determining competitive inhibition of binding are known in
the art and/or described herein. For example, the antibody is exposed to G-CSFR either
in the presence or absence of the protein. If less antibody binds in the presence of the
protein than in the absence of the protein, the protein is considered to competitively
inhibit binding of the antibody. In one example, the competitive inhibition is not due to
steric hindrance.
“Overlapping” in the context of two epitopes shall be taken to mean that two
epitopes share a sufficient number of amino acid residues to permit a protein (or
antigen binding site thereof) that binds to one epitope to competitively inhibit the
binding of a protein (or antigen binding site) that binds to the other epitope. For
example, the “overlapping” epitopes share at least 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or
9 or 20 amino acids.
As used herein, the term “neutralize” shall be taken to mean that a protein is
capable of blocking, reducing or preventing G-CSF-mediated signaling in a cell
through the G-CSFR. Methods for determining neutralization are known in the art
and/or described herein.
As used herein, the term “condition” refers to a disruption of or interference
with normal function, and is not to be limited to any specific condition, and will include
diseases or disorders.
As used herein, a “G-CSF-associated condition” refers to any condition that is
caused by or associated with neutrophils, an excess of G-CSF or cells expressing G-
CSFR or with administration of G-CSF. The skilled artisan will be readily able to
determine such conditions. In this regard, in some examples the condition is an
inflammatory condition, an autoimmune condition or cancer (including metastasis).
As used herein, the terms “preventing”, “prevent” or “prevention” include
administering a protein of the disclosure to thereby stop or hinder the development of at
least one symptom of a condition. This term also encompasses treatment of a subject in
remission to prevent or hinder relapse. For example, a subject suffering from
relapsing-remitting multiple sclerosis is treated during remission to thereby prevent a
relapse.
As used herein, the terms “treating”, “treat” or “treatment” include
administering a protein described herein to thereby reduce or eliminate at least one
symptom of a specified disease or condition.
As used herein, the term “neutropenia” will be understood to encompass mild
neutropenia (1000 <= ANC < 1500), moderate neutropenia (500 <= ANC < 1000) and
Severe neutropenia (ANC < 500) (absolute neutrophil count (ANC) measured in cells
per microliter of blood).
As used herein, the term “subject” shall be taken to mean any animal including
humans, for example a mammal. Exemplary subjects include but are not limited to
humans and non-human primates. For example, the subject is a human.
Antibodies
In one example, a protein as described herein according to any example is an
antibody.
Methods for generating antibodies are known in the art and/or described in
Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, (1988). Generally, in such methods G-CSFR (e.g., hG-CSFR) or a region
thereof (e.g., an extracellular domain) or immunogenic fragment or epitope thereof or a
cell expressing and displaying same (i.e., an immunogen), optionally formulated with
any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is
administered to a non-human animal, for example, a mouse, chicken, rat, rabbit, guinea
pig, dog, horse, cow, goat or pig. The immunogen may be administered intranasally,
intramuscularly, sub-cutaneously, intravenously, intradermally, intraperitoneally, or by
other known route.
The production of polyclonal antibodies may be monitored by sampling blood of
the immunized animal at various points following immunization. One or more further
immunizations may be given, if required to achieve a desired antibody titer. The
process of boosting and titering is repeated until a suitable titer is achieved. When a
desired level of immunogenicity is obtained, the immunized animal is bled and the
serum isolated and stored, and/or the animal is used to generate monoclonal antibodies
(mabs).
Monoclonal antibodies are one exemplary form of antibody contemplated by the
present disclosure. The term “monoclonal antibody" or “mAb” refers to a homogeneous
antibody population capable of binding to the same antigen(s), for example, to the same
epitope within the antigen. This term is not intended to be limited as regards to the
source of the antibody or the manner in which it is made.
For the production of mAbs any one of a number of known techniques may be
used, such as, for example, the procedure exemplified in US4196265 or Harlow and
Lane (1988), supra.
For example, a suitable animal is immunized with an immunogen under
conditions sufficient to stimulate antibody producing cells. Rodents such as rabbits,
mice and rats are exemplary animals. Mice genetically-engineered to express human
antibodies and, for example, do not express murine antibodies, can also be used to
generate an antibody of the present disclosure (e.g., as described in WO2002/066630).
Following immunization, somatic cells with the potential for producing
antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb
generating protocol. These cells may be obtained from biopsies of spleens, tonsils or
lymph nodes, or from a peripheral blood sample. The B cells from the immunized
animal are then fused with cells of an immortal myeloma cell, generally derived from
the same species as the animal that was immunized with the immunogen.
Hybrids are amplified by culture in a selective medium comprising an agent that
blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary
agents are aminopterin, methotrexate and azaserine.
The amplified hybridomas are subjected to a functional selection for antibody
specificity and/or titer, such as, for example, by flow cytometry and/or
immunohistochemstry and/or immunoassay (e.g. radioimmunoassay, enzyme
immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).
Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is
used to produce cell lines secreting MAbs (e.g., as described in Largaespada et al, J.
Immunol. Methods. 197: 85-95, 1996).
Antibodies can also be produced or isolated by screening a display library, e.g.,
a phage display library, e.g., as described in US6300064 and/or US5885793. For
example, the present inventors have isolated fully human antibodies from a phage
display library.
As described herein, some proteins of the present disclosure that bind hG-CSFR
cross-react with cynoG-CSFR and/or bind to some mutant forms of hG-CSFR or
polypeptides comprising regions of hG-CSFR that have been mutated and/or not others
and/or bind to a specific epitope within hG-CSFR. These characteristics can be used in
the generation of an antibody or a protein comprising a binding site thereof.
For example, a phage display library is screened with a polypeptide comprising
the ligand binding domain of hG-CSFR to identify proteins that bind thereto. Mutant
forms of the ligand binding domain of hG-CSFR (e.g., an alanine point mutant of SEQ
ID NO:1 at position 287) to which the protein is not to detectably bind are then used to
remove cross-reactive proteins and mutant forms of the ligand binding domain of hG-
CSFR or regions thereof (e.g., an alanine point mutant of SEQ ID NO:1 at position
168) to which the protein is to bind are used to isolate proteins that are correctly cross-
reactive. A screening process for immunization of a non-human mammal can also be
devised based on the foregoing.
In another example, a phage display library is screened or an animal is
immunized with a polypeptide comprising the ligand binding domain of cynoG-CSFR
and identified proteins and/or antibodies are screened to identify those that are cross-
reactive with hG-CSFR and/or the ligand binding domain thereof.
In a further example, a G-CSFR or a ligand binding domain thereof (optionally a
mutant form to which C1.2 or C1.2G binds) is contacted with C1.2 or C1.2G. A phage
display library is then brought into contact with the G-CSFR or the ligand binding
domain and phage expressing proteins that can compete with C1.2 or C1.2G for
binding selected.
In a still further example, a chimeric protein comprising, e.g., a mouse G-CSFR
in which an epitope of interest from a hG-CSFR is substituted for the corresponding
mouse sequence. This chimeric protein is then used to immunize mice (which are less
likely to induce an immune response against the mouse protein) and/or to screen a
phage display library. The resulting antibodies/proteins are then screened to identify
those that bind to hG-CSFR (particularly at the epitope of interest) and not mouse G-
CSFR.
The antibody of the present disclosure may be a synthetic antibody. For
example, the antibody is a chimeric antibody, a humanized antibody, a human antibody
or a de-immunized antibody.
Chimeric Antibodies
In one example, an antibody described herein is a chimeric antibody. The term
“chimeric antibody” refers to antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in antibodies derived
from a particular species (e.g., murine, such as mouse) or belonging to a particular
antibody class or subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from another species
(e.g., primate, such as human) or belonging to another antibody class or subclass.
Typically chimeric antibodies utilize rodent or rabbit variable regions and human
constant regions, in order to produce an antibody with predominantly human domains.
Methods for producing chimeric antibodies are described in, e.g., US4816567; and
US5807715.
Humanized and Human Antibodies
The antibodies of the present disclosure may be humanized or human.
The term "humanized antibody” shall be understood to refer to a subclass of
chimeric antibodies having an antigen binding site or variable region derived from an
antibody from a non-human species and the remaining antibody structure based upon
the structure and/or sequence of a human antibody. In a humanized antibody, the
antigen-binding site generally comprises the complementarity determining regions
(CDRs) from the non-human antibody grafted onto appropriate FRs in the variable
regions of a human antibody and the remaining regions from a human antibody.
Antigen binding sites may be wild-type (i.e., identical to those of the non-human
antibody) or modified by one or more amino acid substitutions. In some instances, FR
residues of the human antibody are replaced by corresponding non-human residues.
Methods for humanizing non-human antibodies or parts thereof (e.g., variable
regions) are known in the art. Humanization can be performed following the method of
US5225539, or US5585089. Other methods for humanizing an antibody are not
excluded.
The term "human antibody" as used herein refers to antibodies having variable
regions (e.g. V , V ) and, optionally constant regions derived from or corresponding to
sequences found in humans, e.g. in the human germline or somatic cells. The "human"
antibodies can include amino acid residues not encoded by human sequences, e.g.
mutations introduced by random or site directed mutations in vitro (in particular
mutations which involve conservative substitutions or mutations in a small number of
residues of the antibody, e.g. in 1, 2, 3, 4, 5 or 6 of the residues of the antibody, e.g. in
1, 2, 3, 4, 5 or 6 of the residues making up one or more of the CDRs of the antibody).
These “human antibodies” do not actually need to be produced by a human, rather, they
can be produced using recombinant means and/or isolated from a transgenic animal
(e.g., mouse) comprising nucleic acid encoding human antibody constant and/or
variable regions (e.g., as described above). Human antibodies can be produced using
various techniques known in the art, including phage display libraries (e.g., as
described in US5885793).
Human antibodies which recognize a selected epitope can also be generated
using a technique referred to as "guided selection." In this approach a selected non-
human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a
completely human antibody recognizing the same epitope (e.g., as described in
US5565332).
Exemplary human antibodies are described herein and include C1.2 and C1.2G
and/or variable regions thereof. These human antibodies provide an advantage of
reduced immunogenicity in a human compared to non-human antibodies.
Antibody Binding Domain Containing Proteins
Single-Domain Antibodies
In some examples, a protein of the disclosure is or comprises a single-domain
antibody (which is used interchangeably with the term “domain antibody” or “dAb”). A
single-domain antibody is a single polypeptide chain comprising all or a portion of the
heavy chain variable region of an antibody. In certain examples, a single-domain
antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g.,
US6248516).
Diabodies, Triabodies, Tetrabodies
In some examples, a protein of the disclosure is or comprises a diabody,
triabody, tetrabody or higher order protein complex such as those described in
WO98/044001 and/or WO94/007921.
For example, a diabody is a protein comprising two associated polypeptide
chains, each polypeptide chain comprising the structure V -X-V or V -X-V , wherein
L H H L
V is an antibody light chain variable region, V is an antibody heavy chain variable
region, X is a linker comprising insufficient residues to permit the V and V in a
single polypeptide chain to associate (or form an Fv) or is absent, and wherein the V
of one polypeptide chain binds to a V of the other polypeptide chain to form an
antigen binding site, i.e., to form a Fv molecule capable of specifically binding to one
or more antigens. The V and V can be the same in each polypeptide chain or the V
L H L
and V can be different in each polypeptide chain so as to form a bispecific diabody
(i.e., comprising two Fvs having different specificity).
Single Chain Fv (scFv)
The skilled artisan will be aware that scFvs comprise V and V regions in a
single polypeptide chain and a polypeptide linker between the V and V which
enables the scFv to form the desired structure for antigen binding (i.e., for the V and
V of the single polypeptide chain to associate with one another to form a Fv). For
example, the linker comprises in excess of 12 amino acid residues with (Gly Ser)
being one of the more favored linkers for a scFv.
The present disclosure also contemplates a disulfide stabilized Fv (or diFv or
dsFv), in which a single cysteine residue is introduced into a FR of V and a FR of V
and the cysteine residues linked by a disulfide bond to yield a stable Fv.
Alternatively, or in addition, the present disclosure encompasses a dimeric scFv,
i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent
linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively,
two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to
form and to bind to an antigen, e.g., as described in US20060263367.
Heavy Chain Antibodies
Heavy chain antibodies differ structurally from many other forms of antibodies,
in so far as they comprise a heavy chain, but do not comprise a light chain.
Accordingly, these antibodies are also referred to as “heavy chain only antibodies”.
Heavy chain antibodies are found in, for example, camelids and cartilaginous fish (also
called IgNAR).
The variable regions present in naturally occurring heavy chain antibodies are
generally referred to as "V domains" in camelid antibodies and V-NAR in IgNAR, in
order to distinguish them from the heavy chain variable regions that are present in
conventional 4-chain antibodies (which are referred to as "V domains") and from the
light chain variable regions that are present in conventional 4-chain antibodies (which
are referred to as "V domains").
A general description of heavy chain antibodies from camelids and the variable
regions thereof and methods for their production and/or isolation and/or use is found
inter alia in the following references WO94/04678, WO97/49805 and WO 97/49805.
A general description of heavy chain antibodies from cartilaginous fish and the
variable regions thereof and methods for their production and/or isolation and/or use is
found inter alia in WO2005/118629.
Other Antibodies and Antibody Fragments
The present disclosure also contemplates other antibodies and antibody
fragments, such as:
(i) “key and hole” bispecific proteins as described in US5,731,168;
(ii) heteroconjugate proteins, e.g., as described in US4,676,980;
(iii) heteroconjugate proteins produced using a chemical cross-linker, e.g., as
described in US4,676,980; and
(iv) Fab (e.g., as described in EP19930302894).
De-immunized Antibodies and Proteins
The present disclosure also contemplates a de-immunized antibody or protein.
De-immunized antibodies and proteins have one or more epitopes, e.g., B cell epitopes
or T cell epitopes removed (i.e., mutated) to thereby reduce the likelihood that a
mammal will raise an immune response against the antibody or protein. Methods for
producing de-immunized antibodies and proteins are known in the art and described,
for example, in WO00/34317, WO2004/108158 and WO2004/064724.
Methods for introducing suitable mutations and expressing and assaying the
resulting protein will be apparent to the skilled artisan based on the description herein.
Mutations to Proteins
The present disclosure also contemplates mutant forms of a protein of the
disclosure. In this regard, data presented herein indicate sites within a CDR of a
protein of the disclosure that can be changed in addition to exemplary changes that can
be made. The skilled person will understand that changes can additionally or
alternatively be made within a FR of a variable region containing protein without
inhibiting or significantly reducing its function in the context of the present disclosure.
For example, such a mutant protein comprises one or more conservative amino
acid substitutions compared to a sequence set forth herein. In some examples, the
protein comprises 30 or fewer or 20 or fewer or 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5
or 4 or 3 or 2 conservative amino acid substitutions. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced with an amino acid
residue having a similar side chain and/or hydropathicity and/or hydrophilicity.
In one example, a mutant protein has only, or not more than, one or two or three
or four or five or six conservative amino acid changes when compared to a naturally
occurring protein. Details of conservative amino acid changes are provided below. As
the skilled person would be aware, e.g., from the disclosure herein, such minor changes
can reasonably be predicted not to alter the activity of the protein.
Families of amino acid residues having similar side chains have been defined in
the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-
branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine).
The present disclosure also contemplates non-conservative amino acid changes
(e.g., substitutions) in a protein of the present disclosure, e.g., in a CDR, such as CDR3.
For example, the present inventors have identified several non-conservative amino acid
substitutions that can be made while retaining an activity of a protein of the disclosure.
In one example, the protein comprises fewer than 6 or 5 or 4 or 3 or 2 or 1 non-
conservative amino acid substitutions, e.g., in a CDR3, such as in a CDR3.
The present disclosure also contemplates one or more insertions or deletions
compared to a sequence set forth herein. In some examples, the protein comprises 10
or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 insertions and/or deletions.
Constant Regions
The present disclosure encompasses proteins and/or antibodies described herein
comprising a constant region of an antibody. This includes antigen binding fragments
of an antibody fused to a Fc.
Sequences of constant regions useful for producing the proteins of the present
disclosure may be obtained from a number of different sources. In some examples, the
constant region or portion thereof of the protein is derived from a human antibody. The
constant region or portion thereof may be derived from any antibody class, including
IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgG1, IgG2, IgG3 and
IgG4. In one example, the constant region is human isotype IgG4 or a stabilized IgG4
constant region.
In one example, the Fc region of the constant region has a reduced ability to
induce effector function, e.g., compared to a native or wild-type human IgG1 or IgG3
Fc region. In one example, the effector function is antibody-dependent cell-mediated
cytotoxicity (ADCC) and/or antibody-dependent cell-mediated phagocytosis (ADCP)
and/or complement-dependent cytotoxicity (CDC). Methods for assessing the level of
effector function of an Fc region containing protein are known in the art and/or
described herein.
In one example, the Fc region is an IgG4 Fc region (i.e., from an IgG4 constant
region), e.g., a human IgG4 Fc region. Sequences of suitable IgG4 Fc regions will be
apparent to the skilled person and/or available in publically available databases (e.g.,
available from National Center for Biotechnology Information).
In one example, the constant region is a stabilized IgG4 constant region. The
term “stabilized IgG4 constant region” will be understood to mean an IgG4 constant
region that has been modified to reduce Fab arm exchange or the propensity to undergo
Fab arm exchange or formation of a half-antibody or a propensity to form a half
antibody. “Fab arm exchange" refers to a type of protein modification for human IgG4,
in which an IgG4 heavy chain and attached light chain (half-molecule) is swapped for a
heavy-light chain pair from another IgG4 molecule. Thus, IgG4 molecules may acquire
two distinct Fab arms recognizing two distinct antigens (resulting in bispecific
molecules). Fab arm exchange occurs naturally in vivo and can be induced in vitro by
purified blood cells or reducing agents such as reduced glutathione. A “half antibody”
forms when an IgG4 antibody dissociates to form two molecules each containing a
single heavy chain and a single light chain.
In one example, a stabilized IgG4 constant region comprises a proline at
position 241 of the hinge region according to the system of Kabat (Kabat et al.,
Sequences of Proteins of Immunological Interest Washington DC United States
Department of Health and Human Services, 1987 and/or 1991). This position
corresponds to position 228 of the hinge region according to the EU numbering system
(Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United
States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl.
Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is generally a serine.
Following substitution of the serine for proline, the IgG4 hinge region comprises a
sequence CPPC. In this regard, the skilled person will be aware that the “hinge region”
is a proline-rich portion of an antibody heavy chain constant region that links the Fc
and Fab regions that confers mobility on the two Fab arms of an antibody. The hinge
region includes cysteine residues which are involved in inter-heavy chain disulfide
bonds. It is generally defined as stretching from Glu226 to Pro243 of human IgG1
according to the numbering system of Kabat. Hinge regions of other IgG isotypes may
be aligned with the IgG1 sequence by placing the first and last cysteine residues
forming inter-heavy chain disulphide (S-S) bonds in the same positions (see for
example WO2010/080538).
Additional examples of stabilized IgG4 antibodies are antibodies in which
arginine at position 409 in a heavy chain constant region of human IgG4 (according to
the EU numbering system) is substituted with lysine, threonine, methionine, or leucine
(e.g., as described in WO2006/033386). The Fc region of the constant region may
additionally or alternatively comprise a residue selected from the group consisting of:
alanine, valine, glycine, isoleucine and leucine at the position corresponding to 405
(according to the EU numbering system). Optionally, the hinge region comprises a
proline at position 241 (i.e., a CPPC sequence) (as described above).
In another example, the Fc region is a region modified to have reduced effector
function, i.e., a “non-immunostimulatory Fc region”. For example, the Fc region is an
IgG1 Fc region comprising a substitution at one or more positions selected from the
group consisting of 268, 309, 330 and 331. In another example, the Fc region is an
IgG1 Fc region comprising one or more of the following changes E233P, L234V,
L235A and deletion of G236 and/or one or more of the following changes A327G,
A330S and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J
Biol Chem. 276(9):6591-604, 2001). Additional examples of non-immunostimulatory
Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177 : 1129-1138
2006; and/or Hezareh J Virol ;75: 12161-12168, 2001).
In another example, the Fc region is a chimeric Fc region, e.g., comprising at
least one C 2 domain from an IgG4 antibody and at least one C 3 domain from an
IgG1 antibody, wherein the Fc region comprises a substitution at one or more amino
acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307,
309, 323, 399, 409 and 427 (EU numbering) (e.g., as described in WO2010/085682).
Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P,
309K, 309M, 309P, 323F, 399S, and 427F.
Additional Modifications
The present disclosure also contemplates additional modifications to an
antibody.
For example, the antibody comprises one or more amino acid substitutions that
increase the half-life of the protein. For example, the antibody comprises a Fc region
comprising one or more amino acid substitutions that increase the affinity of the Fc
region for the neonatal Fc region (FcRn). For example, the Fc region has increased
affinity for FcRn at lower pH, e.g., about pH 6.0, to facilitate Fc/FcRn binding in an
endosome. In one example, the Fc region has increased affinity for FcRn at about pH 6
compared to its affinity at about pH 7.4, which facilitates the re-release of Fc into blood
following cellular recycling. These amino acid substitutions are useful for extending
the half life of a protein, by reducing clearance from the blood.
Exemplary amino acid substitutions include T250Q and/or M428L or T252A,
T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to
the EU numbering system. Additional or alternative amino acid substitutions are
described, for example, in US20070135620 or US7083784.
Protein Production
In one example, a protein described herein according to any example is
produced by culturing a hybridoma under conditions sufficient to produce the protein,
e.g., as described herein and/or as is known in the art.
Recombinant Expression
In another example, a protein described herein according to any example is
recombinant.
In the case of a recombinant protein, nucleic acid encoding same can be cloned
into expression constructs or vectors, which are then transfected into host cells, such as
E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells,
Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or
myeloma cells that do not otherwise produce the protein. Exemplary cells used for
expressing a protein are CHO cells, myeloma cells or HEK cells. Molecular cloning
techniques to achieve these ends are known in the art and described, for example in
Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub.
Associates and Wiley-Interscience (1988, including all updates until present) or
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods
are suitable for the construction of recombinant nucleic acids. Methods of producing
recombinant antibodies are also known in the art, see, e.g., US4816567 or US5530101.
Following isolation, the nucleic acid is inserted operably linked to a promoter in
an expression construct or expression vector for further cloning (amplification of the
DNA) or for expression in a cell-free system or in cells.
As used herein, the term “promoter” is to be taken in its broadest context and
includes the transcriptional regulatory sequences of a genomic gene, including the
TATA box or initiator element, which is required for accurate transcription initiation,
with or without additional regulatory elements (e.g., upstream activating sequences,
transcription factor binding sites, enhancers and silencers) that alter expression of a
nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue
specific manner. In the present context, the term “promoter” is also used to describe a
recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or
enhances the expression of a nucleic acid to which it is operably linked. Exemplary
promoters can contain additional copies of one or more specific regulatory elements to
further enhance expression and/or alter the spatial expression and/or temporal
expression of said nucleic acid.
As used herein, the term “operably linked to" means positioning a promoter
relative to a nucleic acid such that expression of the nucleic acid is controlled by the
promoter.
Many vectors for expression in cells are available. The vector components
generally include, but are not limited to, one or more of the following: a signal
sequence, a sequence encoding a protein (e.g., derived from the information provided
herein), an enhancer element, a promoter, and a transcription termination sequence. The
skilled artisan will be aware of suitable sequences for expression of a protein.
Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals
(e.g., invertase leader, α factor leader, or acid phosphatase leader) or mammalian
secretion signals (e.g., herpes simplex gD signal).
Exemplary promoters active in mammalian cells include cytomegalovirus
immediate early promoter (CMV-IE), human elongation factor 1- promoter (EF1),
small nuclear RNA promoters (U1a and U1b), -myosin heavy chain promoter, Simian
virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late
promoter, β-actin promoter; hybrid regulatory element comprising a CMV enhancer/ β-
actin promoter or an immunoglobulin promoter or active fragment thereof. Examples of
useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40
(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or
Chinese hamster ovary cells (CHO).
Typical promoters suitable for expression in yeast cells such as for example a
yeast cell selected from the group comprising Pichia pastoris, Saccharomyces
cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1
promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt
promoter, the RPR1 promoter, or the TEF1 promoter.
Means for introducing the isolated nucleic acid or expression construct
comprising same into a cell for expression are known to those skilled in the art. The
technique used for a given cell depends on the known successful techniques. Means for
introducing recombinant DNA into cells include microinjection, transfection mediated
by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine
(Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake,
electroporation and microparticle bombardment such as by using DNA-coated tungsten
or gold particles (Agracetus Inc., WI, USA) amongst others.
The host cells used to produce the protein may be cultured in a variety of media,
depending on the cell type used. Commercially available media such as Ham's Fl0
(Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMl-1640 (Sigma), and
Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing
mammalian cells. Media for culturing other cell types discussed herein are known in
the art.
Isolation of Proteins
Methods for isolating a protein are known in the art and/or described herein.
Where a protein is secreted into culture medium, supernatants from such
expression systems can be first concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A
protease inhibitor such as PMSF may be included in any of the foregoing steps to
inhibit proteolysis and antibiotics may be included to prevent the growth of
adventitious contaminants. Alternatively, or additionally, supernatants can be filtered
and/or separated from cells expressing the protein, e.g., using continuous
centrifugation.
The protein prepared from the cells can be purified using, for example, ion
exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography,
gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity
chromatography or protein G chromatography), or any combination of the foregoing.
These methods are known in the art and described, for example in WO99/57134 or Ed
Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, (1988).
The skilled artisan will also be aware that a protein can be modified to include a
tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine
tag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a
FLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then
purified using methods known in the art, such as, affinity purification. For example, a
protein comprising a hexa-his tag is purified by contacting a sample comprising the
protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag
immobilized on a solid or semi-solid support, washing the sample to remove unbound
protein, and subsequently eluting the bound protein. Alternatively, or in addition a
ligand or antibody that binds to a tag is used in an affinity purification method.
Assaying Activity of a Protein
Binding to G-CSFR and Mutants Thereof
It will be apparent to the skilled artisan from the disclosure herein that some
proteins of the present disclosure bind to the ligand binding domain of hG-CSFR and to
specific mutant forms of the ligand binding domain of hG-CSFR (e.g., SEQ ID NO: 1
without or with certain point mutations) and/or bind to both human and cynomolgus
monkey G-CSFR. Methods for assessing binding to a protein are known in the art, e.g.,
as described in Scopes (In: Protein purification: principles and practice, Third Edition,
Springer Verlag, 1994). Such a method generally involves labeling the protein and
contacting it with immobilized antigen. Following washing to remove non-specific
bound protein, the amount of label and, as a consequence, bound protein is detected.
Of course, the protein can be immobilized and the antigen labeled. Panning-type
assays can also be used. Alternatively, or additionally, surface plasmon resonance
assays can be used.
The assays described above can also be used to detect the level of binding of a
protein to hG-CSFR or a ligand binding domain thereof (e.g., SEQ ID NO: 1) or mutant
form thereof.
In one example, a protein of the present disclosure binds to a polypeptide of
SEQ ID NO: 1 in which an alanine is substituted for the lysine at position 167 of SEQ
ID NO: 1 and/or in which an alanine is substituted for the histidine at position 168 of
SEQ ID NO: 1 at substantially the same level (e.g., within 10% or 5% or 1%) as it
binds to SEQ ID NO: 1.
In one example, a protein of the present disclosure binds to a polypeptide of
SEQ ID NO: 1 in which an alanine is substituted for the arginine at position 287 of
SEQ ID NO: 1 at a level at least about 100 fold or 150 fold or 160 fold or 200 fold
lower than it binds to a polypeptide of SEQ ID NO: 1. In one example, a protein of the
present disclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine is
substituted for the arginine at position 287 of SEQ ID NO: 1 at a level at least about
160 fold lower than it binds to a polypeptide of SEQ ID NO: 1.
In one example, a protein of the present disclosure binds to a polypeptide of
SEQ ID NO: 1 in which an alanine is substituted for the histidine at position 237 of
SEQ ID NO: 1 at a level at least about 20 fold or 40 fold or 50 fold or 60 fold lower
than it binds to a polypeptide of SEQ ID NO: 1. In one example, a protein of the
present disclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine is
substituted for the histidine at position 237 of SEQ ID NO: 1 at a level at least about 50
fold lower than it binds to a polypeptide of SEQ ID NO: 1.
In one example, a protein of the present disclosure binds to a polypeptide of
SEQ ID NO: 1 in which an alanine is substituted for the methionine at position 198 of
SEQ ID NO: 1 at a level at least about 20 fold or 40 fold or 60 fold or 70 fold lower
than it binds to a polypeptide of SEQ ID NO: 1. In one example, a protein of the
present disclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine is
substituted for the methionine at position 198 of SEQ ID NO: 1 at a level at least about
40 fold lower than it binds to a polypeptide of SEQ ID NO: 1.
In one example, a protein of the present disclosure binds to a polypeptide of
SEQ ID NO: 1 in which an alanine is substituted for the tyrosine at position 172 of
SEQ ID NO: 1 at a level at least about 20 fold or 30 fold or 40 fold lower than it binds
to a polypeptide of SEQ ID NO: 1. In one example, a protein of the present disclosure
binds to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the
tyrosine at position 172 of SEQ ID NO: 1 at a level at least about 40 fold lower than it
binds to a polypeptide of SEQ ID NO: 1.
In one example, a protein of the present disclosure binds to a polypeptide of
SEQ ID NO: 1 in which an alanine is substituted for the leucine at position 171 of SEQ
ID NO: 1 at a level at least about 100 fold or 120 fold or 130 fold or 140 fold lower
than it binds to a polypeptide of SEQ ID NO: 1. In one example, a protein of the
present disclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine is
substituted for the leucine at position 171 of SEQ ID NO: 1 at a level at least about 140
fold lower than it binds to a polypeptide of SEQ ID NO: 1.
In one example, a protein of the present disclosure binds to a polypeptide of
SEQ ID NO: 1 in which an alanine is substituted for the leucine at a position 111 of
SEQ ID NO: 1 at a level at least about 20 fold or 40 fold or 60 fold or 70 fold lower
than it binds to a polypeptide of SEQ ID NO: 1. In one example, a protein of the
present disclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine is
substituted for the leucine at a position 111 of SEQ ID NO: 1 at a level at least about 60
fold lower than it binds to a polypeptide of SEQ ID NO: 1.
In one example, a protein of the present disclosure binds to a polypeptide of
SEQ ID NO: 1 in which an alanine is substituted for the histidine at position 168 of
SEQ ID NO: 1 at a level no more than 5 fold or 4 fold or 3 fold or 2 fold or 1 fold
lower than it binds to a polypeptide of SEQ ID NO: 1.
In one example, a protein of the present disclosure binds to a polypeptide of
SEQ ID NO: 1 in which an alanine is substituted for the lysine at position 167 of SEQ
ID NO: 1 at a level no more than 5 fold or 4 fold or 3 fold or 2 fold or 1 fold lower than
it binds to a polypeptide of SEQ ID NO: 1.
The level of binding is conveniently determined using a biosensor.
The present disclosure contemplates any combination of the foregoing
characteristics. In one example, a protein described herein has all of the binding
characteristics set forth in the preceding seven paragraphs.
Epitope Mapping
In another example, the epitope bound by a protein described herein is mapped.
Epitope mapping methods will be apparent to the skilled artisan. For example, a series
of overlapping peptides spanning the hG-CSFR sequence or a region thereof
comprising an epitope of interest, e.g., peptides comprising 10-15 amino acids are
produced. The protein is then contacted to each peptide and the peptide(s) to which it
binds determined. This permits determination of peptide(s) comprising the epitope to
which the protein binds. If multiple non-contiguous peptides are bound by the protein,
the protein may bind a conformational epitope.
Alternatively, or in addition, amino acid residues within hG-CSFR are mutated,
e.g., by alanine scanning mutagenesis, and mutations that reduce or prevent protein
binding are determined. Any mutation that reduces or prevents binding of the protein is
likely to be within the epitope bound by the protein.
A further method is exemplified herein, and involves binding hG-CSFR or a
region thereof to an immobilized protein of the present disclosure and digesting the
resulting complex with proteases. Peptide that remains bound to the immobilized
protein are then isolated and analyzed, e.g., using mass spectrometry, to determine their
sequence.
A further method involves converting hydrogens in hG-CSFR or a region
thereof to deutrons and binding the resulting protein to an immobilized protein of the
present disclosure. The deutrons are then converted back to hydrogen, the hG-CSFR or
region thereof isolated, digested with enzymes and analyzed, e.g., using mass
spectrometry to identify those regions comprising deutrons, which would have been
protected from conversion to hydrogen by the binding of a protein described herein.
Optionally, the dissociation constant (Kd) of a protein for hG-CSFR or an
epitope thereof is determined. The "Kd" or "Kd value" for a hG-CSFR binding protein
is in one example measured by a radiolabeled or fluorescently-labeled hG-CSFR
binding assay. This assay equilibrates the protein with a minimal concentration of
labeled G-CSFR in the presence of a titration series of unlabeled hG-CSFR. Following
washing to remove unbound hG-CSFR, the amount of label is determined, which is
indicative of the Kd of the protein.
According to another example the Kd or Kd value is measured by using surface
plasmon resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore,
Inc., Piscataway, NJ) with immobilized hG-CSFR or a region thereof.
In some examples, proteins having a similar Kd or a higher Kd than C1.2 or
C1.2G are selected, because they are likely to compete for binding to hG-CSFR.
Determining Competitive Binding
Assays for determining a protein that competitively inhibits binding of
monoclonal antibody C1.2 or C1.2G will be apparent to the skilled artisan. For
example, C1.2 or C1.2G is conjugated to a detectable label, e.g., a fluorescent label or a
radioactive label. The labeled antibody and the test protein are then mixed and
contacted with hG-CSFR or a region thereof (e.g., a polypeptide comprising SEQ ID
NO: 1) or a cell expressing same. The level of labeled C1.2 or C1.2G is then
determined and compared to the level determined when the labeled antibody is
contacted with the hG-CSFR, region or cells in the absence of the protein. If the level
of labeled C1.2 or C1.2G is reduced in the presence of the test protein compared to the
absence of the protein, the protein is considered to competitively inhibit binding of
C1.2 or C1.2G to hG-CSFR.
Optionally, the test protein is conjugated to different label to C1.2 or C1.2G.
This alternate labeling permits detection of the level of binding of the test protein to
hG-CSFR or the region thereof or the cell.
In another example, the protein is permitted to bind to hG-CSFR or a region
thereof (e.g., a polypeptide comprising SEQ ID NO: 1) or a cell expressing same prior
to contacting the hG-CSFR, region or cell with C1.2 or C1.2G. A reduction in the
amount of bound C1.2 or C1.2G in the presence of the protein compared to in the
absence of the protein indicates that the protein competitively inhibits C1.2 or C1.2G
binding to hG-CSFR. A reciprocal assay can also be performed using labeled protein
and first allowing C1.2 or C1.2G to bind to G-CSFR. In this case, a reduced amount of
labeled protein bound to hG-CSFR in the presence of C1.2 or C1.2G compared to in the
absence of C1.2 or C1.2G indicates that the protein competitively inhibits binding of
C1.2 or C1.2G to hG-CSFR.
Any of the foregoing assays can be performed with a mutant form of hG-CSFR
and/or SEQ ID NO: 1 and/or a ligand binding region of hG-CSFR to which C1.2 or
C1.2G binds, e.g., as described herein.
Determining Neutralization
In some examples of the present disclosure, a protein is capable of neutralizing
hG-CSFR signaling.
Various assays are known in the art for assessing the ability of a protein to
neutralize signaling of a ligand through a receptor.
In one example, the protein reduces or prevents G-CSF binding to the hG-
CSFR. These assays can be performed as a competitive binding assay as described
herein using labeled G-CSF and/or labeled protein.
In another example, the protein reduces formation of CFU-G when CD34 bone
marrow cells are cultured in the presence of G-CSF. In such assays, CD34 bone
marrow cells are cultured in a semi-solid cell culture medium in the presence of G-CSF
(e.g., about 10ng/ml cell culture medium) and, optionally stem cell factor (e.g., about
10ng/ml cell culture medium) in the presence or absence of a test protein. After a
sufficient time for granulocyte clones (CFU-G) to form, the number of clones or
colonies is determined. A reduction in the number of colonies in the presence of the
protein compared to in the absence of the protein indicates that the protein neutralizes
G-CSF signaling. By testing multiple concentrations of the protein an IC is
determined, i.e., a concentration at which 50% of the maximum inhibition of CFU-G
formation occurs. In one example, the IC is 0.2nM or less, such as 0.1nM or less, for
example, 0.09nM or less, or 0.08nM or less, or 0.07nM or less, or 0.06nM or less
or0.05nM or less. In one example, the IC is 0.04nM or less. In another example, the
IC is 0.02nM or less. The foregoing IC s relate to any CFU-G assay described
50 50
herein.
In a further example, the protein reduces proliferation of cells (e.g., BaF3 cells)
expressing hG-CSFR which are cultured in the presence of G-CSF. Cells are cultured
in the presence of G-CSF (e.g., 0.5ng/ml) and the presence or absence of a test protein.
Methods for assessing cell proliferation are known in the art and include, for example,
MTT reduction and thymidine incorporation. A protein that reduces the level of
proliferation compared to the level observed in the absence of the protein is considered
to neutralize G-CSF signaling. By testing multiple concentrations of the protein an
IC is determined, i.e., a concentration at which 50% of the maximum inhibition of
cell proliferation occurs. In one example, the IC is 6nM or less, such as 5.9nM or
less. In another example, the IC is 2nM or less or 1nM or less or 0.7nM or cell or
0.6nM or less or 0.5nM or less. The foregoing IC s relate to any cell proliferation
assay described herein.
In a further example, the protein reduces mobilization of hematopoietic stem
cells and/or endothelial progenitor cells in vivo following G-CSF administration and/or
reduces the number of neutrophils in vivo, e.g., following G-CSF administration
(however this is not essential). For example, the protein is administered to a subject,
optionally before, at the time of or after administration of G-CSF or a modified form
thereof (e.g., PEGylated G-CSF or filgrastim). The number of hematopoietic stem cells
(e.g., expressing CD34 and/or Thy1) and/or endothelial progenitor cells (e.g.,
expressing CD34 and VEGFR2) and/or neutrophils (identified morphologically and/or
expressing e.g., CD10, CD14, CD31 and/or CD88) is assessed. A protein that reduces
the level of the cell(s) compared to the level observed in the absence of the protein is
considered to neutralize G-CSF signaling. In one example, the protein reduces the
number of neutrophils without inducing neutropenia.
Other methods for assessing neutralization of G-CSF signaling are contemplated
by the present disclosure.
Determining Effector Function
As discussed herein, some proteins of the present disclosure have reduced
effector function. Methods for assessing ADCC activity are known in the art.
In one example, the level of ADCC activity is assessed using a Cr release
assay, an europium release assay or a S release assay. In each of these assays, cells
expressing G-CSFR are cultured with one or more of the recited compounds for a time
and under conditions sufficient for the compound to be taken up by the cell. In the case
35
of a S release assay, cells expressing hG-CSFR can be cultured with S-labeled
methionine and/or cysteine for a time sufficient for the labeled amino acids to be
incorporated into newly synthesized proteins. Cells are then cultured in the presence or
absence of the protein and in the presence of immune effector cells, e.g., peripheral
blood mononuclear cells (PBMC) and/or NK cells. The amount of Cr, europium
and/or S in cell culture medium is then detected, and little or no change in the
presence of the protein compared to in the absence of protein (or a reduced level of the
compound compared to the level observed in the presence of an anti-hG-CSFR
antibody comprising a human IgG1 Fc) indicates that the protein has reduced effector
function. Exemplary publications disclosing assays for assessing the level of ADCC
induced by a protein include Hellstrom, et al. Proc. Natl Acad. Sci. USA 83:7059-7063,
1986 and Bruggemann, et al., J. Exp. Med. 166:1351-1361, 1987.
Other assays for assessing the level of ADCC induced by a protein include
ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc.
CA, USA) or CytoTox 96® non-radioactive cytotoxicity assay (Promega, WI, USA).
C1q binding assays may also be carried out to confirm that the protein is able to
bind C1q and may induce CDC. To assess complement activation, a CDC assay may be
performed (see, for example, Gazzano-Santoro et al, J. Immunol. Methods 202: 163,
1996.
Determining Half Life
Some proteins encompassed by the present disclosure have an improved half-
life, e.g., are modified to extend their half-life compared to proteins that are
unmodified. Methods for determining a protein with an improved half-life will be
apparent to the skilled person. For example, the ability of a protein to bind to a
neonatal Fc receptor (FcRn) is assessed. In this regard, increased binding affinity for
FcRn increased the serum half-life of the molecule (see for example, Kim et al., Eur J
Immunol., 24:2429, 1994).
The half-life of a protein of the disclosure can also be measured by
pharmacokinetic studies, e.g., according to the method described by Kim et al, Eur J of
Immunol 24:542, 1994. According to this method radiolabeled protein is injected
intravenously into mice and its plasma concentration is periodically measured as a
function of time, for example at 3 minutes to 72 hours after the injection. The clearance
curve thus obtained should be biphasic, that is, an alpha phase and beta phase. For the
determination of the in vivo half-life of the protein, the clearance rate in beta-phase is
calculated and compared with that of the wild type or unmodified protein.
Assessing Therapeutic Efficacy
Assays for assessing therapeutic efficacy are described hereinabove in relation
to determining neutralization by a protein.
In another example, the efficacy of a protein to treat a condition is assessed
using an in vivo assay.
For example, the protein is tested in an animal model of arthritis. Exemplary
models include a SKG strain of mouse (Sakaguchi et al., Nature, 426: 454-460), rat
type II collagen arthritis model, mouse type II collagen arthritis model or antigen
induced arthritis models in several species (Bendele J Musculoskel Neuron Interact;
1(4):377-385, 2001). In these assays, arthritis is induced and the ability of the protein
to reduce one or more symptoms of arthritis, e.g., joint inflammation and/or markers of
inflammation in synovial fluid is assessed. A protein that reduces a symptom of
arthritis is considered useful for treating this condition or a G-CSF-mediated condition
(e.g., a G-CSF-mediated inflammatory condition).
The protein can also or alternatively be tested in a model of COPD, e.g., in
which a non-human mammal (e.g., a rodent, such as, a mouse) is exposed to cigarette
smoke. Following exposure, the mammal is administered a protein and the level of
lung inflammation and/or the number of neutrophils in the lung is assessed or estimated
using standard techniques. A protein that reduces lung inflammation and/or the number
of neutrophils is considered useful for treating lung inflammation or COPD or a G-
CSF-mediated condition (e.g., a G-CSF-mediated inflammatory condition, such as a G-
CSF-mediated inflammatory lung condition).
Proteins described herein can also tested in in vivo models of inflammatory
neurological disease. Exemplary models include EAE models in which a mouse or rat
is immunized with a myelin sheath protein or peptide derived therefrom (e.g., MOG,
MBP or PLP) and an immune response is generated against the protein thereby
inducing a model of MS. Alternatively, T cells that are immunoreactive with a myelin
sheath protein are introduced into mice or rats to induce EAE. Exemplary EAE models
are reviewed in, for example Tsunoda and Fujinami, J Neuropathol Exp Neurol.55:673-
686, 1996.
Other models of MS include transgenic animals expressing T cell receptors
specific for a myelin protein, e.g., MOG, MBP or PLP. Exemplary models are
described, for example, in Bettelli et al., JEM 197:1073-1081, 2003; Illés et al., Proc.
Natl. Acad. Sci. USA, 101: 11749-11754, 2004; or Rossi et al., J. Biomolecular
Screening, 12: 481-489, 2007; or are commercially available, e.g., from Jackson
Laboratories USA (e.g. mice 2D2 having transgenic T cell receptors reactive with
MOG).
In a further example, a protein described herein according to any example is
tested in a model of uveitis. Models of uveitis include those induced by immunizing a
non-human mammal with a protein such as retinal arrestin, recoverin or rhodopsin or
administration of bacterial endotoxin to eye. Exemplary models of uveitis are
described, for example, in Caspi, Drug Discovery Today, 3: 3-9, 2006.
A protein of the disclosure can also be tested in models of angiogenesis, e.g.,
Iris Pharma Inc’s models of ocular angiogenesis, or an alginate encapsulated tumor cell
model, and/or by assessing the ability of a cancer cell to metastasize in a subject.
Conditions to be Treated
The present disclosure contemplates treatment or prevention of any condition
that is caused by or exacerbated by G-CSF in a subject. In one example, the condition
is an autoimmune or inflammatory condition.
In one example, the inflammatory or autoimmune condition is an inflammatory
joint condition, such as, inflammatory arthritis, rheumatoid arthritis or idiopathic
arthritis, e.g., juvenile idiopathic arthritis. In one example, the condition is rheumatoid
arthritis.
In one example, the inflammatory or autoimmune condition is an inflammatory
eye condition. For example, the condition is uveitis.
In one example, the inflammatory or autoimmune condition is an inflammatory
lung condition, such as, a pulmonary disease associated with neutrophil infiltration,
e.g., COPD. In one example, the condition is COPD.
In one example, the inflammatory or autoimmune condition is an inflammatory
neurological condition, such as, Devic's disease, a viral infection in the brain or
multiple sclerosis. In one example, the condition is multiple sclerosis, which includes
chronic progressive multiple sclerosis or relapsing-remitting multiple sclerosis.
In another example, the condition is cancer (including angiogenesis thereof) or
metastasis thereof.
In one example, the subject is resistant to, does not adequately respond to, or is
unsuitable for treatment with another compound used to treat the condition. For
example, the subject suffering from an autoimmune or inflammatory condition is
resistant to, does not adequately respond to, or is unsuitable for treatment with a
corticosteroid and/or an immunosuppressant and/or cyclophosphamide and and/or
methotrexate and/or an anti-TNF antibody or soluble TNF receptor and/or an anti-
CD20 antibody and/or an anti-IL6 antibody and/or an anti-CD22 antibody.
Compositions
In some examples, a protein as described herein can be administered orally,
parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally,
bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations
containing conventional non-toxic pharmaceutically-acceptable carriers, or by any
other convenient dosage form. The term “parenteral” as used herein includes
subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular,
intrasternal, and intracranial injection or infusion techniques.
Methods for preparing a protein into a suitable form for administration to a
subject (e.g. a pharmaceutical composition) are known in the art and include, for
example, methods as described in Remington's Pharmaceutical Sciences (18th ed.,
Mack Publishing Co., Easton, Pa., 1990) and U.S. Pharmacopeia: National Formulary
(Mack Publishing Company, Easton, Pa., 1984).
The pharmaceutical compositions of this disclosure are particularly useful for
parenteral administration, such as intravenous administration or administration into a
body cavity or lumen of an organ or joint. The compositions for administration will
commonly comprise a solution of protein dissolved in a pharmaceutically acceptable
carrier, for example an aqueous carrier. A variety of aqueous carriers can be used, e.g.,
buffered saline and the like. The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate physiological conditions
such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride, calcium chloride,
sodium lactate and the like. The concentration of proteins of the present disclosure in
these formulations can vary widely, and will be selected primarily based on fluid
volumes, viscosities, body weight and the like in accordance with the particular mode
of administration selected and the patient's needs. Exemplary carriers include water,
saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous
vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be
used as carriers. The vehicles may contain minor amounts of additives that enhance
isotonicity and chemical stability, e.g., buffers and preservatives.
Upon formulation, proteins of the present disclosure will be administered in a
manner compatible with the dosage formulation and in such amount as is
therapeutically/prophylactically effective. Formulations are easily administered in a
variety of dosage forms, such as the type of injectable solutions described above, but
other pharmaceutically acceptable forms are also contemplated, e.g., tablets, pills,
capsules or other solids for oral administration, suppositories, pessaries, nasal solutions
or sprays, aerosols, inhalants, liposomal forms and the like. Pharmaceutical "slow
release" capsules or compositions may also be used. Slow release formulations are
generally designed to give a constant drug level over an extended period and may be
used to deliver compounds of the present disclosure.
WO2002/080967 describes compositions and methods for administering
aerosolized compositions comprising antibodies for the treatment of, e.g., asthma,
which are also suitable for administration of a protein of the present disclosure.
Combination Therapies
In one example, a protein of the present disclosure is administered in
combination with another compound useful for treating a disease or condition described
herein, either as combined or additional treatment steps or as additional components of
a therapeutic formulation.
For example, the other compound is an anti-inflammatory compound.
Alternatively, or additionally, the other compound is an immunosuppressant.
Alternatively, or additionally, the other compound is a corticosteroid, such as
prednisone and/or prednisolone. Alternatively, or additionally, the other compound is
methotrexate. Alternatively, or additionally, the other compound is cyclophosphamide.
Alternatively, or additionally, the other compound is mycophenolate mofetil.
Alternatively, or additionally, the other compound is an anti-CD20 antibody (e.g.,
rituximab or ofatumumab). Alternatively, or additionally, the other compound is an
anti-CD22 antibody (e.g., epratuzumab). Alternatively, or additionally, the other
compound is an anti-TNF antibody (e.g., infliximab or adalimumab or golimumab) or
soluble TNF receptor (e.g., etanercept). Alternatively, or additionally, the other
compound is a CTLA-4 antagonist (e.g., abatacept, CTLA4-Ig). Alternatively, or
additionally, the other compound is an anti-IL-6 antibody. Alternatively, or
additionally, the other compound is a BLys antagonist, such as an anti-BLys antibody
(e.g., belimumab).
In another example, the other compound is a chemotherapy drug or other drug
used for treating cancer.
In another example, the protein described herein is administered before or after
radiotherapy for the treatment of cancer.
Dosages and Timing of Administration
Suitable dosages of proteins of the present disclosure will vary depending on the
specific protein, the condition to be treated and/or the subject being treated. It is within
the ability of a skilled physician to determine a suitable dosage, e.g., by commencing
with a sub-optimal dosage and incrementally modifying the dosage to determine an
optimal or useful dosage. Alternatively, to determine an appropriate dosage for
treatment/prophylaxis, data from the cell culture assays or animal studies are used,
wherein a suitable dose is within a range of circulating concentrations that include the
ED of the active compound with little or no toxicity. The dosage may vary within this
range depending upon the dosage form employed and the route of administration
utilized. A therapeutically/prophylactically effective dose can be estimated initially
from cell culture assays. A dose may be formulated in animal models to achieve a
circulating plasma concentration range that includes the IC (i.e., the concentration of
the compound which achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to more accurately determine useful doses in
humans. Levels in plasma maybe measured, for example, by high performance liquid
chromatography.
In some examples, a method of the present disclosure comprises administering a
prophylactically or therapeutically effective amount of a protein described herein.
The term “therapeutically effective amount” is the quantity which, when
administered to a subject in need of treatment, improves the prognosis and/or state of
the subject and/or that reduces or inhibits one or more symptoms of a clinical condition
described herein to a level that is below that observed and accepted as clinically
diagnostic or clinically characteristic of that condition. The amount to be administered
to a subject will depend on the particular characteristics of the condition to be treated,
the type and stage of condition being treated, the mode of administration, and the
characteristics of the subject, such as general health, other diseases, age, sex, genotype,
and body weight. A person skilled in the art will be able to determine appropriate
dosages depending on these and other factors. Accordingly, this term is not to be
construed to limit the present disclosure to a specific quantity, e.g., weight or amount of
protein(s), rather the present disclosure encompasses any amount of the protein(s)
sufficient to achieve the stated result in a subject. In one example, a therapeutically
effective amount of the protein does not induce neutropenia.
As used herein, the term “prophylactically effective amount” shall be taken to
mean a sufficient quantity of a protein to prevent or inhibit or delay the onset of one or
more detectable symptoms of a clinical condition. The skilled artisan will be aware
that such an amount will vary depending on, for example, the specific protein(s)
administered and/or the particular subject and/or the type or severity or level of
condition and/or predisposition (genetic or otherwise) to the condition. Accordingly,
this term is not to be construed to limit the present disclosure to a specific quantity,
e.g., weight or amount of protein(s), rather the present disclosure encompasses any
amount of the protein(s) sufficient to achieve the stated result in a subject. In one
example, a prophylactically effective amount of the protein does not induce
neutropenia.
For in vivo administration of the proteins described herein, normal dosage
amounts may vary from about 10ng/kg up to about 100mg/kg of an individual's body
weight or more per day. Exemplary dosages and ranges thereof are described herein.
For repeated administrations over several days or longer, depending on the severity of
the disease or disorder to be treated, the treatment can be sustained until a desired
suppression of symptoms is achieved.
In some examples, the protein is administered at an initial (or loading) dose of
between about 1mg/kg to about 30mg/kg, such as from about 1mg/kg to about
10mg/kg, or about 1mg/kg or about 2mg/kg or 5mg/kg. The protein can then be
administered at a lower maintenance dose of between about 0.01mg/kg to about
2mg/kg, such as from about 0.05mg/kg to about 1mg/kg, for example, from about
0.1mg/kg to about 1mg/kg, such as about 0.1mg/kg or 0.5mg/kg or 1mg/kg. The
maintenance doses may be administered every 7-30 days, such as, every 10-15 days,
for example, every 10 or 11 or 12 or 13 or 14 or 15 days.
In some examples, the protein is administered at a dose of between about
0.01mg/kg to about 50mg/kg, such as between about 0.05mg/kg to about 30mg/kg, for
example, between about 0.1mg/kg to about 20mg/kg, for example, between about
0.1mg/kg to about 10mg/kg, such as between about 0.1mg/kg to about 2mg/kg. For
example, the protein is administered at a dose of between about 0.01mg/kg to about
5mg/kg, such as from about 0.1mg/kg to about 2mg/kg, such as about 0.2mg/kg or
0.3mg/kg or 0.5mg/kg or 1mg/kg or 1.5mg/kg (e.g., without a higher loading dose or a
lower maintenance dose). In some examples, numerous doses are administered, e.g.,
every 7-30 days, such as, every 10-22 days, for example, every 10-15 days, for
example, every 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or
22 days. For example, the protein is administered every 7 days or every 14 days or
every 21 days.
In some examples, at the time of commencing therapy, the mammal is
administered the protein on no more than 7 consecutive days or 6 consecutive days or 5
consecutive days or 4 consecutive days.
In the case of a mammal that is not adequately responding to treatment, multiple
doses in a week may be administered. Alternatively, or in addition, increasing doses
may be administered.
In another example, for mammals experiencing an adverse reaction, the initial
(or loading) dose may be split over numerous days in one week or over numerous
consecutive days.
Administration of a protein according to the methods of the present disclosure
can be continuous or intermittent, depending, for example, on the recipient's
physiological condition, whether the purpose of the administration is therapeutic or
prophylactic, and other factors known to skilled practitioners. The administration of a
protein may be essentially continuous over a preselected period of time or may be in a
series of spaced doses, e.g., either during or after development of a condition.
Non-Limiting Examples
Methods
The Ig and CRH domains of the G-CSFR are involved in ligand binding and
receptor dimerization (Layton et al., J. Biol Chem, 272: 29735-29741, 1997 and
Fukunaga et al, EMBO J. 10: 2855-2865 1991). Soluble forms of G-CSFR (with either
a C-terminal polyhistidine tag or an Fc sequence) comprising these portions of the
receptor have been used in various studies of the receptor, and mutation of the free
cysteines at positions 78, 163, and 228 of the receptor assists in expression and
isolation of the soluble receptor polypeptide (Mine et al., Biochem., 43: 2458-2464,
2004) without affecting ligand binding. In the present studies soluble forms of the
receptor comprising amino acids 25-335 of hG-CSFR with mutations C78A, C163S
and C228S were generally used (e.g. SEQ ID NO:1) and the corresponding segment of
cynoG-CSFR with the cysteine mutations was generally used for studies on the
cynomolgus monkey receptor. Various point mutations of the soluble receptor of SEQ
ID NO:1 have also been utilized. Reference to hG-CSFR-Fc means the polypeptide of
SEQ ID NO:1 wherein the C-terminal polyhistidine tag has been replaced with an Fc
sequence. cynoG-CSFR-Fc means the corresponding segment of cynoG-CSFR with an
Fc sequence attached to its C-terminal. In some instance the corresponding
extracellular domains of the wild type receptor have been used, and in these instances it
is specifically noted. The inventors have shown that antibodies and proteins
comprising antigen binding sites thereof (e.g., Fab) bind to wild type hG-CSF
polypeptides and to these mutant proteins with highly similar affinity. Accordingly,
studies using the mutant proteins are a model of studies using wild type hG-CSFR.
Identification of Fabs from Phage Display Library
A phage display library was screened for clones binding to hG-CSFR which
eluted upon addition of G-CSF ligand. Fabs were assessed for their ability to bind hG-
CSFR, compete G-CSF binding, and cynomolgus G-CSFR cross-reactivity and some
clones reformatted as IgG4 antibodies. Potency was then tested through neutralization
of G-CSF mediated proliferation in a BaF3 cell line stably transfected with hG-CSFR
(described below) and to inhibit CFU-G formation in the presence of G-CSF.
Mammalian Expression Vector Construction for IgG Expression
Mammalian expression vectors were constructed using standard molecular
biology techniques by cloning the entire light chain (variable and constant domains)
and the variable region of the heavy chain from the selected phage-derived Fab
constructs.
Cell Culture and Transient Transfection
Serum-free suspension adapted 293-T cells were obtained from Genechoice Inc.
Cells were cultured in FreeStyle™ Expression Medium (Invitrogen) supplemented with
penicillin/streptomycin/fungizone reagent (Invitrogen). Prior to transfection the cells
were maintained at 37°C in humidified incubators with an atmosphere of 8% CO .
The Transient transfection of the mammalian expression vectors using 293-T
cells was performed using 293fectin transfection reagent (Invitrogen) according to the
manufacturer’s instructions. The cell culture supernatants were harvested after 5 days
incubation by centrifugation at 2500 rpm and were then passed through a 0.45µM filter
(Nalgene) prior to purification using standard methods for IgG purification.
Control Antibodies
Murine monoclonal antibodies 711, 744 and 774 (Layton et al., Growth Factors,
14: 117-130, 1997) were used as control mouse antibodies.
Affinity Measurements of Fabs
To measure affinity of Fab for G-CSFR or G-CSFR-Fc, Fabs were expressed in
E. coli and affinity measured using a Biacore 2000.
Measurement of Binding Kinetics for mAbs
Anti-human (Goat anti-human IgG (gamma) mouse adsorbed, Invitrogen, Cat
No. H10500) or anti mouse Fc specific antibody (Jackson Immuno Research Labs inc.
Cat No. 515071) was chemically immobilized on a CM-5 sensor surface using
amine coupling chemistry.
The immobilized antibodies were then used to capture anti hG-CSFR mAbs
from solution. Soluble hG-CSFR proteins (as described in the methods section) were
then injected over captured mAb at various concentrations. mAbs were captured for
180 seconds at 0.3µg/ml. Soluble hG-CSFR at 0, 1.25, 2.5, 5, 10, 20 and 40nM (in
duplicate ) was injected for 10 minutes and dissociation was monitored for 30 minutes.
Responses from a reference flow cell (in which mAb was not captured, but otherwise
treated identically), were subtracted. The responses from a blank injection were then
subtracted from the resultant sensorgrams.
The final corrected responses were fitted using non-linear regression to a model
describing 1:1 kinetics, including a term for mass transport limitation. The Rmax value
was fitted locally, to account for slight deviations in the level of mAb captured.
Equilibrium dissociation constant (KD) was determined.
Kinetic analysis of Fab C1.2, 5D11, 711 and 744
Fab fragments were generated by papain digestion where 3mg of antibody was
digested (1:500) with pre-activated papain for 40 minutes as per instructions using a
papain digestion kit (Sigma, USA). Resultant Fab was purified, by adsorption, away
from residual Fc and undigested antibody using protein A purification (mAbSelect, GE,
Sweden).
Duplicate biosensor analysis of the Fab were performed using a Biacore 2000
(GE, Sweden) with a doubling dilution of Fab antibodies (100nM to 0.39nM in
0.1mg/ml BSA) at a flow rate of 30μl/min. Binding (100μl) was monitored to
respective flow cells containing control immobilised blank, immobilized cyno G-
CSFR-Fc, immobilized human G-CSFR-Fc and immobilized human G-CSFR.
Receptor proteins (20μg per ml in 20mM Sodium Acetate pH 4.5) were previously
immobilized to a CM5 chip using a NHS/EDC coupling kit as per the manufacturer’s
instructions (Biacore GE, Sweden). Target immobilization values were set at 700, 700
and 500 resonance units for cyno G-CSFR-Fc, hG-CSFR-Fc and hG-CSFR,
respectively. Chip immobilisation was quenched with 50mM ethanolamine pH 8.0.
Dissociation of surface binding was monitored for 1000 seconds prior to desorption of
the remaining complex using 50mM Phosphoric acid. Reference binding was then
subtracted from the control channel and kinetics generated using biaevaluation software
on the Biacore 2000.
Duplicate biosensor analysis of the antibodies c1.2 and 5D11 in IgG4 fomat
were performed using a Biacore 2000 with a doubling dilution of the antibodies
(312nM to 2.4nM in 0.1mg/ml BSA) at a flow rate of 30ul/min. Binding (100μl) was
monitored to respective flow cells containing control immobilized blank, immobilized
cyno-G-CSFR-Fc, immobilized hG-CSFR-Fc and immobilized hG-CSFR. Receptor
proteins (20ug per ml in 20mM Sodium Acetate pH 4.5) were previously immobilized
to a CM5 chip using a NHS/EDC coupling kit as per the manufacturer’s instructions
(Biacore GE, Sweden). Target immobilization values were set at 700, 700 and 500
resonance units for cyno-G-CSFR-Fc, hG-CSFR-Fc and hG-CSFR, respectively. Chip
immobilization was quenched with 50mM ethanolamine pH 8.0. Dissociation of
surface binding was monitored for 1000 seconds prior to desorption of the remaining
complex using 50mM Phosphoric acid. Reference binding was then subtracted from
the control channel and kinetics generated using biaevaluation software on the Biacore
2000.
BIAcore mAb Kinetics of Affinity Matured C1.2G Antibodies
Anti human (Goat anti Human IgG (gamma) mouse adsorbed, Invitrogen, Cat
No. H10500) was chemically immobilised on a CM-5 sensor surface using amine
coupling chemistry and then used to capture the C1.2G affinity matured anti hG-CSFR
mAbs at 1mg/ml for 3 mins. Soluble hG-CSFR was then injected over the captured
mAb at 0, 10 and 40nM. Soluble hG-CSFR was injected for 5 minutes and dissociation
was monitored for 30 minutes. Responses from a reference flow cell (in which mAb
was not captured, but otherwise treated identically), were subtracted. The responses
from a blank injection were then subtracted from the resultant sensorgrams.
The final corrected responses were fitted using non-linear regression to a model
describing 1:1 kinetics, including a term for mass transport limitation. The Rmax value
was fitted locally, to account for slight deviations in the level of mAb captured.
Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K )
were determined.
hG-CSFR/BaF3 Proliferation Bioassay – MTT Reduction
BaF3 cells expressing hG-CSFR were obtained from the Ludwig Institute
Melbourne. To assess the inhibition of G-CSF mediated proliferation by anti hG-CSFR
antibodies, serial dilutions of antibody were added to 2 x10 cells/well in DME medium
with 5% FCS and 0.5ng/ml hGCSF in 96 well plates and incubated for 48 hours at
37 C, 10%CO . Cell proliferation was determined by MTT reduction and measured by
absorbance at 490nM.
hG-CSFR/BaF3 Proliferation Bioassay – 3H-Thymidine Incorporation
BAF/3 cells engineered to express human G-CSFR and which proliferate in
response to human G-CSF were used to measure the ability of various monoclonal
antibodies to neutralize the activity of G-CSF. Cells were plated at 1 x 10 cells in 96
well plates in RPMI/105FCS in the presence of 10ng/mL human G-CSF and increasing
concentrations of of various anti-G-CSFR monoclonal antibodies for 48 hours at 37 C.
Cells were pulsed with H-thymidine for the last 6 hours of culture before being
harvested onto glass fibre filters and the level of radioactive thymidine incorporated
into DNA determined by liquid scintillation counting.
Human CFU-G Progenitor Bioassay
CD34 bone marrow cells were incubated in semi-solid medium in the presence
of 10ng/ml stem cell factor, 10ng/ml hG-CSF and titrating concentrations of test
antibody. CFU-G were enumerated after 14 days of culture.
Epitope Comparison – Competition Binding
This design of this experiment is built on the premise that for 2 antibodies to be
capable of simultaneously binding a single molecule, the epitopes of those 2 antibodies
must be different.
Soluble G-CSFR of SEQ ID NO:1 was captured from solution by a surface
immobilized antibody. A second antibody was then injected over the complex.
Responses from a reference flow cell (in which soluble hG-CSFR was not captured, but
otherwise treated identically), were subtracted. Binding of the second antibody
indicates that the epitopes of the 2 antibodies differ.
Responses measured at the end of the antibody binding phase were divided by
the response at the end of the hG-CSFR capture phase, to correct antibody binding level
for the amount of hG-CSFR captured. These capture corrected responses were then
used to compare the binding of each antibody to hG-CSFR in the presence of the other
antibodies.
Antibodies C1.2, 5D11, 711 and 744 were chemically immobilized on a CM-5
sensor surface using amine coupling chemistry. Soluble hG-CSFR was captured at
100nM for 180 seconds. Each of the antibodies was then injected in duplicate over
captured hG-CSFR at 100nM for 180 seconds. A blank injection of buffer only was
also performed.
Epitope Mapping of C1.2G, 711, 744 and 774
A series of alanine point mutations of SEQ ID NO:1 were generated, expressed
in HEK293 cells and then purified. The binding affinity of these mutants for antibodies
C1.2, 744 and 774 was measured, and compared to that of SEQ ID NO:1. If a mutation
resulted in a change of affinity by more than a factor of 2 from that of SEQ ID NO:1,
that residue was deemed to contribute to the binding interaction, and thus is likely to be
in or near the epitope. A third mAb (711) with an epitope separate and distinct to C1.2,
744 and 774, was included as a control in order to account for any major structural
changes brought about by the mutations.
Anti human (Goat anti Human IgG (gamma) mouse adsorbed, Invitrogen, Cat
No. H10500) or anti mouse Fc specific antibody (Jackson Immuno Research Labs inc.
Cat No. 515071) was chemically immobilized on a CM-5 sensor surface using
amine coupling chemistry. The immobilized antibodies were then used to capture anti
hG-CSFR mAbs from solution. Wild-type hG-CSFR ligand binding domain (SEQ ID
NO:1) and each alanine point mutant were then injected over captured mAbs at various
concentrations. Responses from a reference flow cell (in which mAb was not captured,
but otherwise treated identically), were subtracted. The responses from a blank
injection were then subtracted from the resultant sensorgrams.
The final corrected responses were fitted using non-linear regression to a model
describing 1:1 kinetics, including a term for mass transport limitation. The Rmax value
was fitted locally, to account for slight deviations in the level of mAb captured.
Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD)
were determined.
C1.2 germline mAb was captured at 0.3ug/ml for 180sec, 711 at 1µg/ml for 180
sec, and 744 and 774 at 5µg/ml for 180sec.
For C1.2 and 744 kinetics, WT hG-CSFR and each ala mutant were injected at
0, 2, 10, 50 and 250nM for 300 sec and dissociation monitored for a further 1800 sec.
For antibody 774 kinetics, WT hG-CSFR and each ala mutant were injected at 0,
2, 10, 50 and 250nM for 300 sec and dissociation monitored for a further 600 sec.
For antibody 711 kinetics, WT hG-CSFR and each ala mutant were injected at 0
and 100nM for 180 sec and dissociation monitored for a further 180 sec.
Example 1: Fully Human Anti-hG-CSFR Antibodies Are Potent Inhibitors G-CSF
Signaling
Using affinity measurements and the BaF3 proliferation assay described above,
antibodies 711 and 744 were assessed for affinity to hG-CSFR and G-CSF
neutralization assays. Antibody 711 was found to bind to hG-CSFR-Fc fusion (based
on SEQ ID NO:1 as discussed in the methods) with an affinity greater than antibody
744 (K of 0.86nM and 8.7nM, respectively). Using the MTT-based bioassay
described above, antibody 711 was also found to more potently inhibit G-CSF-
mediated cell proliferation than antibody 744 (IC (nM G-CSF) of 8.8nM and 2.4nM,
respectively).
Using the H-thymidine incorporation assay, antibody 711 was found to inhibit
G-CSF-mediated cell proliferation with an IC of 10.1μg/ml; antibody 774 was found
to inhibit G-CSF-mediated cell proliferation with an IC of 37.4μg/ml and the IC for
50 50
antibody 744 was not determinable (see Figure 1).
Human antibodies isolated from a phage display library (antibodies C1.2 and
5D11) and mouse monoclonal antibody 711 were assessed for their ability to inhibit G-
CSF-mediated proliferation of BaF3 cells using the bioassay described above. Results
showed that antibody C1.2 inhibited BaF3 proliferation with an IC of 0.5nM;
antibody 5D11 inhibited proliferation an IC of 5.9nM; and 711 inhibited proliferation
with an IC of 3.4nM.
The antibodies were also assessed for their ability to reduce or inhibit CFU-G
formation by CD34 bone marrow cells in the presence of G-CSF as described above.
Results showed that antibody C1.2 inhibited CFU-G formation with an IC of
0.016nM; antibody 5D11 inhibited CFU-G formation with an IC of 0.039nM; and
711 inhibited CFU-G formation with an IC of 0.411nM.
Based on these assays, the human antibodies C1.2 and 5D11 are more potent
than 711 in inhibiting CFU-G formation and C1.2 is the most potent antibody in both
bioassays.
Example 2: Affinity of Antibodies for Human G-CSFR and Cynomolgus Monkey G-
CSFR
Affinities of Fabs and IgG4 forms of 5D11 and C1.2 and Fabs of 711 and 744
were also assessed to determine their affinities for the ligand binding domain of hG-
CSFR(SEQ ID NO:1), hG-CSFR-Fc and cynomolgus monkey G-CSFR-Fc (based on
SEQ ID NO:1 as discussed in the methods). In these assays, the regions of G-CSFR
were immobilized and binding of the indicated antibody or fragment to the
immobilized polypeptide determined as described in more detail in the general methods
(Section entitled “Kinetic analysis of Fab C1.2, 5D11, 711 and 744”). Results are
shown in Table 1.
Table 1: Affinities of 5D11 and C1.2 for human and cynomolgus monkey G-CSFR
Chip Immobilised with:
Antibody / Fab hG-CSFR hG-CSFR-Fc cyno G-CSFR-Fc
5D11 Fab 1.30nM 1.20nM 0.42nM
C1.2 Fab 0.37nM 0.33nM 0.39nM
5D11 IgG4 65pM 61pM 37pM
C1.2 IgG4 27pM 77pM 54pM
These data show that 5D11 and C1.2 have high affinities for hG-CSFR and that
these affinities are improved when the Fabs are expressed as complete IgG4 antibodies.
Moreover, the affinities for hG-CSFR and cynoG-CSFR for 5D11 or C1.2 are similar.
C1.2 has higher affinity for hG-CSFR than 5D11.
The Fab of antibody 711 was shown to have an affinity for hG-CSFR-Fc of
0.86nM and for cynoG-CSFR of 1.8μM. The Fab of antibody 744 was shown to have
an affinity for hG-CSFR-Fc of 8.7nM and for cynoG-CSFR of >10μM. Thus, these
Fabs have much poorer affinity for cynoG-CSFR than they do for hG-CSFR.
Reciprocal assays were also performed (i.e., in which antibodies were
immobilized and binding of wild type h-GCSFR or wild type cynoG-CSFR to the
immobilized antibody was determined in accordance with the general methods (Section
entitled “Measurement of Binding Kinetics for mAbs”). Representative results are
shown in Table 2.
Table 2: Representative affinities of antibodies C1.2G, 5D11, 711, 744 and 774 for
wild type hG-CSFR or wild type cynoG-CSFR.
Binding to wt cyno G-CSFR
Captured Antibody Binding to wt hG-CSFR (K )
(K )
C1.2G (human IgG4) 1.4nM 494pM
5D11 (human IgG4) 6.09nM 477pM
mAb 711 (mIgG1) 1.69nM 97nM
mAb744 (mIgG2a) 7.82nM Negligible binding
mAb774 (mIgG1) 23.4nM Negligible binding
These data shown that C1.2G and 5D11 bind to wild type cynoG-CSFR with
higher affinities than 711, 744 and 774. Moreover, the affinity of C1.2G for wild type
hG-CSFR and wild type cynoG-CSFR are within about 3 fold of one another and the
affinity of 5D11 for wild type hG-CSFR and wild type cynoG-CSFR are within about
13 fold of one another.
Example 3: Germlining of C1.2
To minimize potential immunogenicity, the variable region framework of C1.2
was changed to match that of the closest human germline framework. This required a
single change in the framework of the heavy chain and five changes in the light chain
and resulted in a V with a sequence set forth in SEQ ID NO: 4 and a V with a
sequence set forth in SEQ ID NO: 5. Affinity of the germlined antibody (C1.2G) for
4 3 -4 -6
G-CSFR was similar to C1.2 (k 9.54 x 10 ± 5.5 x 10 ; k 1.31 x 10 ± 2.6 x 10 ; K
a d D
1.37 ± 0.07 (N=8)). Affinity of C1.2G for hG-CSFR expressed on the cell surface of
BaF3 cells was shown to be 257pM.
Using the H-thymidine incorporation assay described above, antibody
C1.2G was found to inhibit G-CSF-mediated cell proliferation with an IC of 0.8μg/ml
(Figure 1).
Reformatting of this antibody into a stabilized IgG4 produced an antibody
comprising a heavy chain with a sequence set forth in SEQ ID NO: 64 and a light chain
with a sequence set forth in SEQ ID NO: 65.
When expressed in CHO cells a lysine variant of the antibody was observed, i.e.,
in which one or both of the heavy chains lacked a C-terminal lysine residue (i.e., thus
comprising a sequence set forth in SEQ ID NO: 64).
Example 4: Epitope Mapping
Competition Binding
Published data has shown that mAb711 and mAb744 bound to different domains
of the hG-CSFR. Competition binding experiments showed that mAb711 but not
mAb744 was able to bind to the hG-CSFR subsequent to the binding of C1.2 Ab
suggesting that C1.2 binds to a similar region of the receptor as mAb744 but a different
region to that of mAb711.
Epitope Excision to Identify Peptides Involved in C1.2 Binding
Epitope excision followed by mass spectrometry analysis was used to identify
four peptides of the hG-CSFR that were involved in the binding of C1.2. In this
method, the hG-CSFR protein is first bound to an immobilized C1.2 antibody and then
digested with proteolytic enzymes. The bound peptides are then identified by MALDI
and Electrospray mass spectrometry. The four peptides identified by this approach
mapped to positions 111-115, 170-176, 218-234 and/or 286-300 of hG-CSFR (SEQ ID
NO: 1).
Binding of C1.2 and mAb744 to hG-CSFR Region Mutants
Published data (Tamada et al Proc Natl Acad Sci USA. 103:3135-3140, 2006;
Aritomi et al Acta Crystallogr D Biol Crystallogr; 56:751-753 1999) has identified
surface residues on the hG-CSFR. A number of these residues located within the four
peptides identified by epitope excision experiments were substituted by alanine and the
resulting mutant forms of a region of hG-CSFR were expressed and purified. The
binding of C1.2 and mAb744 to each of these mutants was assessed and the key
residues involved in binding identified. Results are shown in Figure 2A. Alanine
substitution of residues K167 and H168 resulted in a complete loss of binding by
mAb744 whilst binding of C1.2 was unaffected. In contrast, alanine substitution of
residue R287 resulted in a complete loss of binding by C1.2 with no effect on mAb744
binding. Other residues that significantly reduced the binding of C1.2 were L111,
L171, Y172, M198 and H237. MAb744 bound these mutants at a similar affinity to
that of the wild type receptor.
The above assay was repeated with the the same antibodies together with
mAb774/ As shown in Figure 2B, alanine substitution of residues K167, H168 and
L169 resulted in a complete loss of binding by mAb774 whilst binding of C1.2 was
unaffected. As with mAb 744, alanine substitution of residue L111, Y172, H237 and
R287 had little or no effect on mAb774 binding.
Example 5: Affinity Maturation of C1.2G
Affinity maturation was performed by mutating residues in HCDR3 and/or
LCDR3 of C1.2G and screening for Fabs that bound to hG-CSFR. Libraries of mutant
antibodies were panned using biotinylated hG-CSFR-Fc recombinant protein, either at
a constant concentration over several panning rounds or at reducing concentrations.
At the completion of panning, a number of phage clones were selected from
each enriched library and sequenced. Unique clones were then selected based on
sequence and reformatted into fully human IgG4/kappa antibodies for binding analysis
to hG-CSFR using Biacore (following the general methods in the Section entitled
“Measurement of Binding Kinetics for mAbs”) and, in some cases ability to inhibit G-
CSF-mediated proliferation of BaF3 cells. The reformatted antibodies with improved
affinities as compared to the parental C1.2G mAb are listed in Table 3.
Table 3: Characteristics of affinity matured antibodies
mAb LCDR3 Sequence HCDR3 Sequence K (M) IC50 (nM)
C1.2G-987 IQYPQM LGQSSA 4.46E-11 0.88
C1.2G-95 WEYPLV wt 5.78E-11 0.26
C1.2G-79 QVSWEY wt 6.13E-11 0.31
C1.2G-83 WMYALF wt 6.60E-11 0.21
C1.2G-1003 WHYPLT LGSGST 6.82E-11 0.26
C1.2G-44 YSYPQK wt 7.33E-11 0.39
C1.2G-97 FMYPLY wt 9.06E-11 0.21
C1.2G-986 YAYPQQ LGFFQE 9.11E-11 0.28
C1.2G-56 YMYPIK wt 9.93E-11 0.22
C1.2G-77 EQGWNY wt 1.07E-10 0.23
C1.2G-54 MWMPMG LGMFLE 1.10E-10 0.50
C1.2G-802 HFSMQY wt 1.11E-10 0.26
C1.2G-967 WAYGLS LGMYDL 1.34E-10 0.29
C1.2G-989 FYYPFY LGQYMF 1.44E-10 0.34
C1.2G-63 ANSWGY wt 1.61E-10 0.26
C1.2G-1002 WTYGQT LGMYMN 1.67E-10 0.18
C1.2G-994 LEYPQM LGQFMD 1.70E-10 0.49
C1.2G-969 FQYAQH LGQYQF 1.81E-10 0.41
C1.2G-1000 WMYAHM LGQMMY 1.82E-10 0.34
mAb LCDR3 Sequence HCDR3 Sequence K (M) IC50 (nM)
C1.2G-94 WVYPAW wt 1.89E-10 ND
C1.2G-975 WQIKLK LGQSML 2.09E-10 ND
C1.2G-75 EESMNY wt 2.11E-10 ND
C1.2G-814 SQSMEY wt 2.21E-10 ND
C1.2G-973 FKYPMT LGQMVY 2.30E-10 ND
C1.2G-977 WVYHLP LGEIRE 2.46E-10 ND
C1.2G-984 IEYPAH LGMMQS 2.50E-10 ND
C1.2G-61 QQGMWM wt 2.57E-10 ND
C1.2G-852 wt LGHSLA 2.92E-10 ND
C1.2G-996 MWMPIF LGQYMG 3.36E-10 ND
C1.2G-43 IGYPGS LGQFMR 3.36E-10 ND
C1.2G-999 WEYAMF LGMFHK 3.56E-10 ND
C1.2G-870 WMYHKI wt 3.67E-10 ND
C1.2G-877 FRYPFY wt 5.08E-10 ND
C1.2G wt wt 6.33E-10 0.32
Sequence is preceded by the sequence QQS
Sequence is followed by the sequence PLT
Sequence (other than wt) is preceded by the sequence LGE
wt – sequence of CDR3 from C1.2G
ND – not determined
Example 6: C1.2G Reduces Neutrophil Levels Without Inducing Neutropenia
Cynomolgus monkeys were administered pegylated G-CSF and C1.2G
(10mg/kg) administered 12 hours later. As shown in Figure 3, C1.2 significantly
reduced the level of neutrophils compared to control animals, however did not induce
neutropenia.
Similar experiments dosing cynomolgus monkeys with between 0.1 to 10 mg/kg
of C1.2G 2 hours prior to administration with G-CSF also reduced the level of
neutrophils compared to control animals, however did not induce neutropenia.
Claims (39)
1. A protein comprising an antigen binding site of an antibody, wherein the antigen binding site binds to human granulocyte-colony stimulating factor receptor (hG-CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, and wherein the protein inhibits growth of colony forming units – granulocytes (CFU-G) from CD34 bone marrow cells grown in the presence of G-CSF with an IC of at least about 0.2nM.
2. A protein comprising an antigen binding site of an antibody, wherein the antigen binding site binds to both human and cynomolgus monkey granulocyte-colony stimulating factor receptor (G- CSFR) with a similar affinity and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling.
3. A protein comprising an antigen binding site of an antibody wherein the antigen binding site binds specifically to the same epitope in human granulocyte-colony stimulating factor receptor (hG- CSFR) as that bound by C1.2 (comprising a heavy chain variable region (V ) comprising a sequence set forth in SEQ ID NO: 2 and a light chain variable region (V ) comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a V comprising a sequence set forth in SEQ ID NO: 4 and a V comprising a sequence set forth in SEQ ID NO: 5).
4. A protein comprising an antigen binding site of an antibody, wherein (i) the protein competitively inhibits binding of C1.2 (comprising a heavy chain variable region (V ) comprising a sequence set forth in SEQ ID NO: 2 and a light chain variable region (V ) comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a V comprising a sequence set forth in SEQ ID NO: 4 and a V comprising a sequence set forth in SEQ ID NO: 5) to human granulocyte-colony stimulating factor receptor (hG-CSFR); (ii) the protein neutralizes granulocyte colony stimulating factor (G-CSF) signaling; and (iii) the level of binding of the protein to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of: (a) the arginine at position 287 of SEQ ID NO:1; (b) the histidine at position 237 of SEQ ID NO:1; (c) the methionine at position 198 of SEQ ID NO:1; (d) the tyrosine at position 172 of SEQ ID NO:1; (e) the leucine at position 171 of SEQ ID NO:1; or (f) the leucine at position 111 of SEQ ID NO:1: is lower than the level of binding of the protein to a polypeptide of SEQ ID NO: 1.
5. The protein of claim 4, wherein the antigen binding site does not detectably bind to the polypeptide comprising the alanine substitution position 287 of SEQ ID NO: 1.
6. A protein comprising an antigen binding site of an antibody, wherein (i) the protein binds to human granulocyte colony stimulating factor receptor (hG-CSFR); (ii) the protein neutralizes granulocyte colony stimulating factor (G-CSF) signaling; and (iii) the level of binding of the protein to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of: (a) the arginine at position 287 of SEQ ID NO:1; (b) the histidine at position 237 of SEQ ID NO:1; (c) the methionine at position 198 of SEQ ID NO:1; (d) the tyrosine at position 172 of SEQ ID NO:1; (e) the leucine at position 171 of SEQ ID NO:1; or (f) the leucine at position 111 of SEQ ID NO:1: is lower than the level of binding of the protein to a polypeptide of SEQ ID NO: 1.
7. The protein of any one of claims 4 to 6, wherein the polypeptide to which the protein binds at a reduced level additionally comprises one or more of the following: (i) an alanine at a position 111 of SEQ ID NO: 1; (ii) an alanine at a position 171 of SEQ ID NO: 1; (iii) an alanine at a position 172 of SEQ ID NO: 1; and (iv) an alanine at a position 198 of SEQ ID NO: 1.
8. The protein of any one of claims 1 to 7, wherein the antigen binding site cross-reacts with: (i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the lysine at position 167 of SEQ ID NO: 1; and/or (ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the histidine at position 168 of SEQ ID NO: 1.
9. The protein of any one of claims 1 to 8, which competitively inhibits the binding of C1.2 (comprising a heavy chain variable region (V ) comprising a sequence set forth in SEQ ID NO: 2 and a light chain variable region (V ) comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a V comprising a sequence set forth in SEQ ID NO: 4 and a V comprising a sequence set forth in SEQ ID NO: 5) to one or more of: (i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the lysine at position 167 of SEQ ID NO: 1; and/or (ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the histidine at position 168 of SEQ ID NO: 1.
10. A protein that binds to human granulocyte-colony stimulating factor receptor (hG-CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, the protein comprising at least one of: (i) a V comprising a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 6, a CDR2 comprising a sequence set forth in SEQ ID NO: 7 and a CDR3 comprising a sequence at least about 70% identical to the sequence set forth in SEQ ID NO: 8; (ii) a V comprising a sequence at least about 80% identical to a sequence set forth in SEQ ID NO: 2 and/or 4; (iii) a V comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 9, a CDR2 comprising a sequence set forth in SEQ ID NO: 10 and a CDR3 comprising a sequence at least about 70% identical to the sequence set forth in SEQ ID NO: 11; and (iv) a V comprising a sequence at least about 80% identical to a sequence set forth in SEQ ID NO: 3 and/or 5.
11. The protein of claim 10, the V comprises a CDR3 comprising a sequence LGELGX X X X (SEQ ID NO: 12), wherein: 1 2 3 4 X is an amino acid selected from the group consisting of tryptophan, glutamine, methionine, serine, phenylalanine, glutamic acid and histidine; X is an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine, serine, glycine and isoleucine; X is an amino acid selected from the group consisting of aspartic acid, methionine, glutamine, serine, leucine, valine, arginine and histidine; and X is an amino acid selected from the group consisting of proline, glutamic acid, alanine, leucine, phenylalanine, tyrosine, threonine, asparagine, aspartic acid, serine, glycine, arginine, and lysine.
12. The protein of claim 11, wherein: X is a neutral amino acid; X is phenylalanine, tyrosine, methionine or serine; X is aspartic acid, methionine, glutamine, serine or leucine; and X is proline, glutamic acid, alanine, leucine, phenylalanine or tyrosine.
13. The protein of claim 10, wherein the V comprising a CDR3 comprising a sequence X X X X X X X X X (SEQ ID NO: 13), wherein: 1 2 3 4 5 6 7 8 9 X is an amino acid selected from the group consisting of glutamine, glutamic acid, histidine, alanine and serine; X is an amino acid selected from the group consisting of glutamine, valine, phenylalanine, asparagine and glutamic acid; X is an amino acid selected from the group consisting of serine and glycine; X is an amino acid selected from the group consisting of tryptophan, methionine, phenylalanine, tyrosine, isoleucine and leucine; X is an amino acid selected from the group consisting of glutamic acid, methionine, glutamine, tryptophan, serine, valine, asparagine, glycine, alanine, arganine, histidine, tyrosine, lysine and threonine; X is an amino acid selected from the group consisting of tyrosine, methionine, isoleucine and threonine; X is an amino acid selected from the group consisting of proline, alanine, histidine, glycine and lysine; X is an amino acid selected from the group consisting of leucine, glutamine, methionine, alanine, phenylalanine, isoleucine, lysine, histidine and glycine; X is an amino acid selected from the group consisting of threonine, phenylalanine, tyrosine, methionine, lysine, serine, histidine, proline, tryptophan, isoleucine, glutamine, glycine and valine.
14. The protein of claim 13, wherein: X is glutamine or glutamic acid; X is glutamine; X is an amino acid selected from the group consisting of serine and glycine; X is an amino acid selected from the group consisting of tryptophan, methionine, phenylalanine and tyrosine; X is an amino acid selected from the group consisting of glutamic acid, methionine, glutamine, tryptophan, serine, valine, asparagine, glycine and alanine; X is an amino acid selected from the group consisting of tyrosine, threonine and methionine; X is an amino acid selected from the group consisting of proline, alanine, histidine and glycine; X is an amino acid selected from the group consisting of leucine, glutamine, methionine, alanine, phenylalanine and isoleucine; X is an amino acid selected from the group consisting of threonine, phenylalanine, tyrosine, methionine, lysine, serine, histidine and phenylalanine.
15. A protein that binds to human granulocyte-colony stimulating factor receptor (hG-CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, the protein comprising at least one variable region of an antibody selected from the group consisting of: (i) a heavy chain variable region (V ) comprising an amino acid sequence set forth in SEQ ID NO: 2; (ii) a light chain variable region (V ) comprising an amino acid sequence set forth in SEQ ID NO: 3; (iii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4; (iv) a V comprising an amino acid sequence set forth in SEQ ID NO: 5; (v) a V comprising an amino acid sequence set forth in SEQ ID NO: 14; (vi) a V comprising an amino acid sequence set forth in SEQ ID NO: 15; (vii) a V comprising an amino acid sequence set forth in SEQ ID NO: 16; (viii) a V comprising an amino acid sequence set forth in SEQ ID NO: 17; (ix) a V comprising an amino acid sequence set forth in SEQ ID NO: 18; (x) a V comprising an amino acid sequence set forth in SEQ ID NO: 19; (xi) a V comprising an amino acid sequence set forth in SEQ ID NO: 20; (xii) a V comprising an amino acid sequence set forth in SEQ ID NO: 21; (xiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 22; (xiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 23; (xv) a V comprising an amino acid sequence set forth in SEQ ID NO: 24; (xvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 25; (xvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 26; (xviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 27; (xix) a V comprising an amino acid sequence set forth in SEQ ID NO: 28; (xx) a V comprising an amino acid sequence set forth in SEQ ID NO: 29; (xxi) a V comprising an amino acid sequence set forth in SEQ ID NO: 30; (xxii) a V comprising an amino acid sequence set forth in SEQ ID NO: 31; (xxiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 32; (xxiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 33; (xxv) a V comprising an amino acid sequence set forth in SEQ ID NO: 34; (xxvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 35; (xxvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 36; (xxviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 37; (xxix) a V comprising an amino acid sequence set forth in SEQ ID NO: 38; (xxx) a V comprising an amino acid sequence set forth in SEQ ID NO: 39; (xxxi) a V comprising an amino acid sequence set forth in SEQ ID NO: 40; (xxxii) a V comprising an amino acid sequence set forth in SEQ ID NO: 41; (xxxiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 42; (xxxiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 43; (xxxv) a V comprising an amino acid sequence set forth in SEQ ID NO: 44; (xxxvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 45; (xxxvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 46; (xxxviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 47; (xxxix) a V comprising an amino acid sequence set forth in SEQ ID NO: 48; (xl) a V comprising an amino acid sequence set forth in SEQ ID NO: 49; (xli) a V comprising an amino acid sequence set forth in SEQ ID NO: 50; (xlii) a V comprising an amino acid sequence set forth in SEQ ID NO: 51; (xliii) a V comprising an amino acid sequence set forth in SEQ ID NO: 52; (xliv) a V comprising an amino acid sequence set forth in SEQ ID NO: 53; (xlv) a V comprising an amino acid sequence set forth in SEQ ID NO: 54; (xlvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 55; (xlvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 56; (xlviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 57; (xlix) a V comprising an amino acid sequence set forth in SEQ ID NO: 58; (l) a V comprising an amino acid sequence set forth in SEQ ID NO: 59; (li) a V comprising an amino acid sequence set forth in SEQ ID NO: 60; (lii) a V comprising an amino acid sequence set forth in SEQ ID NO: 61; (liii) a V comprising an amino acid sequence set forth in SEQ ID NO: 62; and (liv) a V comprising an amino acid sequence set forth in SEQ ID NO: 63.
16. The protein of any one of claims 1 to 15 comprising at least a heavy chain variable region (V ) and a light chain variable region (V ), wherein the V and V bind to form a Fv comprising an H L H L antigen binding domain.
17. The protein of claim 16, wherein the V and the V are in a single polypeptide chain.
18. The protein of claim 17, which is: (i) a single chain Fv fragment (scFv); (ii) a dimeric scFv (di-scFv); or (iii) at least one of (i) and/or (ii) linked to a constant region of an antibody, a Fc or a heavy chain constant domain (C ) 2 and/or C 3.
19. The protein of claim 16, wherein the V and V are in separate polypeptide chains.
20. The protein of claim 19, which is: (i) a diabody; (ii) a triabody; (iii) a tetrabody; (iv) a Fab; (v) a F(ab’) ; (vi) a Fv; or (vii) one of (i) to (vi) linked to a constant region of an antibody, a Fc or a heavy chain constant domain (C ) 2 and/or C 3.
21. The protein of claim 19, which is an antibody.
22. The protein of any one of claims 1 to 21 which is chimeric, de-immunized, humanized, human or primatized.
23. An antibody to that binds to human granulocyte-colony stimulating factor receptor (hG- CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, the antibody comprising: (i) a heavy chain variable region (V ) comprising an amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region (V ) comprising an amino acid sequence set forth in SEQ ID NO: 3; (ii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 5; (iii) a V comprising an amino acid sequence set forth in SEQ ID NO: 15 and a V comprising an amino acid sequence set forth in SEQ ID NO: 14; (iv) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 16; (v) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 17; (vi) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 18; (vii) a V comprising an amino acid sequence set forth in SEQ ID NO: 20 and a V comprising an amino acid sequence set forth in SEQ ID NO: 19; (viii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 21; (ix) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 22; (x) a V comprising an amino acid sequence set forth in SEQ ID NO: 24 and a V comprising an amino acid sequence set forth in SEQ ID NO: 23; (ix) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 25; (x) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 26; (xi) a V comprising an amino acid sequence set forth in SEQ ID NO: 28 and a V comprising an amino acid sequence set forth in SEQ ID NO: 27; (xii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 29; (xiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 31 and a V comprising an amino acid sequence set forth in SEQ ID NO: 30; (xiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 33 and a V comprising an amino acid sequence set forth in SEQ ID NO: 32; (xv) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 34; (xvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 36 and a V comprising an amino acid sequence set forth in SEQ ID NO: 35; (xvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 38 and a V comprising an amino acid sequence set forth in SEQ ID NO: 37; (xviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 40 and a V comprising an amino acid sequence set forth in SEQ ID NO: 39; (xix) a V comprising an amino acid sequence set forth in SEQ ID NO: 42 and a V comprising an amino acid sequence set forth in SEQ ID NO: 41; (xx) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 43; (xxi) a V comprising an amino acid sequence set forth in SEQ ID NO: 45 and a V comprising an amino acid sequence set forth in SEQ ID NO: 44; (xxii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 46; (xxiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 47; (xxiv) a V comprising an amino acid sequence set forth in SEQ ID NO: 49 and a V comprising an amino acid sequence set forth in SEQ ID NO: 48; (xxv) a V comprising an amino acid sequence set forth in SEQ ID NO: 51 and a V comprising an amino acid sequence set forth in SEQ ID NO: 50; (xxvi) a V comprising an amino acid sequence set forth in SEQ ID NO: 53 and a V comprising an amino acid sequence set forth in SEQ ID NO: 52; (xxvii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 54; (xxviii) a V comprising an amino acid sequence set forth in SEQ ID NO: 55 and a V comprising an amino acid sequence set forth in SEQ ID NO: 5; (xxix) a V comprising an amino acid sequence set forth in SEQ ID NO: 57 and a V comprising an amino acid sequence set forth in SEQ ID NO: 56; (xxx) a V comprising an amino acid sequence set forth in SEQ ID NO: 59 and a V comprising an amino acid sequence set forth in SEQ ID NO: 58; (xxxi) a V comprising an amino acid sequence set forth in SEQ ID NO: 61 and a V comprising an amino acid sequence set forth in SEQ ID NO: 60; (xxxii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 62; (xxxiii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 63; or (xxxiv) a V comprising three CDRs of a V set forth in any one or more of (i) to (xxxiii) and a V H H L comprising three CDRs of a V set forth in any one or more of (i) to (xxxiii).
24. An antibody to that binds to human granulocyte-colony stimulating factor receptor (hG- CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, the antibody comprising: (i) a heavy chain variable region (V ) comprising an amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region (V ) comprising an amino acid sequence set forth in SEQ ID NO: 3; or (ii) a V comprising an amino acid sequence set forth in SEQ ID NO: 4 and a V comprising an amino acid sequence set forth in SEQ ID NO: 5.
25. An antibody comprising: (i) a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 or 68 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65; or (ii) one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 68 and two light chains comprising an amino acid sequence set forth in SEQ ID NO: 65.
26. The protein or antibody of any one of claims 1 to 25, which inhibits G-CSF-induced proliferation of a BaF3 cell expressing hG-CSFR with an IC of at least about 0.5nM.
27. The protein or antibody of any one of claims 1 to 26, which binds to a polypeptide comprising amino acids 1 to 311 of SEQ ID NO: 1 expressed as a fusion with an antibody Fc region with an affinity of at least about 0.5nM, wherein affinity is determined in an assay in which the polypeptide immobilized and the protein or antibody contacted to the immobilized polypeptide.
28. The protein or antibody of any one of claims 1 to 27, which binds to hG-CSFR expressed on the surface of a cell at an affinity of at least about 1nM.
29. A nucleic acid encoding the protein or antibody of any one of claims 1 to 27.
30. An isolated cell modified to contain the nucleic acid of claim 29.
31. An antibody or protein encoded by the nucleic acid of claim 29 or as encoded by the nucleic acid and expressed by the cell of claim 30.
32. The protein of any one of claims 1 to 28 or 31 conjugated to another compound.
33. A composition comprising the protein or antibody of any one of claims 1 to 28 or 31 and a pharmaceutically acceptable carrier.
34. A composition comprising: (i) an antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65; and (ii) (a) an antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65 and/or (b) an antibody comprising one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 68 and two light chains comprising an amino acid sequence set forth in SEQ ID NO: and, optionally, a pharmaceutically acceptable carrier.
35. Use of the protein or antibody of any one of claims 1 to 29 or the composition of claim 30 or 31 in the manufacture of a medicament for treating or preventing a granulocyte-colony stimulating factor (G-CSF)-mediated condition in a subject.
36. The use of claim 35, wherein the G-CSF-mediated condition is an autoimmune disease, an inflammatory disease or cancer.
37. The use of claim 36, wherein the autoimmune disease or the inflammatory disease is arthritis, multiple sclerosis, pulmonary inflammation or chronic obstructive pulmonary disease.
38. The use of any one of claims 35 to 37 wherein the medicament is formulated for administering an amount of the protein or antibody sufficient to reduce the number of neutrophils in a subject without inducing neutropenia.
39. The use of claim 38, wherein the amount of the protein is between about 0.05mg/kg and 30mg/kg.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161496351P | 2011-06-13 | 2011-06-13 | |
US61/496,351 | 2011-06-13 | ||
PCT/AU2012/000675 WO2012171057A1 (en) | 2011-06-13 | 2012-06-13 | Antibodies against g-csfr and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ617725A NZ617725A (en) | 2015-02-27 |
NZ617725B2 true NZ617725B2 (en) | 2015-05-28 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210009701A1 (en) | Antibodies against g-csfr and uses thereof | |
US9796782B2 (en) | Fibronectin III domain of IL-11Rα | |
AU2016361462B2 (en) | CD131 binding proteins and uses thereof | |
AU2015367224B2 (en) | IL-21 antibodies | |
US20220332804A1 (en) | Complement C2 Binding Proteins and Uses Thereof | |
NZ617725B2 (en) | Antibodies against g-csfr and uses thereof |