US20220169733A1 - Immunological Reagents - Google Patents
Immunological Reagents Download PDFInfo
- Publication number
- US20220169733A1 US20220169733A1 US17/485,752 US202117485752A US2022169733A1 US 20220169733 A1 US20220169733 A1 US 20220169733A1 US 202117485752 A US202117485752 A US 202117485752A US 2022169733 A1 US2022169733 A1 US 2022169733A1
- Authority
- US
- United States
- Prior art keywords
- seq
- binding
- antibody
- antibodies
- hiv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000003153 chemical reaction reagent Substances 0.000 title description 15
- 230000001900 immune effect Effects 0.000 title description 2
- 239000011230 binding agent Substances 0.000 claims abstract description 185
- 108010074708 B7-H1 Antigen Proteins 0.000 claims description 51
- 102000008096 B7-H1 Antigen Human genes 0.000 claims description 51
- 230000003993 interaction Effects 0.000 claims description 39
- 102100040678 Programmed cell death protein 1 Human genes 0.000 abstract description 165
- 101710089372 Programmed cell death protein 1 Proteins 0.000 abstract description 165
- 241000725303 Human immunodeficiency virus Species 0.000 abstract description 62
- 238000000034 method Methods 0.000 abstract description 56
- 206010028980 Neoplasm Diseases 0.000 abstract description 23
- 208000015181 infectious disease Diseases 0.000 abstract description 17
- 201000011510 cancer Diseases 0.000 abstract description 16
- 208000035473 Communicable disease Diseases 0.000 abstract description 6
- 230000005784 autoimmunity Effects 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 52
- 230000027455 binding Effects 0.000 description 51
- 108090000765 processed proteins & peptides Proteins 0.000 description 44
- 230000002860 competitive effect Effects 0.000 description 42
- 210000001744 T-lymphocyte Anatomy 0.000 description 37
- 238000003556 assay Methods 0.000 description 36
- 125000003275 alpha amino acid group Chemical group 0.000 description 28
- 241000282414 Homo sapiens Species 0.000 description 25
- 239000000427 antigen Substances 0.000 description 23
- 108091007433 antigens Proteins 0.000 description 23
- 102000036639 antigens Human genes 0.000 description 23
- 102100024952 Protein CBFA2T1 Human genes 0.000 description 22
- 230000035755 proliferation Effects 0.000 description 22
- 239000003795 chemical substances by application Substances 0.000 description 21
- 229920001184 polypeptide Polymers 0.000 description 21
- 102000004196 processed proteins & peptides Human genes 0.000 description 21
- 229960002621 pembrolizumab Drugs 0.000 description 19
- 230000036963 noncompetitive effect Effects 0.000 description 18
- 210000004408 hybridoma Anatomy 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 230000014509 gene expression Effects 0.000 description 16
- 108020004707 nucleic acids Proteins 0.000 description 15
- 102000039446 nucleic acids Human genes 0.000 description 15
- 150000007523 nucleic acids Chemical class 0.000 description 15
- 235000018102 proteins Nutrition 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 150000001413 amino acids Chemical group 0.000 description 14
- 239000000203 mixture Substances 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 238000011225 antiretroviral therapy Methods 0.000 description 10
- 239000011324 bead Substances 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 241000287828 Gallus gallus Species 0.000 description 9
- 239000012472 biological sample Substances 0.000 description 9
- 239000003814 drug Substances 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 230000002163 immunogen Effects 0.000 description 9
- 230000010076 replication Effects 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 241000282465 Canis Species 0.000 description 8
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 8
- -1 Val Chemical compound 0.000 description 8
- 239000005557 antagonist Substances 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 229940079593 drug Drugs 0.000 description 8
- 102000048362 human PDCD1 Human genes 0.000 description 8
- 239000008194 pharmaceutical composition Substances 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 7
- 108091006020 Fc-tagged proteins Proteins 0.000 description 7
- 208000031886 HIV Infections Diseases 0.000 description 7
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 230000009137 competitive binding Effects 0.000 description 7
- 239000003937 drug carrier Substances 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 210000002865 immune cell Anatomy 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 238000010186 staining Methods 0.000 description 7
- 208000024891 symptom Diseases 0.000 description 7
- 241000283707 Capra Species 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 6
- 229940124060 PD-1 antagonist Drugs 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 239000002502 liposome Substances 0.000 description 6
- 229960003301 nivolumab Drugs 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- VDABVNMGKGUPEY-UHFFFAOYSA-N 6-carboxyfluorescein succinimidyl ester Chemical compound C=1C(O)=CC=C2C=1OC1=CC(O)=CC=C1C2(C1=C2)OC(=O)C1=CC=C2C(=O)ON1C(=O)CCC1=O VDABVNMGKGUPEY-UHFFFAOYSA-N 0.000 description 5
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 229940122544 PD-1 agonist Drugs 0.000 description 5
- 239000000556 agonist Substances 0.000 description 5
- 230000003915 cell function Effects 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- 229940000406 drug candidate Drugs 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 238000013507 mapping Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 230000002062 proliferating effect Effects 0.000 description 5
- 238000000159 protein binding assay Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 239000003656 tris buffered saline Substances 0.000 description 5
- HBOMLICNUCNMMY-XLPZGREQSA-N zidovudine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-XLPZGREQSA-N 0.000 description 5
- VPFUWHKTPYPNGT-UHFFFAOYSA-N 3-(3,4-dihydroxyphenyl)-1-(5-hydroxy-2,2-dimethylchromen-6-yl)propan-1-one Chemical compound OC1=C2C=CC(C)(C)OC2=CC=C1C(=O)CCC1=CC=C(O)C(O)=C1 VPFUWHKTPYPNGT-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- BXZVVICBKDXVGW-NKWVEPMBSA-N Didanosine Chemical compound O1[C@H](CO)CC[C@@H]1N1C(NC=NC2=O)=C2N=C1 BXZVVICBKDXVGW-NKWVEPMBSA-N 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 230000006052 T cell proliferation Effects 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 230000001363 autoimmune Effects 0.000 description 4
- 238000010256 biochemical assay Methods 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- WHBIGIKBNXZKFE-UHFFFAOYSA-N delavirdine Chemical compound CC(C)NC1=CC=CN=C1N1CCN(C(=O)C=2NC3=CC=C(NS(C)(=O)=O)C=C3C=2)CC1 WHBIGIKBNXZKFE-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 4
- 239000012678 infectious agent Substances 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 150000003904 phospholipids Chemical class 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 230000001629 suppression Effects 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 3
- XQSPYNMVSIKCOC-NTSWFWBYSA-N Emtricitabine Chemical compound C1=C(F)C(N)=NC(=O)N1[C@H]1O[C@@H](CO)SC1 XQSPYNMVSIKCOC-NTSWFWBYSA-N 0.000 description 3
- 208000037357 HIV infectious disease Diseases 0.000 description 3
- 241000713340 Human immunodeficiency virus 2 Species 0.000 description 3
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 3
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 description 3
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 3
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 description 3
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 description 3
- XNKLLVCARDGLGL-JGVFFNPUSA-N Stavudine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1C=C[C@@H](CO)O1 XNKLLVCARDGLGL-JGVFFNPUSA-N 0.000 description 3
- MCGSCOLBFJQGHM-SCZZXKLOSA-N abacavir Chemical compound C=12N=CN([C@H]3C=C[C@@H](CO)C3)C2=NC(N)=NC=1NC1CC1 MCGSCOLBFJQGHM-SCZZXKLOSA-N 0.000 description 3
- 239000004599 antimicrobial Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000000562 conjugate Substances 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 229960002656 didanosine Drugs 0.000 description 3
- 230000008030 elimination Effects 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
- 230000001747 exhibiting effect Effects 0.000 description 3
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- JTEGQNOMFQHVDC-NKWVEPMBSA-N lamivudine Chemical compound O=C1N=C(N)C=CN1[C@H]1O[C@@H](CO)SC1 JTEGQNOMFQHVDC-NKWVEPMBSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- QWAXKHKRTORLEM-UGJKXSETSA-N saquinavir Chemical compound C([C@@H]([C@H](O)CN1C[C@H]2CCCC[C@H]2C[C@H]1C(=O)NC(C)(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)C=1N=C2C=CC=CC2=CC=1)C1=CC=CC=C1 QWAXKHKRTORLEM-UGJKXSETSA-N 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 230000029812 viral genome replication Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 description 2
- YMZMTOFQCVHHFB-UHFFFAOYSA-N 5-carboxytetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C([O-])=O YMZMTOFQCVHHFB-UHFFFAOYSA-N 0.000 description 2
- IDLISIVVYLGCKO-UHFFFAOYSA-N 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein Chemical compound O1C(=O)C2=CC=C(C(O)=O)C=C2C21C1=CC(OC)=C(O)C(Cl)=C1OC1=C2C=C(OC)C(O)=C1Cl IDLISIVVYLGCKO-UHFFFAOYSA-N 0.000 description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 2
- 108010066676 Abrin Proteins 0.000 description 2
- AXRYRYVKAWYZBR-UHFFFAOYSA-N Atazanavir Natural products C=1C=C(C=2N=CC=CC=2)C=CC=1CN(NC(=O)C(NC(=O)OC)C(C)(C)C)CC(O)C(NC(=O)C(NC(=O)OC)C(C)(C)C)CC1=CC=CC=C1 AXRYRYVKAWYZBR-UHFFFAOYSA-N 0.000 description 2
- 108010019625 Atazanavir Sulfate Proteins 0.000 description 2
- 108010006654 Bleomycin Proteins 0.000 description 2
- 102100031658 C-X-C chemokine receptor type 5 Human genes 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 241000251730 Chondrichthyes Species 0.000 description 2
- 108700032819 Croton tiglium crotin II Proteins 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 101100421450 Drosophila melanogaster Shark gene Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 108010032976 Enfuvirtide Proteins 0.000 description 2
- 101710082714 Exotoxin A Proteins 0.000 description 2
- 101000922405 Homo sapiens C-X-C chemokine receptor type 5 Proteins 0.000 description 2
- KJHKTHWMRKYKJE-SUGCFTRWSA-N Kaletra Chemical compound N1([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=2C=CC=CC=2)NC(=O)COC=2C(=CC=CC=2C)C)CC=2C=CC=CC=2)CCCNC1=O KJHKTHWMRKYKJE-SUGCFTRWSA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 2
- 108010039491 Ricin Proteins 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- WREGKURFCTUGRC-POYBYMJQSA-N Zalcitabine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)CC1 WREGKURFCTUGRC-POYBYMJQSA-N 0.000 description 2
- 229960004748 abacavir Drugs 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 229940100198 alkylating agent Drugs 0.000 description 2
- 239000002168 alkylating agent Substances 0.000 description 2
- 229960001830 amprenavir Drugs 0.000 description 2
- YMARZQAQMVYCKC-OEMFJLHTSA-N amprenavir Chemical compound C([C@@H]([C@H](O)CN(CC(C)C)S(=O)(=O)C=1C=CC(N)=CC=1)NC(=O)O[C@@H]1COCC1)C1=CC=CC=C1 YMARZQAQMVYCKC-OEMFJLHTSA-N 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000003042 antagnostic effect Effects 0.000 description 2
- 229940045799 anthracyclines and related substance Drugs 0.000 description 2
- 230000000340 anti-metabolite Effects 0.000 description 2
- 239000000611 antibody drug conjugate Substances 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 229940049595 antibody-drug conjugate Drugs 0.000 description 2
- 229960005475 antiinfective agent Drugs 0.000 description 2
- 229940100197 antimetabolite Drugs 0.000 description 2
- 239000002256 antimetabolite Substances 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
- 229960003277 atazanavir Drugs 0.000 description 2
- AXRYRYVKAWYZBR-GASGPIRDSA-N atazanavir Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)OC)C(C)(C)C)[C@@H](O)CN(CC=1C=CC(=CC=1)C=1N=CC=CC=1)NC(=O)[C@@H](NC(=O)OC)C(C)(C)C)C1=CC=CC=C1 AXRYRYVKAWYZBR-GASGPIRDSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229960001561 bleomycin Drugs 0.000 description 2
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000001246 colloidal dispersion Methods 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 230000016396 cytokine production Effects 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 206010013023 diphtheria Diseases 0.000 description 2
- XPOQHMRABVBWPR-ZDUSSCGKSA-N efavirenz Chemical compound C([C@]1(C2=CC(Cl)=CC=C2NC(=O)O1)C(F)(F)F)#CC1CC1 XPOQHMRABVBWPR-ZDUSSCGKSA-N 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- PEASPLKKXBYDKL-FXEVSJAOSA-N enfuvirtide Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(C)=O)[C@@H](C)O)[C@@H](C)CC)C1=CN=CN1 PEASPLKKXBYDKL-FXEVSJAOSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108010028531 enomycin Proteins 0.000 description 2
- 229940072253 epivir Drugs 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000002825 functional assay Methods 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000003365 immunocytochemistry Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- CBVCZFGXHXORBI-PXQQMZJSSA-N indinavir Chemical compound C([C@H](N(CC1)C[C@@H](O)C[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H]2C3=CC=CC=C3C[C@H]2O)C(=O)NC(C)(C)C)N1CC1=CC=CN=C1 CBVCZFGXHXORBI-PXQQMZJSSA-N 0.000 description 2
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- CFCUWKMKBJTWLW-BKHRDMLASA-N mithramycin Chemical compound O([C@@H]1C[C@@H](O[C@H](C)[C@H]1O)OC=1C=C2C=C3C[C@H]([C@@H](C(=O)C3=C(O)C2=C(O)C=1C)O[C@@H]1O[C@H](C)[C@@H](O)[C@H](O[C@@H]2O[C@H](C)[C@H](O)[C@H](O[C@@H]3O[C@H](C)[C@@H](O)[C@@](C)(O)C3)C2)C1)[C@H](OC)C(=O)[C@@H](O)[C@@H](C)O)[C@H]1C[C@@H](O)[C@H](O)[C@@H](C)O1 CFCUWKMKBJTWLW-BKHRDMLASA-N 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 230000002085 persistent effect Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 108010076042 phenomycin Proteins 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 229950010773 pidilizumab Drugs 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229960003171 plicamycin Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 229940064914 retrovir Drugs 0.000 description 2
- NCDNCNXCDXHOMX-XGKFQTDJSA-N ritonavir Chemical compound N([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1SC=NC=1)CC=1C=CC=CC=1)C(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-XGKFQTDJSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229960001852 saquinavir Drugs 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 2
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 description 2
- VCMJCVGFSROFHV-WZGZYPNHSA-N tenofovir disoproxil fumarate Chemical compound OC(=O)\C=C\C(O)=O.N1=CN=C2N(C[C@@H](C)OCP(=O)(OCOC(=O)OC(C)C)OCOC(=O)OC(C)C)C=NC2=C1N VCMJCVGFSROFHV-WZGZYPNHSA-N 0.000 description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 2
- QOFZZTBWWJNFCA-UHFFFAOYSA-N texas red-X Chemical compound [O-]S(=O)(=O)C1=CC(S(=O)(=O)NCCCCCC(=O)O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 QOFZZTBWWJNFCA-UHFFFAOYSA-N 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 238000011269 treatment regimen Methods 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 229960000523 zalcitabine Drugs 0.000 description 2
- 229960002555 zidovudine Drugs 0.000 description 2
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 1
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- IOOMXAQUNPWDLL-UHFFFAOYSA-N 2-[6-(diethylamino)-3-(diethyliminiumyl)-3h-xanthen-9-yl]-5-sulfobenzene-1-sulfonate Chemical compound C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=C(S(O)(=O)=O)C=C1S([O-])(=O)=O IOOMXAQUNPWDLL-UHFFFAOYSA-N 0.000 description 1
- OWSRLHPWDZOHCR-UHFFFAOYSA-N 4,4-diaminobutanoic acid Chemical compound NC(N)CCC(O)=O OWSRLHPWDZOHCR-UHFFFAOYSA-N 0.000 description 1
- JARCFMKMOFFIGZ-UHFFFAOYSA-N 4,6-dioxo-n-phenyl-2-sulfanylidene-1,3-diazinane-5-carboxamide Chemical compound O=C1NC(=S)NC(=O)C1C(=O)NC1=CC=CC=C1 JARCFMKMOFFIGZ-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- LHCOVOKZWQYODM-CPEOKENHSA-N 4-amino-1-[(2r,5s)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2-one;1-[(2r,4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1N=C(N)C=CN1[C@H]1O[C@@H](CO)SC1.O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 LHCOVOKZWQYODM-CPEOKENHSA-N 0.000 description 1
- VERWQPYQDXWOGT-LVJNJWHOSA-N 4-amino-5-fluoro-1-[(2r,5s)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2-one;[[(2r)-1-(6-aminopurin-9-yl)propan-2-yl]oxymethyl-(propan-2-yloxycarbonyloxymethoxy)phosphoryl]oxymethyl propan-2-yl carbonate;(e)-but-2-enedioic acid Chemical compound OC(=O)\C=C\C(O)=O.C1=C(F)C(N)=NC(=O)N1[C@H]1O[C@@H](CO)SC1.N1=CN=C2N(C[C@@H](C)OCP(=O)(OCOC(=O)OC(C)C)OCOC(=O)OC(C)C)C=NC2=C1N VERWQPYQDXWOGT-LVJNJWHOSA-N 0.000 description 1
- COCMHKNAGZHBDZ-UHFFFAOYSA-N 4-carboxy-3-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]benzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(C([O-])=O)=CC=C1C(O)=O COCMHKNAGZHBDZ-UHFFFAOYSA-N 0.000 description 1
- BUJRUSRXHJKUQE-UHFFFAOYSA-N 5-carboxy-X-rhodamine triethylammonium salt Chemical compound CC[NH+](CC)CC.[O-]C(=O)C1=CC(C(=O)[O-])=CC=C1C1=C(C=C2C3=C4CCCN3CCC2)C4=[O+]C2=C1C=C1CCCN3CCCC2=C13 BUJRUSRXHJKUQE-UHFFFAOYSA-N 0.000 description 1
- WQZIDRAQTRIQDX-UHFFFAOYSA-N 6-carboxy-x-rhodamine Chemical compound OC(=O)C1=CC=C(C([O-])=O)C=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 WQZIDRAQTRIQDX-UHFFFAOYSA-N 0.000 description 1
- VWOLRKMFAJUZGM-UHFFFAOYSA-N 6-carboxyrhodamine 6G Chemical compound [Cl-].C=12C=C(C)C(NCC)=CC2=[O+]C=2C=C(NCC)C(C)=CC=2C=1C1=CC(C(O)=O)=CC=C1C(=O)OCC VWOLRKMFAJUZGM-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 1
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 1
- YYAVDNKUWLAFCV-ACZMJKKPSA-N Ala-Ser-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O YYAVDNKUWLAFCV-ACZMJKKPSA-N 0.000 description 1
- 101100519159 Arabidopsis thaliana PCR3 gene Proteins 0.000 description 1
- BHSYMWWMVRPCPA-CYDGBPFRSA-N Arg-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CCCN=C(N)N BHSYMWWMVRPCPA-CYDGBPFRSA-N 0.000 description 1
- PTNFNTOBUDWHNZ-GUBZILKMSA-N Asn-Arg-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O PTNFNTOBUDWHNZ-GUBZILKMSA-N 0.000 description 1
- LJUOLNXOWSWGKF-ACZMJKKPSA-N Asn-Asn-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N LJUOLNXOWSWGKF-ACZMJKKPSA-N 0.000 description 1
- KHCNTVRVAYCPQE-CIUDSAMLSA-N Asn-Lys-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O KHCNTVRVAYCPQE-CIUDSAMLSA-N 0.000 description 1
- FANQWNCPNFEPGZ-WHFBIAKZSA-N Asp-Asp-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O FANQWNCPNFEPGZ-WHFBIAKZSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 101100005789 Caenorhabditis elegans cdk-4 gene Proteins 0.000 description 1
- 101100463133 Caenorhabditis elegans pdl-1 gene Proteins 0.000 description 1
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 1
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- NAPULYCVEVVFRB-HEIBUPTGSA-N Cys-Thr-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)CS NAPULYCVEVVFRB-HEIBUPTGSA-N 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 102000003915 DNA Topoisomerases Human genes 0.000 description 1
- 108090000323 DNA Topoisomerases Proteins 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 108700022150 Designed Ankyrin Repeat Proteins Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- XPOQHMRABVBWPR-UHFFFAOYSA-N Efavirenz Natural products O1C(=O)NC2=CC=C(Cl)C=C2C1(C(F)(F)F)C#CC1CC1 XPOQHMRABVBWPR-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108010008165 Etanercept Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010056740 Genital discharge Diseases 0.000 description 1
- QYKBTDOAMKORGL-FXQIFTODSA-N Gln-Gln-Asp Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N QYKBTDOAMKORGL-FXQIFTODSA-N 0.000 description 1
- 206010018364 Glomerulonephritis Diseases 0.000 description 1
- NUSWUSKZRCGFEX-FXQIFTODSA-N Glu-Glu-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(O)=O NUSWUSKZRCGFEX-FXQIFTODSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- 229940126154 HIV entry inhibitor Drugs 0.000 description 1
- 229940033330 HIV vaccine Drugs 0.000 description 1
- 241000560067 HIV-1 group M Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 1
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 1
- 101000578784 Homo sapiens Melanoma antigen recognized by T-cells 1 Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- 102000000521 Immunophilins Human genes 0.000 description 1
- 108010016648 Immunophilins Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- 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 1
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 102100022430 Melanocyte protein PMEL Human genes 0.000 description 1
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 1
- ZZIKIHCNFWXKDY-UHFFFAOYSA-N Myriocin Natural products CCCCCCC(=O)CCCCCCC=CCC(O)C(O)C(N)(CO)C(O)=O ZZIKIHCNFWXKDY-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- YJQPYGGHQPGBLI-UHFFFAOYSA-N Novobiocin Natural products O1C(C)(C)C(OC)C(OC(N)=O)C(O)C1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102100034640 PWWP domain-containing DNA repair factor 3A Human genes 0.000 description 1
- 108050007154 PWWP domain-containing DNA repair factor 3A Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108010009711 Phalloidine Proteins 0.000 description 1
- WEMYTDDMDBLPMI-DKIMLUQUSA-N Phe-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N WEMYTDDMDBLPMI-DKIMLUQUSA-N 0.000 description 1
- KIQUCMUULDXTAZ-HJOGWXRNSA-N Phe-Tyr-Tyr Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O KIQUCMUULDXTAZ-HJOGWXRNSA-N 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- NCDNCNXCDXHOMX-UHFFFAOYSA-N Ritonavir Natural products C=1C=CC=CC=1CC(NC(=O)OCC=1SC=NC=1)C(O)CC(CC=1C=CC=CC=1)NC(=O)C(C(C)C)NC(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-UHFFFAOYSA-N 0.000 description 1
- QMCDMHWAKMUGJE-IHRRRGAJSA-N Ser-Phe-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O QMCDMHWAKMUGJE-IHRRRGAJSA-N 0.000 description 1
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 101800001271 Surface protein Proteins 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- DPOPAJRDYZGTIR-UHFFFAOYSA-N Tetrazine Chemical compound C1=CN=NN=N1 DPOPAJRDYZGTIR-UHFFFAOYSA-N 0.000 description 1
- APJYDQYYACXCRM-UHFFFAOYSA-N Tryptamine Natural products C1=CC=C2C(CCN)=CNC2=C1 APJYDQYYACXCRM-UHFFFAOYSA-N 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 102000007537 Type II DNA Topoisomerases Human genes 0.000 description 1
- 108010046308 Type II DNA Topoisomerases Proteins 0.000 description 1
- ARJASMXQBRNAGI-YESZJQIVSA-N Tyr-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N ARJASMXQBRNAGI-YESZJQIVSA-N 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- PAPWZOJOLKZEFR-AVGNSLFASA-N Val-Arg-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N PAPWZOJOLKZEFR-AVGNSLFASA-N 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 241000863480 Vinca Species 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- APERIXFHHNDFQV-UHFFFAOYSA-N [2-[2-[2-[bis(carboxymethyl)amino]-5-methylphenoxy]ethoxy]-4-[3,6-bis(dimethylamino)xanthen-9-ylidene]cyclohexa-2,5-dien-1-ylidene]-bis(carboxymethyl)azanium;chloride Chemical compound [Cl-].C12=CC=C(N(C)C)C=C2OC2=CC(N(C)C)=CC=C2C1=C(C=1)C=CC(=[N+](CC(O)=O)CC(O)=O)C=1OCCOC1=CC(C)=CC=C1N(CC(O)=O)CC(O)=O APERIXFHHNDFQV-UHFFFAOYSA-N 0.000 description 1
- GLWHPRRGGYLLRV-XLPZGREQSA-N [[(2s,3s,5r)-3-azido-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](N=[N+]=[N-])C1 GLWHPRRGGYLLRV-XLPZGREQSA-N 0.000 description 1
- WMHSRBZIJNQHKT-FFKFEZPRSA-N abacavir sulfate Chemical compound OS(O)(=O)=O.C=12N=CN([C@H]3C=C[C@@H](CO)C3)C2=NC(N)=NC=1NC1CC1.C=12N=CN([C@H]3C=C[C@@H](CO)C3)C2=NC(N)=NC=1NC1CC1 WMHSRBZIJNQHKT-FFKFEZPRSA-N 0.000 description 1
- USZYSDMBJDPRIF-SVEJIMAYSA-N aclacinomycin A Chemical compound O([C@H]1[C@@H](O)C[C@@H](O[C@H]1C)O[C@H]1[C@H](C[C@@H](O[C@H]1C)O[C@H]1C[C@]([C@@H](C2=CC=3C(=O)C4=CC=CC(O)=C4C(=O)C=3C(O)=C21)C(=O)OC)(O)CC)N(C)C)[C@H]1CCC(=O)[C@H](C)O1 USZYSDMBJDPRIF-SVEJIMAYSA-N 0.000 description 1
- 229960004176 aclarubicin Drugs 0.000 description 1
- 229930183665 actinomycin Natural products 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000001270 agonistic effect Effects 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010004469 allophycocyanin Proteins 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940035674 anesthetics Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 230000000798 anti-retroviral effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003972 antineoplastic antibiotic Substances 0.000 description 1
- 229940124977 antiviral medication Drugs 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 239000000823 artificial membrane Substances 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 229940092117 atgam Drugs 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000013378 biophysical characterization Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- PMDQGYMGQKTCSX-HQROKSDRSA-L calcium;[(2r,3s)-1-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-[[(3s)-oxolan-3-yl]oxycarbonylamino]-4-phenylbutan-2-yl] phosphate Chemical compound [Ca+2].C([C@@H]([C@H](OP([O-])([O-])=O)CN(CC(C)C)S(=O)(=O)C=1C=CC(N)=CC=1)NC(=O)O[C@@H]1COCC1)C1=CC=CC=C1 PMDQGYMGQKTCSX-HQROKSDRSA-L 0.000 description 1
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 1
- 229940127093 camptothecin Drugs 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 229940106189 ceramide Drugs 0.000 description 1
- 229930183167 cerebroside Natural products 0.000 description 1
- 150000001784 cerebrosides Chemical class 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 229940014461 combivir Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 229940088900 crixivan Drugs 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229960005319 delavirdine Drugs 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- CFCUWKMKBJTWLW-UHFFFAOYSA-N deoliosyl-3C-alpha-L-digitoxosyl-MTM Natural products CC=1C(O)=C2C(O)=C3C(=O)C(OC4OC(C)C(O)C(OC5OC(C)C(O)C(OC6OC(C)C(O)C(C)(O)C6)C5)C4)C(C(OC)C(=O)C(O)C(C)O)CC3=CC2=CC=1OC(OC(C)C1O)CC1OC1CC(O)C(O)C(C)O1 CFCUWKMKBJTWLW-UHFFFAOYSA-N 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 1
- RUZYUOTYCVRMRZ-UHFFFAOYSA-N doxazosin Chemical compound C1OC2=CC=CC=C2OC1C(=O)N(CC1)CCN1C1=NC(N)=C(C=C(C(OC)=C2)OC)C2=N1 RUZYUOTYCVRMRZ-UHFFFAOYSA-N 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000007878 drug screening assay Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 229960003804 efavirenz Drugs 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 229960000366 emtricitabine Drugs 0.000 description 1
- 229940001018 emtriva Drugs 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 229940073621 enbrel Drugs 0.000 description 1
- 230000002121 endocytic effect Effects 0.000 description 1
- 229960002062 enfuvirtide Drugs 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- YJGVMLPVUAXIQN-UHFFFAOYSA-N epipodophyllotoxin Natural products COC1=C(OC)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YJGVMLPVUAXIQN-UHFFFAOYSA-N 0.000 description 1
- 229940019131 epzicom Drugs 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 229960000556 fingolimod Drugs 0.000 description 1
- KKGQTZUTZRNORY-UHFFFAOYSA-N fingolimod Chemical compound CCCCCCCCC1=CC=C(CCC(N)(CO)CO)C=C1 KKGQTZUTZRNORY-UHFFFAOYSA-N 0.000 description 1
- 150000005699 fluoropyrimidines Chemical class 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 229960003142 fosamprenavir Drugs 0.000 description 1
- MLBVMOWEQCZNCC-OEMFJLHTSA-N fosamprenavir Chemical compound C([C@@H]([C@H](OP(O)(O)=O)CN(CC(C)C)S(=O)(=O)C=1C=CC(N)=CC=1)NC(=O)O[C@@H]1COCC1)C1=CC=CC=C1 MLBVMOWEQCZNCC-OEMFJLHTSA-N 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 229940099052 fuzeon Drugs 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 150000002270 gangliosides Chemical class 0.000 description 1
- 239000003193 general anesthetic agent Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 239000002835 hiv fusion inhibitor Substances 0.000 description 1
- 230000000652 homosexual effect Effects 0.000 description 1
- 229940048921 humira Drugs 0.000 description 1
- 229920001600 hydrophobic polymer Polymers 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 229960001936 indinavir Drugs 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229940088976 invirase Drugs 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229960005386 ipilimumab Drugs 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229940112586 kaletra Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 229960001627 lamivudine Drugs 0.000 description 1
- 229940113354 lexiva Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- IOOMXAQUNPWDLL-UHFFFAOYSA-M lissamine rhodamine anion Chemical compound C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=C(S([O-])(=O)=O)C=C1S([O-])(=O)=O IOOMXAQUNPWDLL-UHFFFAOYSA-M 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229960004525 lopinavir Drugs 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical class ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 1
- 229960001428 mercaptopurine Drugs 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 238000012737 microarray-based gene expression Methods 0.000 description 1
- 210000002500 microbody Anatomy 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 238000012243 multiplex automated genomic engineering Methods 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 229940014456 mycophenolate Drugs 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- ZZIKIHCNFWXKDY-GNTQXERDSA-N myriocin Chemical compound CCCCCCC(=O)CCCCCC\C=C\C[C@@H](O)[C@H](O)[C@@](N)(CO)C(O)=O ZZIKIHCNFWXKDY-GNTQXERDSA-N 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- QAGYKUNXZHXKMR-HKWSIXNMSA-N nelfinavir Chemical compound CC1=C(O)C=CC=C1C(=O)N[C@H]([C@H](O)CN1[C@@H](C[C@@H]2CCCC[C@@H]2C1)C(=O)NC(C)(C)C)CSC1=CC=CC=C1 QAGYKUNXZHXKMR-HKWSIXNMSA-N 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- NQDJXKOVJZTUJA-UHFFFAOYSA-N nevirapine Chemical compound C12=NC=CC=C2C(=O)NC=2C(C)=CC=NC=2N1C1CC1 NQDJXKOVJZTUJA-UHFFFAOYSA-N 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- OSTGTTZJOCZWJG-UHFFFAOYSA-N nitrosourea Chemical compound NC(=O)N=NO OSTGTTZJOCZWJG-UHFFFAOYSA-N 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229940072250 norvir Drugs 0.000 description 1
- 229960002950 novobiocin Drugs 0.000 description 1
- YJQPYGGHQPGBLI-KGSXXDOSSA-N novobiocin Chemical compound O1C(C)(C)[C@H](OC)[C@@H](OC(N)=O)[C@@H](O)[C@@H]1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-KGSXXDOSSA-N 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 229940005483 opioid analgesics Drugs 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 125000001095 phosphatidyl group Chemical group 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 229960001237 podophyllotoxin Drugs 0.000 description 1
- YJGVMLPVUAXIQN-XVVDYKMHSA-N podophyllotoxin Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YJGVMLPVUAXIQN-XVVDYKMHSA-N 0.000 description 1
- YVCVYCSAAZQOJI-UHFFFAOYSA-N podophyllotoxin Natural products COC1=C(O)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YVCVYCSAAZQOJI-UHFFFAOYSA-N 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 229940116176 remicade Drugs 0.000 description 1
- 201000010174 renal carcinoma Diseases 0.000 description 1
- 229940063627 rescriptor Drugs 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 229960000311 ritonavir Drugs 0.000 description 1
- 239000003419 rna directed dna polymerase inhibitor Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 229940115586 simulect Drugs 0.000 description 1
- 229960002930 sirolimus Drugs 0.000 description 1
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 150000003408 sphingolipids Chemical class 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 229960001203 stavudine Drugs 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 229940054565 sustiva Drugs 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 229960001967 tacrolimus Drugs 0.000 description 1
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 description 1
- 229960001278 teniposide Drugs 0.000 description 1
- 229960001355 tenofovir disoproxil Drugs 0.000 description 1
- 229960004693 tenofovir disoproxil fumarate Drugs 0.000 description 1
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940021747 therapeutic vaccine Drugs 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 230000036964 tight binding Effects 0.000 description 1
- 229960000303 topotecan Drugs 0.000 description 1
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 1
- 229940111527 trizivir Drugs 0.000 description 1
- 229940008349 truvada Drugs 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 229940118696 vibrio cholerae Drugs 0.000 description 1
- 229940111505 videx ec Drugs 0.000 description 1
- 229940023080 viracept Drugs 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 229940087450 zerit Drugs 0.000 description 1
- 229940052255 ziagen Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- 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
- A61P37/02—Immunomodulators
-
- 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
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- A61K2039/507—Comprising a combination of two or more separate antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- 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
- C07K2317/74—Inducing 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
- C07K2317/75—Agonist effect on antigen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- This disclosure relates to binding agents with specificity for programmed cell death 1 (PD-1) (e.g., human PD-1) and to methods for using the same to treat and/or prevent infection (e.g., by human immunodeficiency virus (HIV)), cancer and/or autoimmunity.
- PD-1 programmed cell death 1
- HAV human immunodeficiency virus
- CD4 T-cells latently infected with HIV has been identified in the blood as an important component of the HIV cell reservoir and as the primary cause of HIV persistence.
- the life-span of this latent cell reservoir is estimated to be approximately 70 years in the presence of full HIV suppression with ART.
- two populations of CD4 T-cells resident in lymph nodes serve as the primary CD4 T-cell compartment for HIV infection, replication and production.
- These two CD4 T-cell populations are defined by the expression of PD-1 and CXCR5 and include the PD-1+CXCR5+, i.e. T follicular helper cells (Tfh) and PD-1+CXCR5 ⁇ CD4 T-cell populations.
- ART A number of mechanisms responsible for the establishment and maintenance of the HIV latent cell reservoir(s) have been proposed.
- One of the mechanisms is the persistent of minimal virus replication under ART which may replenish the HIV cell reservoir. Therefore, ART is unable to induce full suppression of HIV replication and the “natural” HIV-1 specific immune response under ART is also unable to totally suppress and eliminate ongoing residual virus replication.
- the failures of ART and of the HIV-specific immune response provide the rationale for investigating alternative interventions to target also the persistent HIV cell reservoir.
- infiltrating tumor-specific CD8 T-cells are dysfunctional with regard their ability to proliferate and to mediate cytotoxic activity.
- the large majority of infiltrating tumor-specific CD8 T-cells are in a so-called exhaustion functional state.
- the primary mechanism responsible for the exhaustion of infiltrating tumor-specific CD8 T-cells is the increased expression of a number of regulatory receptors and particularly PD-1 regulatory receptor.
- the observation that the blockade of the PD-1/PDL-1/2 (PD-1 ligands) is associated with the recovery of CD8 T-cells from exhaustion has provided the rationale for developing intervention strategies targeting the PD-1 molecule expressed by exhausted CD8 T-cells.
- FIG. 1 FIG. 1A . Mouse PD-1.
- FIG. 1B Human PD-1.
- FIG. 2 CFSE assay to evaluate the functional effect of anti-PD1 antibodies on the proliferation of HIV specific CD8 T cells.
- FIG. 3 Concentration response binding of anti-PD-1 antibodies to cell surface PD-1 on activated CD4 T-cells.
- FIG. 4 Antibody classes.
- FIG. 5 Proliferation relative to controls (NEG and Peptide 8).
- FIG. 5C Class 2 mAb 136B4: non-competitive.
- FIG. 5D Class 1 mAb 137F2: strongly competitive.
- FIG. 5F Class 1 mAb 136B4: non-competitive.
- FIG. 6 Restoration of HIV peptide specific CD8 T-cell proliferation mediated by anti-PD-1 antibodies binding to different epitopes in a functional exhaustion recovery assay.
- FIG. 6A HIV peptide 8+BMS-5C4, MK3475, 135H12, 122H2, 139D6, 135E10, or 136B5.
- FIG. 6B HIV peptide 8+MK3475, 135H12, 122H2, 137F2, 135C12, 135D11, or 136B5.
- FIG. 7 Enhanced restoration of HIV peptide specific CD8 T-cell proliferation mediated by the combination of anti-PD-1 antibodies binding to different PD-1 epitopes in a functional exhaustion recovery assay.
- FIG. 8 Synergy between a first binding agent that blocks the interaction of PD-1 and PD-L1 with a second binding agent that does not block the interaction of PD-1 and PD-L1.
- This disclosure relates to binding agents with specificity for programmed cell death 1 (PD-1) (e.g., human PD-1) and to methods for using the same such as to treat, prevent and/or ameliorate infection (e.g., by human immunodeficiency virus (HIV)), cancer and/or an autoimmune condition.
- PD-1 programmed cell death 1
- Functional assays for identifying binding agents that interact with PD-1 are also provided.
- Combinations of binding agents such as a first binding agent that blocks the interaction of PD-1 and PD-L1 with a second binding agent that does not block the interaction of PD-1 and PD-L1, are also provided that act synergistically to rescue T cells from exhaustion.
- binding agents that bind programmed cell death (PD-1) protein (e.g., SEQ ID NO:1, FIG. 1A, FIG. 1B of U.S. Pat. No. 5,698,520 (Honjo, et al.) which is hereby incorporated by reference in its entirety) (e.g., human PD-1) on the surface of cells in vitro and/or in vivo.
- the binding agents may also bind isolated PD-1 polypeptide (e.g., human PD-1) and/or fragments and/or derivatives thereof, typically in vitro. Also provided are methods for using such binding agents to diagnose, treat, prevent and/or ameliorate one or more diseases associated with the existence of cells expressing PD-1.
- the binding agents may be antibodies (e.g., monoclonal antibodies) that may react with and/or bind to the epitopes of PD-1.
- the “binding agents” described herein may include, for example, an agonist or an antagonist of PD-1.
- An agonist binding agent is one that is not typically capable of restoring T-cell function and/or expression of PD-1.
- An agonist PD-1 binding agent may be useful for treating autoimmune diseases and others in which PD-1 expressing cells are involved in disease progression.
- an antagonist binding agent is one capable for restoring T-cell function and/or expression of PD-1.
- a PD-1 antagonist binding agent may be capable of restoring the function of PD-1 expressing T-cells from functional exhaustion as is known to occur in HIV infection and in a variety of tumors. Restoration of T cell function may be determined by, for instance, measuring proliferation, cytokine production, cytotoxic activity or other characteristics of such cells.
- Another use for the binding agents described herein is the selective targeting and elimination of HIV-infected CD4 T-cell populations containing replication competent HIV (e.g., in a latent and/or replication state).
- replication competent HIV e.g., in a latent and/or replication state.
- Such PD-1 expressing cells expressing PD-1 are known to serve as a major cell reservoir for replication competent HIV.
- a potential mechanism for the elimination of these CD4 T-cell populations is antibody-dependent cellular cytotoxicity (ADCC) using the binding agents described herein (e.g., mono- and/or bi-specific PD-1 antibodies).
- ADCC antibody-dependent cellular cytotoxicity
- one or more PD-1 antagonistic binding agents having, for instance, different specificities (e.g., recognizing different epitopes) may be combined to induce rescue of antigen-specific CD8 T-cells from functional exhaustion caused by PD-1 expression in those cells (e.g., restoring or improving proliferation, cytokine production and/or cytotoxic activity).
- the binding agents described herein may also provide for the selective elimination and/or suppression of PD-1 expressing cells.
- the PD-1 agonist binding agents described herein may be used to supress and/or eliminate PD-1 expressing cells to treat, for instance, infectious diseases (e.g., HIV), cancer, and/or, especially, autoimmune conditions.
- infectious diseases e.g., HIV
- cancer e.g., cancer
- autoimmune conditions e.g., rhinitis
- the binding agents may be antibodies such as monoclonal antibodies that may comprise, for instance, any one or more of the amino acid sequences shown in Table 1 (and/or one or more fragments and/or derivatives thereof).
- This disclosure also provides for the use of such monoclonal antibodies to isolate, identify, and/or target cells expressing PD-1.
- these monoclonal antibodies may be reactive against PD-1 expressed on the surface of cells.
- antibody or “antibodies” may refer to whole or fragmented antibodies in unpurified or partially purified form (e.g., hybridoma supernatant, ascites, polyclonal antisera) or in purified form.
- the antibodies may be of any suitable origin or form including, for example, murine (e.g., produced by murine hybridoma cells), or expressed as humanized antibodies, chimeric antibodies, human antibodies, and the like.
- antibodies may be wholly or partially dervied from human (e.g., IgG (IgG1, IgG2, IgG2a, Ig2b, IgG3, IgG4), IgM, IgA (IgA1 and IgA2), IgD, and IgE), canine (e.g., IgGA, IgGB, IgGC, IgGD), chicken (e.g., IgA, IgD, IgE, IgG, IgM, IgY), goat (e.g., IgG), mouse (e.g., IgG, IgD, IgE, IgG, IgM), and/or pig (e.g., IgG, IgD, IgE, IgG, IgM
- the antibodies may be contained within hybridoma supernatant or ascites and utilized either directly as such or following concentration using standard techniques.
- the antibodies may be further purified using, for example, salt fractionation and ion exchange chromatography, or affinity chromatography using Protein A, Protein G, Protein A/G, and/or Protein L ligands covalently coupled to a solid support such as agarose beads, or combinations of these techniques.
- the antibodies may be stored in any suitable format, including as a frozen preparation (e.g., ⁇ 20° C. or ⁇ 70° C.), in lyophilized form, or under normal refrigeration conditions (e.g., 4° C.).
- the binding agent When stored in liquid form, for instance, it is preferred that a suitable buffer such as Tris-buffered saline (TBS) or phosphate buffered saline (PBS) is utilized.
- TBS Tris-buffered saline
- PBS phosphate buffered saline
- the binding agent may be prepared as an injectable preparation, such as in suspension in a non-toxic parenterally acceptable diluent or solvent.
- Suitable vehicles and solvents that may be utilized include water, Ringer's solution, and isotonic sodium chloride solution, TBS and/or PBS, among others.
- Such preparations may be suitable for use in vitro or in vivo may be prepared as is known in the art and the exact preparation may depend on the particular application.
- the binding agents described are not in any way limited to antibodies.
- the binding agent may be any compound exhibiting similar binding properties as another (e.g., a mimetic).
- an exemplary binding agent may be one that binds PD-1 and/or can compete with binding agent having specificity therefor (e.g., a monoclonal antibody).
- the mimetic may exhibit substantially the same affinity in binding assays as the binding agent (e.g., monoclonal antibody) to which it is being compared.
- the affinity a particular binding agent may be measured by any suitable assay including but not limited to FACS staining of endogenous cell surface PD-1 on activated CD4 T cells as described in the Examples.
- One binding agent may be said to have “substantially the same affinity” as another where the measurements (e.g., nm) are within about any of 1-20, 1-5, 5-10, 10-15, or 15-20 percent of one another.
- exemplary mimetics may include, for example, organic compounds that specifically bind PD-1, or an affibody (Nygren, et al. FEBS J. 275 (11): 2668-76 (2008)), affilin (Ebersbach, et al. J. Mol. Biol. 372 (1): 172-85 (2007)), affitin (Krehenbrink, et al. J. Mol. Biol. 383 (5): 1058-68 (2008)), anticalin (Skerra, A. FEBS J.
- Other mimetics may include, for example, a derivative of an antibody (of, for example, the monoclonal antibody 1E4,1G10, and/or 1G1) such as, for example, an F ab , F ab2 , Fab′ single chain antibody, F v , single domain antibody, mono-specific antibody, bi-specific antibody, tri-specific antibody, multi-valent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized, CDR-grafted antibody, shark antibody, nanobody, canelid antibody, microbody, and/or intrabody, or derivative thereof.
- Other binding agents are also provided herein as would be understood by one of ordinary skill in the art.
- any method known to those of ordinary skill in the art may be used to generate binding agents having specificity for (e.g., binding to) PD-1.
- an animal such as a mouse may be administered (e.g., immunized) with one or more PD-1 proteins (e.g., PD-1 Fc fusion protein and/or PD-1 His tag protein).
- Animals exhibiting serum reactivity to PD-1 expressed on activated human T lymphocytes (as determined by, for instance, flow cytometry and/or microscopy) may then be selected for generation of anti-PD-1 hybridoma cell lines. This may be repeated for multiple rounds.
- the primary criteria for the first round of binding agent selection may be include but are not limited to: i) level of staining of PD-1 on activated human T lymphocytes by flow cytometry; (ii) diversity of CDR VH and VL sequences as compared to those of the existing anti-PD-1 antibodies; and, (iii) epitope mapping performed by competitive binding studies with PD-1 conjugated Luminex beads pre-coupled with PD-L1 or one of several commercially available anti-PD-1 antibodies binding to different epitopes on PD-1.
- An exemplary first or second round of selection may also include, for instance, affinity binding (not a primary criteria since it may not correlate with the stimulatory potential of anti-PD-1 antibodies); and/or, functional characterization to identify the binding agent as an agonist or an antagonist.
- test binding agents may be assayed for the ability to rescue immune cells such as T cells from exhaustion. This may be determined by measuring the ability of a binding agent to restore proliferation to such cells in the presence of an antigen, such as a test peptide derived from a virus such as human immunodeficiency virus (HIV). Proliferation is measured in a CFSE assay in comparison to a control, such as the test peptide alone or a positive control anti-PD-1 antibody such as MK-3475 (pembrolizumab).
- an antigen such as a test peptide derived from a virus such as human immunodeficiency virus (HIV).
- HIV human immunodeficiency virus
- a binding agent is determined to restore proliferation where the comparison shows a significant difference (such as a P value of ⁇ 0.001) compared to either a peptide alone control or peptide with an isotype control mouse IgG1 antibody.
- This assay may be used to identify binding agents (such as antibodies) that compete with other binding agents for binding to PD-1 (such as PD-L1 or PD-L2) and/or lead to the functional restoration of immune cells.
- Example 1 also describes two methods of epitope mapping the antibodies listed in Table 2 using Luminex-based assays.
- Example 1 describes four classes of monoclonal antibodies binding to distinct epitopes on PD-1 that were: class 1 (competitive with a first monoclonal antibody that blocks the interaction of PD-1 with PD-L1), class 2 (competitive with a second monoclonal antibody that binds PD-1 but does not block the interaction of PD-1 with PD-L1), class 3 (competitive with both the first and second monoclonal antibodies), and class 4 (non-competitive with either the first or second antibodies).
- Combinations of binding agents may also be identified, such as those described in Example 2.
- the combinations may be identified to provide statistically significant differences from results obtained using only one or more of the binding agents and not others.
- combinations exhibiting synergistic ability to restore immune cell function may be identified.
- the combination may comprise a first binding agent that blocks the interaction of PD-1 and PD-L1 with a second binding agent that does not block the interaction of PD-1 and PD-L1.
- the first and second binding agents may be different entities such as two or more different monoclonal antibodies or derivatives thereof, or may be found on the same entity such as a bi-functional antibody (a single antibody or derivative thereof comprising multiple binding specificities).
- an exemplary bi-functional antibody may comprise a first binding region that blocks the interaction of PD-1 and PD-L1 and a second binding region that does not block the interaction of PD-1 and PD-L1.
- combinations that provide multiple types of each binding agent.
- the combination may comprise multiple types of binding agents that block the interaction of PD-1 and PD-L1 with one or more that does not block the interaction of PD-1 and PD-L1.
- the combination may comprise one or more of binding agents that block the interaction of PD-1 and PD-L1 with multiple binding agents that do not block the interaction of PD-1 and PD-L1.
- the combination may comprise multiple binding agents that block the interaction of PD-1 and PD-L1 with multiple binding agents that do not block the interaction of PD-1 and PD-L1.
- Such combinations as described herein may also be combined with one or more other agents that may effect immune cell function such as antibodies against CTLA-4 and the like.
- One of ordinary skill in the art would recognize that many such combinations may be suitable for use as described herein.
- the binding agent is an antibody
- it may be identified with reference to the nucleotide and/or amino acid sequence corresponding to the variability and/or complementarity determining regions (“CDRs”) thereof.
- the variable region/CDR sequences may be used in combination with one or more other variable region/CDR amino acid sequences.
- the variable region/CDR amino acid sequences may alternatively and/or also be adjoined to one or more types of constant region polypeptides of an antibody molecule.
- the CDR amino acid sequences shown in Tables 1A and 1B may be adjoined to or associated with the constant regions of any antibody molecule of the same or a different species (e.g., human, goat, rat, sheep, chicken) and/or antibody subtype of that from which the CDR amino acid sequence was derived.
- an exemplary binding agent may be, or may be derived from, or may be related to the monoclonal antibody produced by the hybridomas listed in, and/or may have about the same affinity and/or proliferation effect, and/or exhibit the same binding class shown in Table 2, and/or may have any one or more of the amino acid sequences of SEQ ID NOS. 1-138 and/or as shown in Tables 1A and 1B.
- the binding agent may comprise an antibody heavy and/or a light chain that each comprises one or more constant and/or variable regions.
- the variable regions typically comprise one or more CDRs that may determine the binding specificity of the antibody.
- the monoclonal antibodies may also be identified by analysis of the amino acid sequences of (e.g., which may be encoded by such nucleotide sequences) such variable regions.
- exemplary amino acid sequences of the heavy chain CDRs of binding agents that bind PD-1 may include any one or more of comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOS. 1-138, and/or any other shown in Tables 1A and/or 1B.
- any of the amino acid sequences described herein, and/or any fragments and/or derivatives thereof may also be combined with any other variable region and/or CDR in any order and/or combination to form hybrid and/or fusion binding agents and/or inserted into other heavy and/or light chain variable regions using standard techniques.
- Exemplary combinations of CDRs e.g., combination of heavy and/or light chain CDR1, CDR2 and CDR3 amino acid sequences
- the heavy chain CDRs of each clone are combined with their respective light chain CDRs into a binding agent.
- the binding agent may comprise the heavy chain CDRs and light chain CDRs shown below:
- Binding agents comprising the CDRs of Tables 1A and/or 1B, or those of the immediately preceding paragraph, may also exhibit the following characteristics:
- Binding affinity may be determined by any technique available to those of ordinary skill in the art.
- the binding affinity data presented in Table 2 was evaluated by flow cytometry staining of endogenous cell surface PD-1 on CD4 T cells that were stimulated for a period of 3 to 6 days with phytohaemagglutinin (PHA). Binding class may also be determined by any technique available to those of ordinary skill in the art.
- the binding class data presented in Table 2 was determined by Luminex assay competitive binding studies. In Table 2, binding class 1 mAb antibodies are those determined to be competitive with the EH12.2H7 clone commercial antibody (available from BioLegend, San Diego, Calif. (e.g., Cat. No.
- class 2 antibodies are those determined to be competitive with the J116 clone commercial antibody (available from Affymetrix eBioscience, San Diego, Calif. (e.g., Cat. No. 16-9989-80)); and class 3 antibodies are those determined to be competitive with both EH12.2H7 and J116 antibodies; and class 4 mAb clone antibodies are those determined to bind PD-1 in the presence of both EH12.2H7 and J116 antibodies.
- Proliferative effect may be determined by any technique available to those of ordinary skill in the art. For instance, the EFRA system described above and used in Example 1 may be used. Such an assay was used to determine the proliferative effect data presented in Table 2. Briefly, a carboxyfluorescein succinimidyl ester (CFSE) assay in which peripheral blood mononuclear cells (PBMCs) were isolated from a chronically infected HIV subject and stimulated with an HIV-specific peptide in the presence and absence of an anti-PD-1 antibody. A control anti-PD1 antibody (the Merck antibody MK-3475) was also tested as a positive control. Following a six-day incubation, proliferation of HIV-specific CD8 T cells was evaluated in the anti-PD-1 treated samples relative to the peptide alone control and the result expressed as a percentage above control (“Proliferation effect”).
- CFSE carboxyfluorescein succinimidyl ester
- the techniques used to identify and characterize PD-1 binding agents such as antibodies may be combined to provide a system for identifying and characterizing such binding agents.
- one or more candidate binding agents such one or more monoclonal antibodies may be assayed by EFRA or a similar assay to determine the ability of the candidate binding agent to restore function to immune cells as measured by, for instance, proliferation in the presence of an immunogenic peptide.
- this type of assay may be used as an initial screen to ensure the candidate binding agents to be further studied are capable of restoring immune cell function.
- these types of assays may be followed by one for determining the binding affinity to immune cells such as activated peripheral blood mononuclear cells (PBMCs).
- PBMCs peripheral blood mononuclear cells
- this assay may use a technique such as fluorescence activated cell sorting (FACS).
- FACS fluorescence activated cell sorting
- the assay may include the presence or absence of non-specific binding and/or competitive binding studies using known binding reagents such as anti-PD1 antibody (e.g., the Merck antibody MK-3475, also know as pembrolizumab). These assays may then be followed by sequencing of the CDRs of the candidate binding agents such as provided in Tables 1A and/or 1B above. Together, then, the EFRA, affinity determination, epitope mapping studies and CDR identification methods described herein provide a system with which a candidate binding agent may be identified.
- any of the amino acid sequences of Tables 1A and/or 1B may be also substituted by any other amino acid as desired by one of ordinary skill in the art.
- one of skill in the art may make conservative substitutions by replacing particular amino acids with others as shown in Table 3 below.
- the specific amino acid substitution selected may depend on the location of the site selected.
- conservative amino acid substitutions may involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position and, in particular, does not result in decreased PD-1 binding.
- this disclosure provides binding agents with multiple specificities such that PD-1 and at least one other secondary antigen (e.g., a cell surface protein) may be bound by a single binding agent.
- the secondary antigen may be one expressed by cells infected by an infectious agent.
- an exemplary secondary antigen may be HIV Env antigen.
- binding agents may bind the secondary antigen and/or may serve to neutralize the infectious agent.
- a bi-specific binding agent having dual specificity for PD-1 and an HIV antigen such as env and/or another antigen, for instance.
- the HIV immunogen may be derived from any of the subtypes described herein, or any other.
- such binding agents may include: PD-1 agonist/Env binding; PD-1 agonist PD-1/Env binding and neutralization; PD-1 antagonist/Env binding; and/or PD-1 antagonist/PD-1/Env binding and neutralization.
- PD-1 subtypes B and/or C it may be preferable to select antigens from HIV-1 subtypes B and/or C. It may also be desirable to include binding agents having specificity for antigens from multiple HIV subtypes (e.g., HIV-1 subtypes B and C, HIV-2 subtypes A and B, or a combination of HIV-1 and HIV-2 subtypes) in a single composition.
- PD-1 specificities e.g., bi-specific PD-1a/PD1b antagonist PD-1 antibodies specific to two different epitopes
- tumor antigens e.g., cancer-testis (CT) antigen (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigen (i.e., Melan A/MART-1, tyrosinase, gp100); mutational antigen (i.e., MUM-1, p53, CDK-4); overexpressed ‘self’ antigen (i.e., HER-2/neu, p53); and/or viral antigens (i.e., HPV, EBV)).
- CT cancer-testis
- melanocyte differentiation antigen i.e., Melan A/MART-1, tyrosinase, gp100
- mutational antigen i.e., MUM-1, p53, CDK-4
- overexpressed ‘self’ antigen i.e., HER
- binding agents e.g., monoclonal antibodies
- the specificities of such binding agents may be recombined into a single binding agent using techniques that are widely available to those of ordinary skill in the art.
- multiple single specifity binding agents may also be combined and used (e.g., administered) to provide an effective multiple specificity reagent.
- the binding agents described herein may be conjugated to active agents to target and inhibit the function of and/or eliminate cell populations expressing PD-1 (and/or another antigen in the case of binding agents with multiple specificities).
- CD4 + T-cell populations containing replication competent HIV may be targeted and eliminated using binding agent/drug conjugates (e.g., antibody-drug conjugates (ADC)).
- ADC antibody-drug conjugates
- Mono- and/or bi-specific candidate binding agents may be conjugated with one or more types of drugs (e.g., drugs damaging DNA, targeting microtubules).
- the binding agents described herein and/or derivatives thereof may also be adjoined to and/or conjugated to functional agents for in vitro and/or in vivo use.
- the binding agent may be adjoined to and/or conjugated to functional moieties such as cytotoxic drugs or toxins, and/or active fragments thereof such as diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, among others.
- Suitable functional moieties may also include radiochemicals.
- Binding agents, such as antibodies, may be adjoined to and/or conjugated to the one or more functional agents using standard techniques in the art.
- the binding agents may be administered in conjunction with other agents such as anti-infective agents (e.g., antibiotics, anti-viral medications).
- anti-infective agents e.g., antibiotics, anti-viral medications
- the binding agents described herein may be combined with monoclonal antibodies and/or other reagents such as Nivolumab (also known as MDX-1106, BMS-936558 (Topalian, et al. N. Eng. J. Med.
- the PD-1 binding agents described herein may be used to treat and/or prevent and/or ameliorate the symptoms of infection by HIV.
- HIV isolates are now classified into discrete genetic subtypes. HIV-1 is known to comprise at least ten subtypes (A1, A2, A3, A4, B, C, D, E, F1, F2, G, H, J and K) (Taylor et al, NEJM, 359(18):1965-1966 (2008)). HIV-2 is known to include at least five subtypes (A, B, C, D, and E). Subtype B has been associated with the HIV epidemic in homosexual men and intravenous drug users worldwide.
- HIV-1 immunogens laboratory adapted isolates, reagents and mapped epitopes belong to subtype B.
- subtype B In sub-Saharan Africa, India and China, areas where the incidence of new HIV infections is high, HIV-1 subtype B accounts for only a small minority of infections, and subtype HIV-1 C appears to be the most common infecting subtype. Any of these types of isolates may be addressed using the binding agents described herein.
- One or more binding agents may also be administered with or in conjunction with one or more agents used to prevent, treat and/or ameliorate HIV such as for example, a protease inhibitor, an HIV entry inhibitor, a reverse transcriptase inhibitor, and/or an anti-retroviral nucleoside analog.
- Suitable compounds include, for example, Agenerase (amprenavir), Combivir (Retrovir/Epivir), Crixivan (indinavir), Emtriva (emtricitabine), Epivir (3tc/lamivudine), Epzicom, Fortovase/Invirase (saquinavir), Fuzeon (enfuvirtide), Hivid (ddc/zalcitabine), Kaletra (lopinavir), Lexiva (Fosamprenavir), Norvir (ritonavir), Rescriptor (delavirdine), Retrovir/AZT (zidovudine), Reyatax (atazanavir, BMS-232632), Sustiva (efavirenz), Trizivir (abacavir/zidovudine/lamivudine), Truvada (Emtricitabine/Tenofovir DF), Videx (ddl/didanosine), Videx EC (ddl, didanosine),
- the PD-1 binding agents described herein may be used to treat and/or prevent and/or ameliorate the symptoms of cancer.
- exemplary cancers may include, for instance, any of the breast, blood, colon, stomach, rectum, skeletal tissue, skin (e.g., melanoma) brain, lung, bladder, kidney, ovary, and/or liver, among others.
- binding agents may also be combined with and/or administered with or in conjunction with one or more agents used to prevent, treat and/or ameliorate cancer such as for example, an alkylating agent (e.g., any nitrogen mustard, nitrosourea, tetrazine, aziridine, cisplatin and/or derivative thereof), anti-metabolite (e.g., any of the methotrexates, pemetrexeds, fluoropyrimidines and/or derivative thereof), anti-microbtubule agent (e.g., vinca alkyloids, taxanes, podophyllotoxin and/or derivative thereof), topoisomerase I and/or II inhibitors (e.g., a camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, aclarubicin and/or derivative thereof) and/or
- the one or more binding agents may also, or alternatively, be combined with one or more other binding agents available to those of ordinary skill in the art for treating, preventing and/or ameliorating cancer such as, for example, Nivolumab, Lambrolizumab, Pidilizumab and/or other similar agents and/or derivatives thereof.
- Other suitable agents are known to those of skill in the art and may be suitable for use as described herein. Such agents may either be used prior to, during, or after administration of the binding agents and/or use of the methods described herein.
- the PD-1 binding agents described herein may be used to treat and/or prevent and/or ameliorate the symptoms of autoimmunity.
- exemplary autoimmune conditions may include, for instance, any in which PD-1 is involved in maintaining self-tolerance and/or one involving inflammatory T cells (e.g., autoreactive or self antigen-specific T cells) such as, for instance, systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, glomerulonephritis, and multiple sclerosis.
- inflammatory T cells e.g., autoreactive or self antigen-specific T cells
- SLE systemic lupus erythematosus
- type I diabetes rheumatoid arthritis
- glomerulonephritis glomerulonephritis
- multiple sclerosis e.g., multiple sclerosis.
- anti-CTLA-4 agents e.g., ipilimumab
- binding agents may also be combined with and/or administered with or in conjunction with one or more agents used to prevent, treat and/or ameliorate autoimmunity such as, for example, glucocorticoids, cytostatics (e.g., alkylating agent, anti-metabolite, methotrexate, azathioprine, mercaptopurine, cytotoxic antibiotics (e.g., dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin), antibodies (e.g., Atgam, Thymoglobuline, Simulect, Zenapax), drugs acting on immunophilins (e.g., ciclosporin, tacrolimus, sirolimus), interferons, opioids, TNF-binding agents (e.g., Remicade, Enbrel, Humira), mycophenolate, fingolimod, myriocin, and/or derivatives thereof.
- the binding agents may be adjoined to and/or conjugated to one or more detectable labels.
- suitable detectable labels may include, for instance, fluorosceins (e.g., DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); FITC; 6-carboxy-1,4-dichloro-2′,7′-dichlorofluorescein (TET); 6-carboxy-1,4-dichloro-2′,4′,5′,7′-tetra-chlorofluorescein (HEX); 6-carboxy-4′,5′-dichloro-2′,7′-dimeth
- a nucleic acid molecule encoding one or more binding agents described herein may be inserted into one or more expression vectors, as discussed below in greater detail.
- the binding agent may be encoded by nucleotides corresponding to the amino acid sequence.
- the particular combinations of nucleotides (codons) that encode the various amino acids (AA) are well known in the art, as described in various references used by those skilled in the art (e.g., Lewin, B. Genes V, Oxford University Press, 1994).
- the nucleotide sequences encoding the amino acids of said binding agents may be ascertained with reference to Table 4, for example.
- Nucleic acid variants may use any combination of nucleotides that encode the binding agent.
- nucleotide sequences encoding SEQ ID NOS. 2-138 may be deduced in the same way, and such nucleotide sequences are contemplated herein.
- binding agents are antibodies
- nucleotide sequences encoding the variable regions thereof may also be isolated from the phage and/or hybridoma cells expressing the same cloned into expression vectors to produce certain preparations (e.g., humanized antibodies). Methods for producing such preparations are well-known in the art.
- hybridoma cells from mice immunized with a PD-1 antigen/immunogen may be selected using the functional assays described herein and cloning techniques that are readily available to those of ordinary skill in the art. For instance, to isolate and sequence nucleic acids encoding the heavy and light chain variable regions of the selected hybridomas, total RNA may be extracted from fresh hybridoma cells using TRIzol reagent according to the manufacturer's protocol. cDNA may be synthesized from the RNA using isotype-specific anti-sense primers or universal primers using standard techniques (e.g., following the technical manual of PrimeScriptTM 1st Strand cDNA Synthesis Kit).
- PCR Polymerase chain reaction
- PCR Polymerase chain reaction
- Colony PCR screening may then be performed to identify clones with inserts of correct sizes. Preferably, no less than five single colonies with inserts of correct sizes are sequenced for each antibody variable region. Standard protocols may then be used for the expression and purification of the anti-PD-1 antibodies. For instance, hybridoma clones may be grown in serum-free medium and the cell culture broth centrifuged and then filtered.
- the filtered supernatant containing the antibody may then be loaded onto an affinity column (e.g., Protein A) column, washed and eluted with an appropriate buffer (e.g., Pierce IgG elute buffer).
- an appropriate buffer e.g., Pierce IgG elute buffer
- the eluted fractions may then be pooled and buffer-exchanged into PBS, pH 7.2.
- the purified antibody may then be analyzed by SDS-PAGE and Western blot by using standard protocols for molecular weight, yield and purity. Size exclusion chromatography HPLC may then be performed on an appropriate column (e.g., TSK GEL-G3000 SWXL column (Tosoh)) for biophysical characterization in order to ensure high antibody purity (generally >90%) with low presence of protein aggregates.
- TSK GEL-G3000 SWXL column Tosoh
- Nucleic acid molecules encoding one or more PD-1 binding agents may be contained within a viral and/or a non-viral vector.
- a DNA vector is utilized to deliver nucleic acids encoding one or more PD-1 binding agents to the patient.
- various strategies may be utilized to improve the efficiency of such mechanisms including, for example, the use of self-replicating viral replicons (Caley, et al. 1999 . Vaccine, 17: 3124-2135; Dubensky, et al. 2000 . Mol. Med. 6: 723-732; Leitner, et al. 2000 . Cancer Res. 60: 51-55), codon optimization (Liu, et al. 2000 . Mol.
- Various viral vectors that have been successfully utilized for introducing a nucleic acid to a host include retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, and poxvirus, among others.
- the vectors may be constructed using standard recombinant techniques widely available to one skilled in the art.
- Non-viral plasmid vectors may also be suitable in certain embodiments. Preferred plasmid vectors are compatible with bacterial, insect, and/or mammalian host cells.
- Such vectors include, for example, PCR-ii, PCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSii (Stratagene, La Jolla, Calif.), pet15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFp-n2 (Clontech, Palo Alto, Calif.), pETI (Bluebacii, Invitrogen), pDSR-alpha (PCT pub. No.
- telomeres a high copy number COLe1-based phagemid, Stratagene Cloning Systems, La Jolla, Calif.
- PCR cloning plasmids designed for cloning TAQ-amplified PCR products e.g., TOPOTM TA Cloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, Calif.
- Bacterial vectors may also be used.
- vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus , Bacille Calmette Guérin (BCG), and Streptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376).
- BCG Bacille Calmette Guérin
- Streptococcus see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376).
- Other delivery techniques may also suffice including, for example, DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO 4 precipitation, gene gun techniques, electroporation, and colloidal dispersion systems.
- Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- the preferred colloidal system is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo.
- RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., et al., 1981 , Trends Biochem. Sci., 6: 77).
- the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
- the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
- diacylphosphatidylglycerols where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
- Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
- a cultured cell comprising the vector is also provided.
- the cultured cell may be a cultured cell transfected with the vector or a progeny of the cell, wherein the cell expresses the immunogenic polypeptide.
- Suitable cell lines are known to those of skill in the art and are commercially available, for example, through the American Type Culture Collection (ATCC).
- ATCC American Type Culture Collection
- the transfected cells can be used in a method of producing an immunogenic polypeptide.
- the method comprises culturing a cell comprising the vector under conditions that allow expression of the immunogenic polypeptide, optionally under the control of an expression sequence.
- the immunogenic polypeptide can be isolated from the cell or the culture medium using standard protein purification methods.
- antibodies may be utilized to isolate PD-1 using, for example, immunoprecipitation or other capture-type assay.
- This well-known technique is performed by attaching the antibody to a solid support or chromatographic material (e.g., a bead coated with Protein A, Protein G and/or Protein L).
- the bound antibody is then introduced into a solution either containing or believed to contain the PD-1 (e.g., an HIV-infected T cell lysate).
- PD-1 may then bind to the antibody and non-binding materials are washed away under conditions in which the PD-1 remains bound to the antibody.
- the bound protein may then be separated from the antibody and analyzed as desired. Similar methods for isolating a protein using an antibody are well-known in the art.
- the binding agents e.g., antibodies
- the binding agents may also be utilized to detect PD-1 within a biological sample.
- the antibodies may be used in assays such as, for example, flow cytometric analysis, ELISA, immunoblotting (e.g., western blot), in situ detection, immunocytochemistry, and/or immunhistochemistry. Methods of carrying out such assays are well-known in the art.
- the binding agents described herein may be also be used to determine the presence of a disease state in a patient, to predict prognosis, or to determine the effectiveness of a chemotherapeutic or other treatment regimen.
- Expression profile assays performed as described herein or as is otherwise known in the art, may be used to determine the relative level of expression of PD-1. The level of expression may then be correlated with base (e.g., control) levels to determine whether a particular disease is present within the patient, the patient's prognosis, or whether a particular treatment regimen is effective.
- an increased or decreased level of expression of PD-1 in the patient's tissues may indicate the regimen is worsening or improving the load of the infectious agent in that host.
- the increase or decrease in expression may indicate the regimen is having or not having the desired effect and another therapeutic modality may therefore be selected.
- the binding agents described herein may be used to ascertain the effect of a drug candidate on the expression of the immunogenic target in a cell line, or a cell or tissue of a patient.
- the expression profiling technique may be combined with high throughput screening techniques to allow rapid identification of useful compounds and monitor the effectiveness of treatment with a drug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8 (1998)).
- Drug candidates may be chemical compounds, nucleic acids, proteins, antibodies, or derivatives therefrom, whether naturally occurring or synthetically derived. Drug candidates thus identified may be utilized, among other uses, as pharmaceutical compositions for administration to patients or for use in further screening assays.
- the binding agents are in purified form.
- a “purified” binding agent e.g., antibody
- a purified binding agent may be one that is separated from at least about 50% of the proteins and/or other components with which it is initially found (e.g., as part of a hybridoma supernatant or ascites preparation in the case of a monoclonal antibody).
- a purified binding agent e.g., antibody
- a pharmaceutically acceptable carrier is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
- the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Suitable pharmaceutical carriers and their formulations are described in, for example, Remington's: The Science and Practice of Pharmacy, 21 st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005).
- an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
- the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally from about 5 to about 8 or from about 7 to about 7.5.
- Other carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing polypeptides or fragments thereof. Matrices may be in the form of shaped articles, e.g., films, liposomes or microparticles.
- Carriers are those suitable for administration of polypeptides and/or fragments thereof to humans or other subjects.
- Pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives, surface active agents, adjuvants, immunostimulants, in addition to the immunogenic polypeptide.
- Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents and anesthetics.
- the pharmaceutical composition may be administered orally, parentally, by inhalation spray, rectally, intranodally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
- pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a nucleic acid, polypeptide, or peptide as a pharmaceutical composition.
- a “pharmaceutical composition” is a composition comprising a therapeutically effective amount of a nucleic acid or polypeptide.
- effective amount and “therapeutically effective amount” each refer to the amount of a binding agent, nucleic acid or the like used to observe the desired therapeutic effect (e.g., restore T cell function).
- binding agent is a monoclonal antibody or fragment or derivative thereof comprising one or more of SEQ ID NOS. 1-138 and/or shown in Tables 1A and 1B.
- the one or more binding agents may be administered in a dosage amount of about 1 to about 50 mg/kg, about 1 to about 30 mg/kg, or about 5 to about 30 mg/kg (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 mg/kg).
- the one or more binding agents may be administered to the mammal (e.g., intradermally, intravenously, orally, rectally) at about 10 mg/kg one or more times.
- the doses may comprise about the same or different amount of binding agent in each dose.
- the doses may also be separated in time from one another by the same or different intervals.
- the doses may be separated by about any of 6, 12, 24, 36, 48, 60, 72, 84, or 96 hours, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 3 years, 4 years, 5 years, or any time period before, after, and/or between any of these time periods.
- the binding agents may be administered in conjunction with other agents (e.g., anti-infective agents and/or chemotherapeutic agent). Such other agents may be administered about simultaneously with the binding agents, or at a different time and/or frequency. Other embodiments of such methods may also be appropriate as could be readily determined by one of ordinary skill in the art.
- kit format A kit including such antibodies and optionally other components necessary for using the antibodies to detect cells expressing PD-1 is provided.
- the antibodies of the kit may be provided in any suitable form, including frozen, lyophilized, or in a pharmaceutically acceptable buffer such as TBS or PBS.
- the kit may also include other reagents required for utilization of the antibodies in vitro or in vivo such as buffers (e.g., TBS, PBS), blocking agents (solutions including nonfat dry milk, normal sera, Tween-20 Detergent, BSA, or casein), and/or detection reagents (e.g., goat anti-mouse IgG biotin, streptavidin-HRP conjugates, allophycocyanin, B-phycoerythrin, R-phycoerythrin, peroxidase, detectable labels, and other labels and/or staining kits (e.g., ABC Staining Kit, Pierce)).
- buffers e.g., TBS, PBS
- blocking agents solutions including nonfat dry milk, normal sera, Tween-20 Detergent, BSA, or casein
- detection reagents e.g., goat anti-mouse IgG biotin, streptavidin-HRP conjugates, allophycocyanin,
- kits may also include other reagents and/or instructions for using the antibodies in commonly utilized assays described above such as, for example, flow cytometric analysis, ELISA, immunoblotting (e.g., western blot), in situ detection, immunocytochemistry, immunhistochemistry.
- the kit provides a binding agent in purified form.
- the binding agent may be provided in biotinylated form either alone or along with an avidin-conjugated detection reagent (e.g., antibody).
- the kit includes a binding agents comprising one or more detectable labels that may be used to directly detect PD-1.
- kits and the like required for using any of these systems are well-known in the art and/or may be prepared by the end-user or provided as a component of the kit.
- the kit may also include a solid support containing positive- and negative-control protein and/or tissue samples.
- kits for performing spotting or western blot-type assays may include control cell or tissue lysates for use in SDS-PAGE or nylon or other membranes containing pre-fixed control samples with additional space for experimental samples.
- Kits for visualization of PD-1 in cells on slides may include pre-formatted slides containing control cell or tissue samples with additional space for experimental samples.
- Other embodiments of kits are also contemplated herein as would be understood by those of ordinary skill in the art.
- the binding agent is a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOS. 1-138 and/or shown in Tables 1A and 1B. In some embodiments, the binding agent is a polypeptide comprising one or more combinations of SEQ ID NOS. 1-138 (e.g., as shown in Tables 1A and/or 1B). In some embodiments, the binding agent is an antibody. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR1 amino acid sequence selected from the group consisting of SEQ ID NOS. 1-23.
- the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR2 amino acid sequence selected from the group consisting of SEQ ID NOS. 24-46. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR3 amino acid sequence selected from the group consisting of 47-69. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a light chain CDR1 amino acid sequence selected from the group consisting of SEQ ID NOS. 70-92. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR2 amino acid sequence selected from the group consisting of SEQ ID NOS. 93-115.
- the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR3 amino acid sequence selected from the group consisting of 116-138.
- the binding agent comprises the combinations of CDRs shown in Tables 1A and/or 1B and/or has the properties described in Table 2.
- the binding agent is derived from or related to (e.g., by sequence or derivation) a human antibody, human IgG, human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgA1, human IgA2, human IgD, human IgE, canine antibody, canine IgGA, canine IgGB, canine IgGC, canine IgGD, chicken antibody, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, goat antibody, goat IgG, mouse antibody, mouse IgG, pig antibody, and/or rat antibody, and/or a derivative thereof.
- a human antibody canine IgG, human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgA1,
- the derivative may be selected from the group consisting of an F ab , F ab2 , Fab′ single chain antibody, F v , single chain, mono-specific antibody, bispecific antibody, trimeric antibody, multi-specific antibody, multivalent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized antibody, CDR-grafted antibody, shark antibody, nanobody, and/or canelid antibody.
- the binding agent comprises at least a least a first and second specificity, the first being against PD-1 and the second being against a different antigen (e.g., an antigen of an infectious agent such as HIV (e.g., env) and/or a tumor antigen).
- a different antigen e.g., an antigen of an infectious agent such as HIV (e.g., env) and/or a tumor antigen.
- the binding agent and/or derivative thereof may comprise a detectable label fixably attached thereto.
- the binding agent of any one and/or derivative thereof comprises an effector moiety (e.g., a cytotoxic drug, toxin, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and radiochemical) fixably attached thereto.
- an effector moiety e.g., a cytotoxic drug, toxin, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and radiochemical fixably attached thereto.
- polynucleotides encoding one or more binding agents are also provided (e.g., as an expression vector).
- Host cells comprising and/or expressing the polypeptide products of such polynucleotides are also provided.
- compositions comprising at least one binding agent or derivative; at least one isolated polynucleo
- This disclosure also provides methods for detecting PD-1 on a cell, the method comprising contacting a test biological sample with a binding agent or derivative described herein and detecting the binding agent bound to the biological sample or components thereof.
- Such methods may be an in vivo method or an in vitro method.
- the method may comprise comparing the amount of binding to the test biological sample or components thereof to the amount of binding to a control biological sample or components thereof, wherein increased binding to the test biological sample or components thereof relative to the control biological sample or components thereof indicates the presence of a cell expressing PD-1 in the test biological sample (e.g., mammalian blood).
- a system for identifying a PD-1 antibody binding agent by assaying the candidate binding agent by the exhaustion functional recovery assay (EFRA); determining the affinity of the candidate binding agent for PD-1; and, determining the nucleotide sequence of the CDR of the candidate binding agent is provided.
- kits for detecting the expression of PD-1 in or on a cell comprising a binding agent or derivative thereof and instructions for use.
- the binding agent and/or derivative thereof is in lyophilized form.
- this disclosure provides methods for treating, preventing and/or ameliorating an infectious disease, cancer and/or autoimmunity in a mammal comprising administering to the mammal at least one effective dose of a pharmaceutical composition comprising a binding agent or derivative thereof.
- the infectious disease is human immunodeficiency virus (HIV).
- the binding agent and/or derivative thereof used to treat infectious disease and/or cancer is a PD-1 antagonist.
- the binding agent and/or derivative thereof used to treat an autoimmune condition is a PD-1 agonist.
- multiple doses are administered to the animal.
- the binding agent and/or derivative thereof may be administered in a dosage amount of about 1 to 50 mg/kg.
- the combination comprises a first binding agent that blocks the interaction of PD-1 and PD-L1 and a second binding agent that does not block the interaction of PD-1 and PD-L1.
- Such combinations may be used for any use described herein or as may be otherwise ascertained by those of ordinary skill in the art. For instance, such combinations may be used in the methods for treating, preventing and/or ameliorating an infectious disease, cancer and/or autoimmunity in a mammal described herein.
- This disclosure also provides methods for producing the binding agents described herein by expressing the binding agent in a cell and isolating the binding agent from the cell or a culture supernatant of the cell. In some embodiments, such methods may further comprise expressing a nucleic acid encoding such binding agent(s). In some embodiments, such methods may also include combining the binding agent(s) following isolation with one or more pharmaceutically acceptable excipients.
- Methods for producing a combination(s) of binding agents such as a first binding agent that blocks the interaction of PD-1 and PD-L1 and a second binding agent that does not block the interaction of PD-1 and PD-L1, are also provided by this disclosure.
- the second binding agent binds PD-1.
- the first and/or second binding agents are antibodies such monoclonal antibodies or fragments or derivatives thereof.
- the second binding agent comprises the amino acid sequences SEQ ID NOS. 2, 25, 48, 71, 94 and 117; or SEQ ID NOS. 17, 40, 63, 86, 109, and 132. In some embodiments, these methods may further include the addition of a pharmaceutically acceptable excipient.
- a subject or a host is meant to be an individual.
- the subject can include domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs) and birds.
- livestock e.g., cattle, horses, pigs, sheep, and goats
- laboratory animals e.g., mice, rabbits, rats, guinea pigs
- the subject is a mammal such as a primate or a human.
- composition can comprise a combination means that the composition may comprise a combination of different molecules or may not include a combination such that the description includes both the combination and the absence of the combination (i.e., individual members of the combination).
- Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximately, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Ranges (e.g., 90-100%) are meant to include the range per se as well as each independent value within the range as if each value was individually listed.
- combined or “in combination” or “in conjunction” may refer to a physical combination of agents that are administered together or the use of two or more agents in a regimen (e.g., administered separately, physically and/or in time) for treating, preventing and/or ameliorating a particular disease.
- treat, prevent, and/or ameliorate or derivatives thereof are used herein in connection with a given treatment for a given condition (e.g., preventing cancer infection by HIV), it is meant to convey that the treated patient either does not develop a clinically observable level of the condition at all, or develops it more slowly and/or to a lesser degree than he/she would have absent the treatment.
- a treatment will be said to have prevented the condition if it is given during exposure of a patient to a stimulus that would have been expected to produce a given manifestation of the condition, and results in the patient's experiencing fewer and/or milder symptoms of the condition than otherwise expected.
- a treatment can “prevent” infection by resulting in the patient's displaying only mild overt symptoms of the infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism.
- reduce, reducing, and reduction as used herein in connection with prevention, treatment and/or amelioration of a given condition by a particular treatment typically refers to a subject developing an infection more slowly or to a lesser degree as compared to a control or basal level of developing an infection in the absence of a treatment (e.g., administration of one or more PD-1 binding agents).
- a reduction in the risk of infection may result in the patient's displaying only mild overt symptoms of the infection or delayed symptoms of infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism.
- mice strains (total of 16 mice) have been immunized with two PD-1 proteins, i.e. human PD-1 Fc fusion protein and a human PD-1 monomeric protein.
- Mice showing serum reactivity to PD-1 expressed on activated human T lymphocytes have been selected for generation of anti-PD-1 hybridoma cell lines.
- a total of 240 PD-1 hybridoma cell lines were selected for producing antibodies that bind to recombinant PD-1 protein.
- the primary criteria for the first round of antibodies selection were: i) staining of PD-1 on activated human T lymphocytes by flow cytometry; ii) diversity of CDR VH and VL sequences as compared to those of the existing anti-PD-1 antibodies; and, iii) epitope mapping performed by competitive binding studies with PD-1 conjugated Luminex beads with two commercially available anti-PD-1 antibodies binding to different epitopes on PD-1.
- a second round of selection was then carried out by: iv) affinity binding assays (not a primary criteria as it does not correlate with the stimulatory potential of anti-PD-1 antibodies); v) evaluation of anti-PD-1 antibodies that bind PD-1 and are either competitive, partially competitive or non-competitive with the binding of PD-L1 in a Luminex biochemical assay; and, vi) functional characterization of antibodies as agonist (not able to restore T-cells from functional exhaustion) or antagonist (able to restore T-cells from functional exhaustion). In these studies, the antibodies were tested and differentiated based on their ability to rescue proliferation in HIV-specific exhausted CD8 T-cells.
- the EFRA assay was carried out to evaluate the functional effect of anti-PD1 antibodies on the proliferation of HIV-specific CD8 T cells ( FIG. 2 ).
- Antibody clones in the upper box (E8-3, C2-3, E1-3, F3-3, H8-3, C10-2, G2-1, G3-2, H2-1, and H4-2) act as PD-1 antagonists and stimulate proliferation while antibody clones in the lower box (C8-1 and G10-2) are agonistic and promote the PD-1 negative regulatory effect.
- the level of proliferation induced by the peptide control (Pep 8) is indicated by the lower horizontal line (just below 1%) and the induced proliferation by the Merck MK-3475 anti-PD1 antibody is shown in the upper horizontal line (just above 2%).
- Antibodies of interest identified by these processes underwent a second round of subcloning and the resulting hybridoma clones were used for the production and purification of the antibodies in Table 2. Binding assays were carried out with the purified anti-PD-1 antibodies to ensure that the subclones retained their affinity for PD-1.
- the concentration response binding of anti-PD-1 antibodies to cell surface PD-1 was evaluated on activated CD4 T-cells ( FIG. 3 ).
- the EFRA provides for the selection of binding agents that restore T-cells from functional exhaustion but are not necessarily antagonistic, meaning those binding agents that do not necessarily interfere with the interaction between PD-1 and its biological ligand(s) (e.g., PD-L1 or PD-L2).
- An embodiment of the EFRA was to identify such binding agents (antibodies) that bind PD-1.
- Epitope mapping of antibody binding to PD-1 was performed with two separate biochemical assays. In one assay, competitive binding to PD-1 Fc fusion protein labeled beads was evaluated between one of two commercial anti-PD-1 antibodies (BMS-5C4 and MK3475) and the anti-PD-1 antibodies listed (also described in Table 2).
- class 1 competitive with the EH12.2H7 commercial monoclonal antibody clone that blocks the interaction of PD-1 with PD-L1
- class 2 competitive with the J116 commercial monoclonal antibody clone that binds PD-1 but does not effectively block the interaction of PD-1 with PD-L1
- class 3 competitive with both the EH12.2H7 and J116 commercial monoclonal antibodies
- class 4 non-competitive with either the EH12.2H7 or J116 commercial antibodies.
- the relative binding of these antibodies for cell surface PD-1 is represented by the mean fluorescence intensity (MFI) relative to a control anti-PD-1 antibody in FIG. 4 .
- MFI mean fluorescence intensity
- FIG. 5 d - f shows that a completive anti-PD-1 antibody completely blocks the interaction between PD-1 and PD-L1.
- Partially competitive and non-competitive anti-PD-1 antibodies listed in Table 2 results in a shift in the binding affinity of PD-L1 for PD-1, but does not block the interaction when concentration of PD-L1 are increased.
- FIGS. 6 a and 6 b Some of these antibodies were also shown to exhibit statistically significant increases in proliferation relative to controls (peptide 8 alone control or the peptide and IgG1 isotype control antibody in FIGS. 6 a and 6 b ).
- Class 1 antibodies competitive with the EH12.2H7 commercial monoclonal antibody that blocks the interaction of PD-1 with PD-L1 or competitive in the PD-1/PD-L1 interaction assay were generally determined to provide improved proliferative restoration.
- FIG. 7 shows that select antibodies described in Table 2 exhibited equivalent or statistically improved activity (p ⁇ 0.007) compared to the benchmark MK-3475 antibody.
- the combination of a first binding agent that blocks the interaction of PD-1 and PD-L1 with a second binding agent that does not block the interaction of PD-1 and PD-L1 has been determined to act synergistically to rescue T cells from exhaustion.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- AIDS & HIV (AREA)
- Communicable Diseases (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Transplantation (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
- This application is a continuation application of U.S. Ser. No. 15/981,977 filed May 17, 2018, now U.S. Pat. No. 11,130,807, which is a continuation application of U.S. Ser. No. 15/329,760 filed Jan. 27, 2017, now U.S. Pat. No. 9,982,053 B2, which was filed under 35 U.S.C. § 371 and claims priority to International Application No. PCT/IB2015/055943 filed Aug. 5, 2015, and claims priority to U.S. Ser. No. 62/033,177 filed Aug. 5, 2014; U.S. Ser. No. 62/053,366 filed Sep. 22, 2014; and U.S. Ser. No. 62/093,368 filed Dec. 17, 2014; each of which is hereby incorporated by reference into this disclosure in their entirety.
- This disclosure relates to binding agents with specificity for programmed cell death 1 (PD-1) (e.g., human PD-1) and to methods for using the same to treat and/or prevent infection (e.g., by human immunodeficiency virus (HIV)), cancer and/or autoimmunity.
- As we enter the fourth decade of the HIV epidemic, significant advances have been made in the understanding of HIV pathogenesis and in the development of potent and safe antiviral drugs. More than 30 antiviral drugs have been registered and the impact of combination antiretroviral therapy (ART) on both morbidity and mortality has been remarkable. However, despite the long-term suppression of HIV replication achieved in patients with optimal adherence to ART, HIV invariably rebounds after interruption of therapy. Furthermore, successful therapy does not induce or allow restoration/development of virus-specific immune responses capable of controlling HIV replication in the absence of ART. Thus, life-long ART is needed to control HIV replication and associated disease in the large majority of HIV infected subjects.
- A population of long-lived central memory CD4 T-cells latently infected with HIV has been identified in the blood as an important component of the HIV cell reservoir and as the primary cause of HIV persistence. The life-span of this latent cell reservoir is estimated to be approximately 70 years in the presence of full HIV suppression with ART. However, recent studies have demonstrated that two populations of CD4 T-cells resident in lymph nodes serve as the primary CD4 T-cell compartment for HIV infection, replication and production. These two CD4 T-cell populations are defined by the expression of PD-1 and CXCR5 and include the PD-1+CXCR5+, i.e. T follicular helper cells (Tfh) and PD-1+CXCR5− CD4 T-cell populations.
- A number of mechanisms responsible for the establishment and maintenance of the HIV latent cell reservoir(s) have been proposed. One of the mechanisms is the persistent of minimal virus replication under ART which may replenish the HIV cell reservoir. Therefore, ART is unable to induce full suppression of HIV replication and the “natural” HIV-1 specific immune response under ART is also unable to totally suppress and eliminate ongoing residual virus replication. The failures of ART and of the HIV-specific immune response provide the rationale for investigating alternative interventions to target also the persistent HIV cell reservoir.
- A number of immunological interventions have been investigated in the past and currently being further developed with the goal to achieve HIV functional cure, wherein viral replication is suppressed without sustained antiviral therapy (9). Therapeutic vaccine strategies have been the primary intervention strategy investigated but the results have shown modest efficacy in experimental animal models and patients with the exception of a CMV-based vector HIV vaccine (50% efficacy in the NHP model; 10). Recent studies have generated interesting results on the possibility of using anti-envelope broad neutralizing antibodies (bNabs) as therapeutic agents in HIV infection (11,12). Furthermore, antagonist PD-1 Abs have been shown to restore T-cell functions in HIV infected patients and the possibility to use these Abs as a therapeutic strategy to augment the potency of HIV-specific T-cell responses has been proposed (13,14).
- It is well established that infiltrating tumor-specific CD8 T-cells are dysfunctional with regard their ability to proliferate and to mediate cytotoxic activity. The large majority of infiltrating tumor-specific CD8 T-cells are in a so-called exhaustion functional state. The primary mechanism responsible for the exhaustion of infiltrating tumor-specific CD8 T-cells is the increased expression of a number of regulatory receptors and particularly PD-1 regulatory receptor. The observation that the blockade of the PD-1/PDL-1/2 (PD-1 ligands) is associated with the recovery of CD8 T-cells from exhaustion has provided the rationale for developing intervention strategies targeting the PD-1 molecule expressed by exhausted CD8 T-cells. Recent studies have shown very promising results with the use of PD-1 antibodies with antagonist activity in patients with advanced cancer-associated disease. Studies have show substantial rates of response, ranging from 18 to 40%, in patients with advanced melanoma, non-small cell lung carcinoma and renal carcinoma. Anti-PD-1 antibodies in these studies have been used either alone or in combination with an anti-CTL-A4 antibody. After these initial studies, the current studies are being performed in patients with a variety of tumors including also hematological tumors.
- There is a need in the art for additional reagents for targeting PD-1 and methods for using the same. This disclosure addresses those needs by providing reagents and methods that may be used to target PD-1 and cells and/or tissues expressing the same.
-
FIG. 1 .FIG. 1A . Mouse PD-1.FIG. 1B . Human PD-1. -
FIG. 2 . CFSE assay to evaluate the functional effect of anti-PD1 antibodies on the proliferation of HIV specific CD8 T cells. -
FIG. 3 . Concentration response binding of anti-PD-1 antibodies to cell surface PD-1 on activated CD4 T-cells. -
FIG. 4 . Antibody classes. -
FIG. 5 . Proliferation relative to controls (NEG and Peptide 8).FIG. 5A .Class 1 mAb 137F2: strongly competitive (IC50˜200 pM; Imax=100%).FIG. 5B .Class 2 mAb 139D6: partially competitive (IC50˜300 pM; Imax=50%).FIG. 5C .Class 2 mAb 136B4: non-competitive.FIG. 5D .Class 1 mAb 137F2: strongly competitive.FIG. 5E .Class 1 mAb 132F7: partially competitive (IC50˜200 pM; Imax=100%).FIG. 5F .Class 1 mAb 136B4: non-competitive. -
FIG. 6 . Restoration of HIV peptide specific CD8 T-cell proliferation mediated by anti-PD-1 antibodies binding to different epitopes in a functional exhaustion recovery assay.FIG. 6A .HIV peptide 8+BMS-5C4, MK3475, 135H12, 122H2, 139D6, 135E10, or 136B5.FIG. 6B .HIV peptide 8+MK3475, 135H12, 122H2, 137F2, 135C12, 135D11, or 136B5. -
FIG. 7 . Enhanced restoration of HIV peptide specific CD8 T-cell proliferation mediated by the combination of anti-PD-1 antibodies binding to different PD-1 epitopes in a functional exhaustion recovery assay. -
FIG. 8 . Synergy between a first binding agent that blocks the interaction of PD-1 and PD-L1 with a second binding agent that does not block the interaction of PD-1 and PD-L1. - This disclosure relates to binding agents with specificity for programmed cell death 1 (PD-1) (e.g., human PD-1) and to methods for using the same such as to treat, prevent and/or ameliorate infection (e.g., by human immunodeficiency virus (HIV)), cancer and/or an autoimmune condition. Functional assays for identifying binding agents that interact with PD-1 are also provided. Combinations of binding agents, such as a first binding agent that blocks the interaction of PD-1 and PD-L1 with a second binding agent that does not block the interaction of PD-1 and PD-L1, are also provided that act synergistically to rescue T cells from exhaustion.
- This disclosure relates to binding agents that bind programmed cell death (PD-1) protein (e.g., SEQ ID NO:1, FIG. 1A, FIG. 1B of U.S. Pat. No. 5,698,520 (Honjo, et al.) which is hereby incorporated by reference in its entirety) (e.g., human PD-1) on the surface of cells in vitro and/or in vivo. The binding agents may also bind isolated PD-1 polypeptide (e.g., human PD-1) and/or fragments and/or derivatives thereof, typically in vitro. Also provided are methods for using such binding agents to diagnose, treat, prevent and/or ameliorate one or more diseases associated with the existence of cells expressing PD-1. For instance, the binding agents may be antibodies (e.g., monoclonal antibodies) that may react with and/or bind to the epitopes of PD-1. The “binding agents” described herein may include, for example, an agonist or an antagonist of PD-1. An agonist binding agent is one that is not typically capable of restoring T-cell function and/or expression of PD-1. An agonist PD-1 binding agent may be useful for treating autoimmune diseases and others in which PD-1 expressing cells are involved in disease progression. In contrast, an antagonist binding agent is one capable for restoring T-cell function and/or expression of PD-1. For instance, a PD-1 antagonist binding agent may be capable of restoring the function of PD-1 expressing T-cells from functional exhaustion as is known to occur in HIV infection and in a variety of tumors. Restoration of T cell function may be determined by, for instance, measuring proliferation, cytokine production, cytotoxic activity or other characteristics of such cells. Another use for the binding agents described herein is the selective targeting and elimination of HIV-infected CD4 T-cell populations containing replication competent HIV (e.g., in a latent and/or replication state). Such PD-1 expressing cells expressing PD-1 are known to serve as a major cell reservoir for replication competent HIV. A potential mechanism for the elimination of these CD4 T-cell populations is antibody-dependent cellular cytotoxicity (ADCC) using the binding agents described herein (e.g., mono- and/or bi-specific PD-1 antibodies). In some embodiments, one or more PD-1 antagonistic binding agents having, for instance, different specificities (e.g., recognizing different epitopes) may be combined to induce rescue of antigen-specific CD8 T-cells from functional exhaustion caused by PD-1 expression in those cells (e.g., restoring or improving proliferation, cytokine production and/or cytotoxic activity). In some embodiments, the binding agents described herein may also provide for the selective elimination and/or suppression of PD-1 expressing cells. In some embodiments, the PD-1 agonist binding agents described herein may be used to supress and/or eliminate PD-1 expressing cells to treat, for instance, infectious diseases (e.g., HIV), cancer, and/or, especially, autoimmune conditions. Other embodiments, uses and the like are described below.
- The binding agents may be antibodies such as monoclonal antibodies that may comprise, for instance, any one or more of the amino acid sequences shown in Table 1 (and/or one or more fragments and/or derivatives thereof). This disclosure also provides for the use of such monoclonal antibodies to isolate, identify, and/or target cells expressing PD-1. In certain embodiments, these monoclonal antibodies may be reactive against PD-1 expressed on the surface of cells. The term “antibody” or “antibodies” may refer to whole or fragmented antibodies in unpurified or partially purified form (e.g., hybridoma supernatant, ascites, polyclonal antisera) or in purified form. The antibodies may be of any suitable origin or form including, for example, murine (e.g., produced by murine hybridoma cells), or expressed as humanized antibodies, chimeric antibodies, human antibodies, and the like. For instance, antibodies may be wholly or partially dervied from human (e.g., IgG (IgG1, IgG2, IgG2a, Ig2b, IgG3, IgG4), IgM, IgA (IgA1 and IgA2), IgD, and IgE), canine (e.g., IgGA, IgGB, IgGC, IgGD), chicken (e.g., IgA, IgD, IgE, IgG, IgM, IgY), goat (e.g., IgG), mouse (e.g., IgG, IgD, IgE, IgG, IgM), and/or pig (e.g., IgG, IgD, IgE, IgG, IgM), rat (e.g., IgG, IgD, IgE, IgG, IgM) antibodies, for instance. Methods of preparing, utilizing and storing various types of antibodies are well-known to those of skill in the art and would be suitable in practicing the present invention (see, for example, Harlow, et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Harlow, et al. Using Antibodies: A Laboratory Manual, Portable Protocol No. 1, 1998; Kohler and Milstein, Nature, 256:495 (1975)); Jones et al. Nature, 321:522-525 (1986); Riechmann et al. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol., 2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536 (1988); Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991); Marks et al., Bio/
Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al.,Nature Biotechnology 14, 845-51 (1996); Neuberger,Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos. 4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and, 5,661,016). In certain applications, the antibodies may be contained within hybridoma supernatant or ascites and utilized either directly as such or following concentration using standard techniques. In other applications, the antibodies may be further purified using, for example, salt fractionation and ion exchange chromatography, or affinity chromatography using Protein A, Protein G, Protein A/G, and/or Protein L ligands covalently coupled to a solid support such as agarose beads, or combinations of these techniques. The antibodies may be stored in any suitable format, including as a frozen preparation (e.g., −20° C. or −70° C.), in lyophilized form, or under normal refrigeration conditions (e.g., 4° C.). When stored in liquid form, for instance, it is preferred that a suitable buffer such as Tris-buffered saline (TBS) or phosphate buffered saline (PBS) is utilized. In some embodiments, the binding agent may be prepared as an injectable preparation, such as in suspension in a non-toxic parenterally acceptable diluent or solvent. Suitable vehicles and solvents that may be utilized include water, Ringer's solution, and isotonic sodium chloride solution, TBS and/or PBS, among others. Such preparations may be suitable for use in vitro or in vivo may be prepared as is known in the art and the exact preparation may depend on the particular application. - However, the binding agents described are not in any way limited to antibodies. For example, the binding agent may be any compound exhibiting similar binding properties as another (e.g., a mimetic). For example, an exemplary binding agent may be one that binds PD-1 and/or can compete with binding agent having specificity therefor (e.g., a monoclonal antibody). In some embodiments, the mimetic may exhibit substantially the same affinity in binding assays as the binding agent (e.g., monoclonal antibody) to which it is being compared. The affinity a particular binding agent may be measured by any suitable assay including but not limited to FACS staining of endogenous cell surface PD-1 on activated CD4 T cells as described in the Examples. One binding agent may be said to have “substantially the same affinity” as another where the measurements (e.g., nm) are within about any of 1-20, 1-5, 5-10, 10-15, or 15-20 percent of one another. Exemplary mimetics may include, for example, organic compounds that specifically bind PD-1, or an affibody (Nygren, et al. FEBS J. 275 (11): 2668-76 (2008)), affilin (Ebersbach, et al. J. Mol. Biol. 372 (1): 172-85 (2007)), affitin (Krehenbrink, et al. J. Mol. Biol. 383 (5): 1058-68 (2008)), anticalin (Skerra, A. FEBS J. 275 (11): 2677-83 (2008)), avimer (Silverman, et al. Nat. Biotechnol. 23 (12): 1556-61 (2005)), DARPin (Stumpp, et al. Drug Discov. Today 13 (15-16): 695-701 (2008)), Fynomer (Grabulovski, et al. J. Biol. Chem. 282 (5): 3196-3204 (2007)), Kunitz domain peptide (Nixon, et al. Curr. Opin. Drug Discov. Devel. 9 (2): 261-8 (2006)), and/or a monobody (Koide, et al. Methods Mol. Biol. 352: 95-109 (2007)). Other mimetics may include, for example, a derivative of an antibody (of, for example, the monoclonal antibody 1E4,1G10, and/or 1G1) such as, for example, an Fab, Fab2, Fab′ single chain antibody, Fv, single domain antibody, mono-specific antibody, bi-specific antibody, tri-specific antibody, multi-valent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized, CDR-grafted antibody, shark antibody, nanobody, canelid antibody, microbody, and/or intrabody, or derivative thereof. Other binding agents are also provided herein as would be understood by one of ordinary skill in the art.
- Any method known to those of ordinary skill in the art may be used to generate binding agents having specificity for (e.g., binding to) PD-1. For instance, to generate and isolate monoclonal antibodies an animal such as a mouse may be administered (e.g., immunized) with one or more PD-1 proteins (e.g., PD-1 Fc fusion protein and/or PD-1 His tag protein). Animals exhibiting serum reactivity to PD-1 expressed on activated human T lymphocytes (as determined by, for instance, flow cytometry and/or microscopy) may then be selected for generation of anti-PD-1 hybridoma cell lines. This may be repeated for multiple rounds. For instance, the primary criteria for the first round of binding agent selection may be include but are not limited to: i) level of staining of PD-1 on activated human T lymphocytes by flow cytometry; (ii) diversity of CDR VH and VL sequences as compared to those of the existing anti-PD-1 antibodies; and, (iii) epitope mapping performed by competitive binding studies with PD-1 conjugated Luminex beads pre-coupled with PD-L1 or one of several commercially available anti-PD-1 antibodies binding to different epitopes on PD-1. An exemplary first or second round of selection may also include, for instance, affinity binding (not a primary criteria since it may not correlate with the stimulatory potential of anti-PD-1 antibodies); and/or, functional characterization to identify the binding agent as an agonist or an antagonist.
- As described in Example 1 herein, for instance, the Exhaustion Functional Recovery Assay (EFRA) may be used. In this assay, test binding agents may be assayed for the ability to rescue immune cells such as T cells from exhaustion. This may be determined by measuring the ability of a binding agent to restore proliferation to such cells in the presence of an antigen, such as a test peptide derived from a virus such as human immunodeficiency virus (HIV). Proliferation is measured in a CFSE assay in comparison to a control, such as the test peptide alone or a positive control anti-PD-1 antibody such as MK-3475 (pembrolizumab). In some embodiments, a binding agent is determined to restore proliferation where the comparison shows a significant difference (such as a P value of <0.001) compared to either a peptide alone control or peptide with an isotype control mouse IgG1 antibody. This assay may be used to identify binding agents (such as antibodies) that compete with other binding agents for binding to PD-1 (such as PD-L1 or PD-L2) and/or lead to the functional restoration of immune cells. Example 1 also describes two methods of epitope mapping the antibodies listed in Table 2 using Luminex-based assays. In one biochemical assay, a PD-1 Fc fusion protein is bound to beads and competitive binding studies are performed between the anti-PD-1 antibodies described in Table 2 and one of two different commercially available anti-PD-1 antibodies. Example 1 describes four classes of monoclonal antibodies binding to distinct epitopes on PD-1 that were: class 1 (competitive with a first monoclonal antibody that blocks the interaction of PD-1 with PD-L1), class 2 (competitive with a second monoclonal antibody that binds PD-1 but does not block the interaction of PD-1 with PD-L1), class 3 (competitive with both the first and second monoclonal antibodies), and class 4 (non-competitive with either the first or second antibodies). In a separate assay, competition for binding to a recombinant PD-1 protein was evaluated for the anti-PD-1 antibodies listed in Table 2 and a biotinylated PD-L1 recombinant protein. Antibodies that induced proliferation in the EFRA were identified from all four binding classes that are proposed of binding to different epitopes on PD-1. Likewise, the EFRA allowed for the identification of anti-PD-1 antibodies that were either competitive, partially competitive or non-competitive with the PD-1/PD-L1 interaction and specifically restored proliferative function to HIV specific CD8 T-cell.
- Combinations of binding agents may also be identified, such as those described in Example 2. In some embodiments, the combinations may be identified to provide statistically significant differences from results obtained using only one or more of the binding agents and not others. In some embodiments, combinations exhibiting synergistic ability to restore immune cell function may be identified. In some embodiments, the combination may comprise a first binding agent that blocks the interaction of PD-1 and PD-L1 with a second binding agent that does not block the interaction of PD-1 and PD-L1. The first and second binding agents may be different entities such as two or more different monoclonal antibodies or derivatives thereof, or may be found on the same entity such as a bi-functional antibody (a single antibody or derivative thereof comprising multiple binding specificities). For instance, an exemplary bi-functional antibody may comprise a first binding region that blocks the interaction of PD-1 and PD-L1 and a second binding region that does not block the interaction of PD-1 and PD-L1. Also contemplated are combinations that provide multiple types of each binding agent. For instance, the combination may comprise multiple types of binding agents that block the interaction of PD-1 and PD-L1 with one or more that does not block the interaction of PD-1 and PD-L1. In some embodiments, the combination may comprise one or more of binding agents that block the interaction of PD-1 and PD-L1 with multiple binding agents that do not block the interaction of PD-1 and PD-L1. In some embodiments, the combination may comprise multiple binding agents that block the interaction of PD-1 and PD-L1 with multiple binding agents that do not block the interaction of PD-1 and PD-L1. Such combinations as described herein may also be combined with one or more other agents that may effect immune cell function such as antibodies against CTLA-4 and the like. One of ordinary skill in the art would recognize that many such combinations may be suitable for use as described herein.
- Where the binding agent is an antibody, it may be identified with reference to the nucleotide and/or amino acid sequence corresponding to the variability and/or complementarity determining regions (“CDRs”) thereof. The variable region/CDR sequences may be used in combination with one or more other variable region/CDR amino acid sequences. The variable region/CDR amino acid sequences may alternatively and/or also be adjoined to one or more types of constant region polypeptides of an antibody molecule. For instance, the CDR amino acid sequences shown in Tables 1A and 1B may be adjoined to or associated with the constant regions of any antibody molecule of the same or a different species (e.g., human, goat, rat, sheep, chicken) and/or antibody subtype of that from which the CDR amino acid sequence was derived. For instance, an exemplary binding agent may be, or may be derived from, or may be related to the monoclonal antibody produced by the hybridomas listed in, and/or may have about the same affinity and/or proliferation effect, and/or exhibit the same binding class shown in Table 2, and/or may have any one or more of the amino acid sequences of SEQ ID NOS. 1-138 and/or as shown in Tables 1A and 1B. The binding agent may comprise an antibody heavy and/or a light chain that each comprises one or more constant and/or variable regions. The variable regions typically comprise one or more CDRs that may determine the binding specificity of the antibody. The monoclonal antibodies may also be identified by analysis of the amino acid sequences of (e.g., which may be encoded by such nucleotide sequences) such variable regions. For instance, exemplary amino acid sequences of the heavy chain CDRs of binding agents that bind PD-1 may include any one or more of comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOS. 1-138, and/or any other shown in Tables 1A and/or 1B. Any of the amino acid sequences described herein, and/or any fragments and/or derivatives thereof may also be combined with any other variable region and/or CDR in any order and/or combination to form hybrid and/or fusion binding agents and/or inserted into other heavy and/or light chain variable regions using standard techniques. Exemplary combinations of CDRs (e.g., combination of heavy and/or light chain CDR1, CDR2 and CDR3 amino acid sequences) that may be found in a PD-1 (e.g., human PD-1) binding agent of this disclosure may include, for instance, the embodiments shown in Tables 1A and/or 1B.
-
TABLE 1A Heavy chain: Amino acids sequence Clone CDR1 CDR2 CDR3 122F10 DDFLH RIDPANGESRYAPKFQD TDYRGYYYAMDY (SEQ ID NO: 1) (SEQ ID NO: 24) (SEQ ID NO: 47) 139D6 NYYIH SIYPNYGDTNYNQKVKD GYSYAMDY (SEQ ID NO: 2) (SEQ ID NO: 25) (SEQ ID NO: 48) 135D1 NYYIH SIYPNYGETNYNQEFKG GYSYAMDY (SEQ ID NO: 3) (SEQ ID NO: 26) (SEQ ID NO: 49) 134D2 SNWMH AVNPGNSDTTYNQKFKG GRSYDGSFDY (SEQ ID NO: 4) (SEQ ID NO: 27) (SEQ ID NO: 50) 121G1 RYWMH NIDPSDSTTHYNPKFRD DLDDFYVGSHEDFDY (SEQ ID NO: 5) (SEQ ID NO: 28) (SEQ ID NO: 51) 136B5 SNWMH AVYPGNSDTTYNQNFKG GRSYDGSFDY (SEQ ID NO: 6) (SEQ ID NO: 29) (SEQ ID NO: 52) 127C2 NSYIH WISPGDGSTNYNEKFKG EEYDYDNY (SEQ ID NO: 7) (SEQ ID NO: 30) (SEQ ID NO: 53) 137F2 NYWIG DIYPGGGYTNYNEKFKG GYDFVLDR (SEQ ID NO: 8) (SEQ ID NO: 31) (SEQ ID NO: 54) 138H5 SYAMS TISGGGADTYYLDNVKG QRGENLFAH (SEQ ID NO: 9) (SEQ ID NO: 32) (SEQ ID NO: 55) 140A1 SDYAWN YINYSGYTNYNPFLKS YGGSYPWNFDV (SEQ ID NO: 10) (SEQ ID NO: 33) (SEQ ID NO: 56) 135H12 SYWIN NIYPGSSSIDYNEKFKS GLYWYFDV (SEQ ID NO: 11) (SEQ ID NO: 34) (SEQ ID NO: 57) 131D11 SSYIH WIFPGDGKTNYNEKFRD NDFDRGVY (SEQ ID NO: 12) (SEQ ID NO: 35) (SEQ ID NO: 58) 132F7 NHGMS SINTGGYSTYYPDNVKG DDYNWFAY (SEQ ID NO: 13) (SEQ ID NO: 36) (SEQ ID NO: 59) 126E4 NYWIG DIYPGSEYENYNEKFKG GYDFVLDH (SEQ ID NO: 14) (SEQ ID NO: 37) (SEQ ID NO: 60) 135G1 DSYIH RIDPAHGNVIYASKFRD IYYDYGEGDF (SEQ ID NO: 15) (SEQ ID NO: 38) (SEQ ID NO: 61) 136E10 DTYIH RIDLANDDILYASKFQG IYYDYGEGDY (SEQ ID NO: 16) (SEQ ID NO: 39) (SEQ ID NO: 62) 135C12 NFYIH SIYPNYGDTAYNQKFKD GYSYAMDY (SEQ ID NO: 17) (SEQ ID NO: 40) (SEQ ID NO: 63) 136F4 DSYIH RIDPARDNIIYASKFRD IYYDYGEGDY (SEQ ID NO: 18) (SEQ ID NO: 41) (SEQ ID NO: 64) 136B4 DDFLH RIDPANGESRYAPQFQD TDYRGYYYAMDY (SEQ ID NO: 19) (SEQ ID NO: 42) (SEQ ID NO: 65) 135E10 SYFMS GISTGGADTYYADSMKG LSHYYDGIPLDC (SEQ ID NO: 20) (SEQ ID NO: 43) (SEQ ID NO: 66) 140G5 NHGMS SISGGGDNTYYPDNLKG VRQLGLHRAAMDY (SEQ ID NO: 21) (SEQ ID NO: 44) (SEQ ID NO: 67) 122H2 NYWIG DIYPGGDHKNYNEKFKD GFDFVLDY (SEQ ID NO: 22) (SEQ ID NO: 45) (SEQ ID NO: 68) 139F11 SFAMS TITGGGVNTYYPDTVKG QAIYDGHYVLDY (SEQ ID NO: 23) (SEQ ID NO: 46) (SEQ ID NO: 69) -
TABLE 1B Light chain: Amino acids sequence Clone CDR1 CDR2 CDR3 122F10 KSSQSVLYSSNQKNYLA WASTRES HQYLSSYT (SEQ ID NO: 70) (SEQ ID NO: 93) (SEQ ID NO: 116) 139D6 SASQGISDGLN HTSTLHS QQYSKFPLT (SEQ ID NO: 71) (SEQ ID NO: 94) (SEQ ID NO: 117) 135D1 SASQGISNGLN HTSTLHS QQYSKFPLT (SEQ ID NO: 72) (SEQ ID NO: 95) (SEQ ID NO: 118) 134D2 KASQDINKYIA YTSTLRP LQYDNLWT (SEQ ID NO: 73) (SEQ ID NO: 96) (SEQ ID NO: 119) 121G1 RSSQSIVYSNGNTYLE KVSHRFS FQGSHVPYT (SEQ ID NO: 74) (SEQ ID NO: 97) (SEQ ID NO: 120) 136B5 KASQDINKYMA YTSTLRP LQYDNLWT (SEQ ID NO: 75) (SEQ ID NO: 98) (SEQ ID NO: 121) 127C2 KASQNVGTNVG SASYRYN QQYNTYPWT (SEQ ID NO: 76) (SEQ ID NO: 99) (SEQ ID NO: 122) 137F2 KSSQSLFNSETQKNYLA WASTRES KQSYTLRT (SEQ ID NO: 77) (SEQ ID NO: 100) (SEQ ID NO: 123) 138H5 LASQTIGTWLA AATSLAD QQLYSTPWT (SEQ ID NO: 78) (SEQ ID NO: 101) (SEQ ID NO: 124) 140A1 RSSQTIVHNNGDTYLE KISNRFF FQGSHVPYT (SEQ ID NO: 79) (SEQ ID NO: 102) (SEQ ID NO: 125) 135H12 KSSQSLENSGTRKNYLA WASTRDS KQSYNLYT (SEQ ID NO: 80) (SEQ ID NO: 103) (SEQ ID NO: 126) 131D11 KASQNVDTNVA SASYRYN QQYNNYPYT (SEQ ID NO: 81) (SEQ ID NO: 104) (SEQ ID NO: 127) 132F7 KSSQSLLNSGNQKNYLT WASTRES QSDYSYPLT (SEQ ID NO: 82) (SEQ ID NO: 105) (SEQ ID NO: 128) 126E4 KSSQSLENSGTRKSYLA WASTRET MQSYNLRT (SEQ ID NO: 83) (SEQ ID NO: 106) (SEQ ID NO: 129) 135G1 HASQNINVWLS KASNLHT QQGQSWPLT (SEQ ID NO: 84) (SEQ ID NO: 107) (SEQ ID NO: 130) 136E10 HASQNINVWLS KASNLHT QQGQSYPLT (SEQ ID NO: 85) (SEQ ID NO: 108) (SEQ ID NO: 131) 135C12 SASQGISGDLN HTSSLHS QYYSKDLLT (SEQ ID NO: 86) (SEQ ID NO: 109) (SEQ ID NO: 132) 136F4 HASQNINVWLS KASNLHT QQGQSWPLT (SEQ ID NO: 87) (SEQ ID NO: 110) (SEQ ID NO: 133) 136B4 KSSQSVLYSSNQKNYLA WASTRES HQYLSSYT (SEQ ID NO: 88) (SEQ ID NO: 111) (SEQ ID NO: 134) 135E10 RASESVDNSGVSFLT AASNQGS QQTKEVPWT (SEQ ID NO: 89) (SEQ ID NO: 112) (SEQ ID NO: 135) 140G5 KASQSVSDDVS SAFFRYP QQDYSSPLT (SEQ ID NO: 90) (SEQ ID NO: 113) (SEQ ID NO: 136) 122H2 KSSQSLENSGTRKNYLA WASTRES MQSFNLRT (SEQ ID NO: 91) (SEQ ID NO: 114) (SEQ ID NO: 137) 139F11 RTSGNIHNYLA NVKTLTD QQFWSIPWT (SEQ ID NO: 92) (SEQ ID NO: 115) (SEQ ID NO: 138)
In addition, any of SEQ ID NOS. 1-69 may be combined with any one or more of SEQ ID NOS. 70-138 into a binding agent. In preferred embodiments, the heavy chain CDRs of each clone are combined with their respective light chain CDRs into a binding agent. In some embodiments, the binding agent may comprise the heavy chain CDRs and light chain CDRs shown below: -
- 122F10 (SEQ ID NOS. 1, 24, 47, 70, 93, and 116);
- 139D6 (SEQ ID NOS. 2, 25, 48, 71, 94, and 117);
- 135D1 (SEQ ID NOS. 3, 26, 49, 72, 95, and 118);
- 134D2 (SEQ ID NOS. 4, 27, 50, 73, 96, and 119);
- 121G1 (SEQ ID NOS. 5, 28, 51, 74, 97, and 120);
- 136B5 (SEQ ID NOS. 6, 29, 52, 75, 98, and 121);
- 127C2 (SEQ ID NOS. 7, 30, 53, 76, 99, and 122);
- 137F2 (SEQ ID NOS. 8, 31, 54, 77, 100, and 123);
- 138H5 (SEQ ID NOS. 9, 32, 55, 78, 101, and 124);
- 140A1 (SEQ ID NOS. 10, 33, 56, 79, 102, and 125);
- 135H12 (SEQ ID NOS. 11, 34, 57, 80, 103, and 126);
- 131D11 (SEQ ID NOS. 12, 35, 58, 81, 104, and 127);
- 132F7 (SEQ ID NOS. 13, 36, 59, 82, 105, and 128);
- 126E4 (SEQ ID NOS. 14, 37, 60, 83, 106, and 129);
- 135G1 (SEQ ID NOS. 15, 38, 61, 84, 107, and 130);
- 136E10 (SEQ ID NOS. 16, 39, 62, 85, 108, and 131);
- 135C12 (SEQ ID NOS. 17, 40, 63, 86, 109, and 132);
- 136F4 (SEQ ID NOS. 18, 41, 64, 87, 110, and 133);
- 136B4 (SEQ ID NOS. 19, 42, 65, 88, 111, and 134);
- 135E10 (SEQ ID NOS. 20, 43, 66, 89, 112, and 135);
- 140G5 (SEQ ID NOS. 21, 44, 67, 90, 113, and 136);
- 122H2 (SEQ ID NOS. 22, 45, 68, 91, 114, and 137); or
- 139F11 (SEQ ID NOS. 23, 46, 69, 92, 115, and 138).
Other combinations may also be useful as may ascertained by one of ordinary skill in the art.
- Binding agents comprising the CDRs of Tables 1A and/or 1B, or those of the immediately preceding paragraph, may also exhibit the following characteristics:
-
TABLE 2 EFRA Antibody competition % relative Affinity* Binding with the PD-1/PD-L1 to peptide Clone (nM) Class** interaction*** stimulation alone‡ 122F10 2.2 4 Non-competitive 146% 139D6 2.4 2 Partial competition 195% 135D1 6.5 2 Partial competition 187% 134D2 4.8 4 Competitive 205% 121G1 11.9 4 Non-competitive 120% 136B5 7.7 4 Competitive 200% 127C2 1.0 2 Non-competitive 100% 137F2 1.5 1 Competitive 250% 138H5 1.6 3 Competitive 210% 140A1 1.4 3 Competitive 160% 135H12 1.9 1 Competitive 190% 131D11 2.7 1 Competitive 180 % 132F7 100 2 Non-competitive 210% 126E4 0.5 4 Competitive 130% 135G1 32 4 NA 138% 136E10 7.1 4 Non-competitive 148% 135C12 1.7 2 Partial competition 195% 136F4 8.3 4 Non-competitive 108% 136B4 1.4 2 Non-competitive 185% 135E10 1.5 3 Competitive 165% 140G5 1.6 1 Competitive 205% 122H2 4.3 1 Competitive 200% 139F11 3.1 1 Competitive 250% *Binding affinity for the antibodies listed in Table 1 was evaluated by FACS staining of endogenous cell surface PD-1 on activated CD4 T cells. **Binding class was determined by Luminex assay competitive binding studies. Binding class 1 mAb clones are competitive with the EH12.2H7 clone commercial antibody,class 2 mAb clones are competitive with the J116 clone commercial antibody,class 3 mAb clones are competitive with both EH12.2H7 and J116 antibodies andclass 4 mAb clones bind in the presence of both EH12.2H7 and J116 antibodies.***Antibody competition with the PD-1/PD-L1 interaction was determined in a second Luminex binding assay. In this assays, PD-1 Fc fusion protein coated beads were incubated in the absence or presence of an anti-PD-1 antibody from Table 2 at a concentration of 20 nM. A fixed concentration of 1.25 nM biotinylated PD-L1, approximately equivalent to the IC50 of the PD-1/PD-L1 interaction, was then incubated with the PD-1/antibody complex and PD-L1 binding was detected by fluorescence with phycoerythrin labeled streptavidin. Based on PD-L1 binding to the PD-1/antibody complex, antibodies were defined as being competitive, partially competitive or non-competitive with the PD-1/PD-L1 interaction. ‡Proliferative effect is evaluated using a CFSE assay (an embodiment of the Exhaustion Functional Recovery Assay, “EFRA”). PBMCs isolated from a chronically infected HIV subject were stimulated with an HIV specific peptide in the presence and absence of an anti-PD-1 antibody. Following a 6 day incubation, proliferation of HIV specific CD8 T cells was evaluated in the anti-PD-1 treated samples relative to the peptide alone control. NA = not available - Binding affinity may be determined by any technique available to those of ordinary skill in the art. The binding affinity data presented in Table 2 was evaluated by flow cytometry staining of endogenous cell surface PD-1 on CD4 T cells that were stimulated for a period of 3 to 6 days with phytohaemagglutinin (PHA). Binding class may also be determined by any technique available to those of ordinary skill in the art. The binding class data presented in Table 2 was determined by Luminex assay competitive binding studies. In Table 2,
binding class 1 mAb antibodies are those determined to be competitive with the EH12.2H7 clone commercial antibody (available from BioLegend, San Diego, Calif. (e.g., Cat. No. 329905));class 2 antibodies are those determined to be competitive with the J116 clone commercial antibody (available from Affymetrix eBioscience, San Diego, Calif. (e.g., Cat. No. 16-9989-80)); andclass 3 antibodies are those determined to be competitive with both EH12.2H7 and J116 antibodies; andclass 4 mAb clone antibodies are those determined to bind PD-1 in the presence of both EH12.2H7 and J116 antibodies. - Proliferative effect may be determined by any technique available to those of ordinary skill in the art. For instance, the EFRA system described above and used in Example 1 may be used. Such an assay was used to determine the proliferative effect data presented in Table 2. Briefly, a carboxyfluorescein succinimidyl ester (CFSE) assay in which peripheral blood mononuclear cells (PBMCs) were isolated from a chronically infected HIV subject and stimulated with an HIV-specific peptide in the presence and absence of an anti-PD-1 antibody. A control anti-PD1 antibody (the Merck antibody MK-3475) was also tested as a positive control. Following a six-day incubation, proliferation of HIV-specific CD8 T cells was evaluated in the anti-PD-1 treated samples relative to the peptide alone control and the result expressed as a percentage above control (“Proliferation effect”).
- In some embodiments, the techniques used to identify and characterize PD-1 binding agents such as antibodies may be combined to provide a system for identifying and characterizing such binding agents. For instance, one or more candidate binding agents such one or more monoclonal antibodies may be assayed by EFRA or a similar assay to determine the ability of the candidate binding agent to restore function to immune cells as measured by, for instance, proliferation in the presence of an immunogenic peptide. In some embodiments, this type of assay may be used as an initial screen to ensure the candidate binding agents to be further studied are capable of restoring immune cell function. In some embodiments, these types of assays may be followed by one for determining the binding affinity to immune cells such as activated peripheral blood mononuclear cells (PBMCs). In some embodiments, this assay may use a technique such as fluorescence activated cell sorting (FACS). In some embodiments, the assay may include the presence or absence of non-specific binding and/or competitive binding studies using known binding reagents such as anti-PD1 antibody (e.g., the Merck antibody MK-3475, also know as pembrolizumab). These assays may then be followed by sequencing of the CDRs of the candidate binding agents such as provided in Tables 1A and/or 1B above. Together, then, the EFRA, affinity determination, epitope mapping studies and CDR identification methods described herein provide a system with which a candidate binding agent may be identified.
- Any of the amino acid sequences of Tables 1A and/or 1B (and/or any one or more fragments and/or derivatives thereof) may be also substituted by any other amino acid as desired by one of ordinary skill in the art. For example, one of skill in the art may make conservative substitutions by replacing particular amino acids with others as shown in Table 3 below. The specific amino acid substitution selected may depend on the location of the site selected. Such conservative amino acid substitutions may involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position and, in particular, does not result in decreased PD-1 binding.
-
TABLE 3 Preferred Conservative Substitution of Original Amino Acid Exemplary Conservative Substitutions of the Original Amino Acid Residues in SEQ ID the Original Amino Acid Residues of Residues of SEQ ID NOS. NOS. 1-138 SEQ ID NOS. 1-138 1-138 Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyric Acid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu - In some embodiments, this disclosure provides binding agents with multiple specificities such that PD-1 and at least one other secondary antigen (e.g., a cell surface protein) may be bound by a single binding agent. In some embodiments, the secondary antigen may be one expressed by cells infected by an infectious agent. For instance, an exemplary secondary antigen may be HIV Env antigen. Such binding agents may bind the secondary antigen and/or may serve to neutralize the infectious agent. In certain embodiments, such as for a bi-specific binding agent having dual specificity for PD-1 and an HIV antigen such as env and/or another antigen, for instance. The HIV immunogen may be derived from any of the subtypes described herein, or any other. In some embodiments, such binding agents may include: PD-1 agonist/Env binding; PD-1 agonist PD-1/Env binding and neutralization; PD-1 antagonist/Env binding; and/or PD-1 antagonist/PD-1/Env binding and neutralization. Given the prevelance of the various subtypes, it may be preferable to select antigens from HIV-1 subtypes B and/or C. It may also be desirable to include binding agents having specificity for antigens from multiple HIV subtypes (e.g., HIV-1 subtypes B and C, HIV-2 subtypes A and B, or a combination of HIV-1 and HIV-2 subtypes) in a single composition. For treating a disease such as cancer, it may be beneficial to obtain binding agents with multiple PD-1 specificities (e.g., bi-specific PD-1a/PD1b antagonist PD-1 antibodies specific to two different epitopes) and/or specificity to both PD-1 and one or more tumor antigens (e.g., cancer-testis (CT) antigen (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigen (i.e., Melan A/MART-1, tyrosinase, gp100); mutational antigen (i.e., MUM-1, p53, CDK-4); overexpressed ‘self’ antigen (i.e., HER-2/neu, p53); and/or viral antigens (i.e., HPV, EBV)). The binding agents (e.g., monoclonal antibodies) may be generated as generally described above. The specificities of such binding agents may be recombined into a single binding agent using techniques that are widely available to those of ordinary skill in the art. In some embodiments, multiple single specifity binding agents may also be combined and used (e.g., administered) to provide an effective multiple specificity reagent.
- In some embodiments, the binding agents described herein may be conjugated to active agents to target and inhibit the function of and/or eliminate cell populations expressing PD-1 (and/or another antigen in the case of binding agents with multiple specificities). For instance, CD4+ T-cell populations containing replication competent HIV may be targeted and eliminated using binding agent/drug conjugates (e.g., antibody-drug conjugates (ADC)). Mono- and/or bi-specific candidate binding agents may be conjugated with one or more types of drugs (e.g., drugs damaging DNA, targeting microtubules). The binding agents described herein and/or derivatives thereof may also be adjoined to and/or conjugated to functional agents for in vitro and/or in vivo use. For instance, the binding agent may be adjoined to and/or conjugated to functional moieties such as cytotoxic drugs or toxins, and/or active fragments thereof such as diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, among others. Suitable functional moieties may also include radiochemicals. Binding agents, such as antibodies, may be adjoined to and/or conjugated to the one or more functional agents using standard techniques in the art.
- In some embodiments, the binding agents may be administered in conjunction with other agents such as anti-infective agents (e.g., antibiotics, anti-viral medications). For instance, the binding agents described herein may be combined with monoclonal antibodies and/or other reagents such as Nivolumab (also known as MDX-1106, BMS-936558 (Topalian, et al. N. Eng. J. Med. 2012; 366(26): 2443-2454), MDX-1106, ONO-4538, a fully human IgG4 mAb available from Bristol-Myers Squibb), Lambrolizumab (also known as MK-3475 and SCH 900475, a humanized IgG4 monoclonal antibody available from Merck), Pidilizumab (a humanized IgG1 monoclonal antibody available from CureTech), AMP-224 (a B7-DC/IgG1 fusion protein available from GlaxoSmithKline/Amplimmune), and/or an antibody or other reagent or method described in any of U.S. Pat. No. 8,354,509B2 (Carven, et al), U.S. Pat. No. 8,008,449B2 (Korman, et al), WO 2012/135408A1 (Manoj, et al.), US 2010/026617 (Carven, et al.), WO 2011/110621A1 (Tyson, et al), U.S. Pat. No. 7,488,802B2 (Collins, et al.), WO 2010/029435A1 (Simon, et al.), WO 2010/089411A2 (Olive, D.), WO 2012/145493A1 (Langermann, et al.), WO 2013/0435569A1 (Rolland, et al.), WO 2011/159877A2 (Kuchroo, et al.), U.S. Pat. No. 7,563,869B2 (Ono Pharm.), U.S. Pat. No. 7,858,746B2 (Honjo, et al.), U.S. Pat. No. 8,728,474B2 (Ono Pharm.), U.S. Pat. No. 9,067,999 (Ono Pharm.), and/or U.S. Pat. No. 9,067,999, each of which is hereby incorporated in its entirety into this disclosure.
- As mentioned above, the PD-1 binding agents described herein (e.g., a PD-1 antagonist) may be used to treat and/or prevent and/or ameliorate the symptoms of infection by HIV. As is well-known in the art, HIV isolates are now classified into discrete genetic subtypes. HIV-1 is known to comprise at least ten subtypes (A1, A2, A3, A4, B, C, D, E, F1, F2, G, H, J and K) (Taylor et al, NEJM, 359(18):1965-1966 (2008)). HIV-2 is known to include at least five subtypes (A, B, C, D, and E). Subtype B has been associated with the HIV epidemic in homosexual men and intravenous drug users worldwide. Most HIV-1 immunogens, laboratory adapted isolates, reagents and mapped epitopes belong to subtype B. In sub-Saharan Africa, India and China, areas where the incidence of new HIV infections is high, HIV-1 subtype B accounts for only a small minority of infections, and subtype HIV-1 C appears to be the most common infecting subtype. Any of these types of isolates may be addressed using the binding agents described herein. One or more binding agents may also be administered with or in conjunction with one or more agents used to prevent, treat and/or ameliorate HIV such as for example, a protease inhibitor, an HIV entry inhibitor, a reverse transcriptase inhibitor, and/or an anti-retroviral nucleoside analog. Suitable compounds include, for example, Agenerase (amprenavir), Combivir (Retrovir/Epivir), Crixivan (indinavir), Emtriva (emtricitabine), Epivir (3tc/lamivudine), Epzicom, Fortovase/Invirase (saquinavir), Fuzeon (enfuvirtide), Hivid (ddc/zalcitabine), Kaletra (lopinavir), Lexiva (Fosamprenavir), Norvir (ritonavir), Rescriptor (delavirdine), Retrovir/AZT (zidovudine), Reyatax (atazanavir, BMS-232632), Sustiva (efavirenz), Trizivir (abacavir/zidovudine/lamivudine), Truvada (Emtricitabine/Tenofovir DF), Videx (ddl/didanosine), Videx EC (ddl, didanosine), Viracept (nevirapine), Viread (tenofovir disoproxil fumarate), Zerit (d4T/stavudine), and Ziagen (abacavir) may be utilized. Other suitable agents are known to those of skill in the art and may be suitable for use as described herein. Such agents may either be used prior to, during, or after administration of the binding agents and/or use of the methods described herein.
- As mentioned above, the PD-1 binding agents described herein (e.g., a PD-1 antagonist) may be used to treat and/or prevent and/or ameliorate the symptoms of cancer. Exemplary cancers may include, for instance, any of the breast, blood, colon, stomach, rectum, skeletal tissue, skin (e.g., melanoma) brain, lung, bladder, kidney, ovary, and/or liver, among others. One or more of the binding agents may also be combined with and/or administered with or in conjunction with one or more agents used to prevent, treat and/or ameliorate cancer such as for example, an alkylating agent (e.g., any nitrogen mustard, nitrosourea, tetrazine, aziridine, cisplatin and/or derivative thereof), anti-metabolite (e.g., any of the methotrexates, pemetrexeds, fluoropyrimidines and/or derivative thereof), anti-microbtubule agent (e.g., vinca alkyloids, taxanes, podophyllotoxin and/or derivative thereof), topoisomerase I and/or II inhibitors (e.g., a camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, aclarubicin and/or derivative thereof) and/or cytotoxic antibiotic (e.g., any anthracyclines, actinomycin, bleomycin, plicamycin, and mitomycin and/or derivative thereof). The one or more binding agents may also, or alternatively, be combined with one or more other binding agents available to those of ordinary skill in the art for treating, preventing and/or ameliorating cancer such as, for example, Nivolumab, Lambrolizumab, Pidilizumab and/or other similar agents and/or derivatives thereof. Other suitable agents are known to those of skill in the art and may be suitable for use as described herein. Such agents may either be used prior to, during, or after administration of the binding agents and/or use of the methods described herein.
- As mentioned above, the PD-1 binding agents described herein (e.g., a PD-1 agonist) may be used to treat and/or prevent and/or ameliorate the symptoms of autoimmunity. Exemplary autoimmune conditions may include, for instance, any in which PD-1 is involved in maintaining self-tolerance and/or one involving inflammatory T cells (e.g., autoreactive or self antigen-specific T cells) such as, for instance, systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, glomerulonephritis, and multiple sclerosis. Such PD-1 binding agents may also be combined with other agents such as anti-CTLA-4 agents (e.g., ipilimumab). One or more of the binding agents may also be combined with and/or administered with or in conjunction with one or more agents used to prevent, treat and/or ameliorate autoimmunity such as, for example, glucocorticoids, cytostatics (e.g., alkylating agent, anti-metabolite, methotrexate, azathioprine, mercaptopurine, cytotoxic antibiotics (e.g., dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin), antibodies (e.g., Atgam, Thymoglobuline, Simulect, Zenapax), drugs acting on immunophilins (e.g., ciclosporin, tacrolimus, sirolimus), interferons, opioids, TNF-binding agents (e.g., Remicade, Enbrel, Humira), mycophenolate, fingolimod, myriocin, and/or derivatives thereof. Other suitable agents are known to those of skill in the art and may be suitable for use as described herein. Such agents may either be used prior to, during, or after administration of the binding agents and/or use of the methods described herein.
- In some embodiments, the binding agents may be adjoined to and/or conjugated to one or more detectable labels. For instance, suitable detectable labels may include, for instance, fluorosceins (e.g., DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); FITC; 6-carboxy-1,4-dichloro-2′,7′-dichlorofluorescein (TET); 6-carboxy-1,4-dichloro-2′,4′,5′,7′-tetra-chlorofluorescein (HEX); 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE); Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores (e.g., 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568, 564/570, 576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP, FI-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE)), rhodamines (e.g., 110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red, Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, TAMRA (6-carboxytetramethyl-rhodamine), Tetramethylrhodamine (TRITC), WT), Texas Red, and/or Texas Red-X. Other detectable labels known in the art may also be suitable for use. Binding agents, such as antibodies, may be adjoined to and/or conjugated to the one or more detectable labels using standard techniques in the art.
- In certain embodiments, a nucleic acid molecule encoding one or more binding agents described herein may be inserted into one or more expression vectors, as discussed below in greater detail. In such embodiments, the binding agent may be encoded by nucleotides corresponding to the amino acid sequence. The particular combinations of nucleotides (codons) that encode the various amino acids (AA) are well known in the art, as described in various references used by those skilled in the art (e.g., Lewin, B. Genes V, Oxford University Press, 1994). The nucleotide sequences encoding the amino acids of said binding agents may be ascertained with reference to Table 4, for example. Nucleic acid variants may use any combination of nucleotides that encode the binding agent.
-
TABLE 4 Codons Encoding Amino Acids (AA) of SEQ ID NOS. 1-138 of Variants Thereof AA Codon AA Codons AA Codons AA Codons Phe (F) TTT Ser (S) TCT Tyr (Y) TAT Cys (C) TGT TTC TCC TAC TGC Leu (L) TTA TCA TERM TAA TERM TGA TTG TCG TAG Trp (W) TGG CTT Pro (P) CCT His (H) CAT Arg (R) CGT CTC CCC CAC CGC CTA CCA Gln (Q) CAA CGA CTG CCG CAG CGG Ile (I) ATT Thr (T) ACT Asn (N) AAT Ser (S) AGT ATC ACC AAC AGC ATA ACA Lys (K) AAA Arg (R) AGA Met (M) ATG ACG AAG AGG Val (V) GTT Ala (A) GCT Asp (D) GAT Gly (G) GGT GTC GCC GAC GGC GTA GCA Glu (E) GAA GGA GTG GCG GAG GGG
Those of ordinary skill in the art understand that the nucleotide sequence encoding a particular amino acid sequence may be easily derived from the amino acid sequence and the information presented in Table 4. For instance, it may be deduced from the amino acid sequence DDFLH (SEQ ID NO.: 1) and the information presented in Table 4 that the amino acid sequence may be encoded by the nucleotide sequence GAT GAT TTT TTA CAT (SEQ ID NO.:139). Those of ordinary skill in the art would understand that nucleotide sequences encoding SEQ ID NOS. 2-138 may be deduced in the same way, and such nucleotide sequences are contemplated herein. Where the binding agents are antibodies, nucleotide sequences encoding the variable regions thereof may also be isolated from the phage and/or hybridoma cells expressing the same cloned into expression vectors to produce certain preparations (e.g., humanized antibodies). Methods for producing such preparations are well-known in the art. - To determine the amino acid sequences of the variable regions (e.g., CDRs) of interest, hybridoma cells from mice immunized with a PD-1 antigen/immunogen may be selected using the functional assays described herein and cloning techniques that are readily available to those of ordinary skill in the art. For instance, to isolate and sequence nucleic acids encoding the heavy and light chain variable regions of the selected hybridomas, total RNA may be extracted from fresh hybridoma cells using TRIzol reagent according to the manufacturer's protocol. cDNA may be synthesized from the RNA using isotype-specific anti-sense primers or universal primers using standard techniques (e.g., following the technical manual of PrimeScript™ 1st Strand cDNA Synthesis Kit). Polymerase chain reaction (PCR) may then be performed to amplify the nucleic acids encoding the variable regions (heavy and light chains) of the antibody produced by the selected hybridoma, which may then be cloned into a standard cloning vector separately and sequenced. Colony PCR screening may then be performed to identify clones with inserts of correct sizes. Preferably, no less than five single colonies with inserts of correct sizes are sequenced for each antibody variable region. Standard protocols may then be used for the expression and purification of the anti-PD-1 antibodies. For instance, hybridoma clones may be grown in serum-free medium and the cell culture broth centrifuged and then filtered. The filtered supernatant containing the antibody may then be loaded onto an affinity column (e.g., Protein A) column, washed and eluted with an appropriate buffer (e.g., Pierce IgG elute buffer). The eluted fractions may then be pooled and buffer-exchanged into PBS, pH 7.2. The purified antibody may then be analyzed by SDS-PAGE and Western blot by using standard protocols for molecular weight, yield and purity. Size exclusion chromatography HPLC may then be performed on an appropriate column (e.g., TSK GEL-G3000 SWXL column (Tosoh)) for biophysical characterization in order to ensure high antibody purity (generally >90%) with low presence of protein aggregates. These procedures were used in isolating and sequencing nucleic acids encoding SEQ ID NOS. 1-138 from selected hybridoma cells. These techniques, variations thereof, and/or other may also be of use for these purposes as would be understood by those of ordinary skill in the art.
- Nucleic acid molecules encoding one or more PD-1 binding agents may be contained within a viral and/or a non-viral vector. In one embodiment, a DNA vector is utilized to deliver nucleic acids encoding one or more PD-1 binding agents to the patient. In doing so, various strategies may be utilized to improve the efficiency of such mechanisms including, for example, the use of self-replicating viral replicons (Caley, et al. 1999. Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al. 2000. Cancer Res. 60: 51-55), codon optimization (Liu, et al. 2000. Mol. Ther., 1: 497-500; Dubensky, supra; Huang, et al. 2001. J. Virol. 75: 4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol. 164: 4635-3640), incorporation of nucleic acids encoding co-stimulatory molecules, cytokines and/or chemokines (Xiang, et al. 1995. Immunity, 2: 129-135; Kim, et al. 1998. Eur. J. Immunol., 28: 1089-1103; Iwasaki, et al. 1997. J. Immunol. 158: 4591-3601; Sheerlinck, et al. 2001. Vaccine, 19: 2647-2656), incorporation of stimulatory motifs such as CpG (Gurunathan, supra; Leitner, supra), sequences for targeting of the endocytic or ubiquitin-processing pathways (Thomson, et al. 1998. J. Virol. 72: 2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373), prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature, 408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al. 2001. Science, 292: 69-74), proteasome-sensitive cleavage sites, and the use of mucosal delivery vectors such as Salmonella (Darji, et al. 1997. Cell, 91: 765-775; Woo, et al. 2001. Vaccine, 19: 2945-2954). Other methods are known in the art, some of which are described below. Various viral vectors that have been successfully utilized for introducing a nucleic acid to a host include retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, and poxvirus, among others. The vectors may be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques may be found in common molecular biology references such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, ca). “Non-viral” plasmid vectors may also be suitable in certain embodiments. Preferred plasmid vectors are compatible with bacterial, insect, and/or mammalian host cells. Such vectors include, for example, PCR-ii, PCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSii (Stratagene, La Jolla, Calif.), pet15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFp-n2 (Clontech, Palo Alto, Calif.), pETI (Bluebacii, Invitrogen), pDSR-alpha (PCT pub. No. WO 90/14363) and pFASTBACdual (Gibco-BRL, Grand island, NY) as well as Bluescript® plasmid derivatives (a high copy number COLe1-based phagemid, Stratagene Cloning Systems, La Jolla, Calif.), PCR cloning plasmids designed for cloning TAQ-amplified PCR products (e.g., TOPO™ TA Cloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, Calif.). Bacterial vectors may also be used. These vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille Calmette Guérin (BCG), and Streptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376). Many other non-viral plasmid expression vectors and systems are known in the art and may be use. Other delivery techniques may also suffice including, for example, DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO4 precipitation, gene gun techniques, electroporation, and colloidal dispersion systems. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., et al., 1981, Trends Biochem. Sci., 6: 77). The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
- A cultured cell comprising the vector is also provided. The cultured cell may be a cultured cell transfected with the vector or a progeny of the cell, wherein the cell expresses the immunogenic polypeptide. Suitable cell lines are known to those of skill in the art and are commercially available, for example, through the American Type Culture Collection (ATCC). The transfected cells can be used in a method of producing an immunogenic polypeptide. The method comprises culturing a cell comprising the vector under conditions that allow expression of the immunogenic polypeptide, optionally under the control of an expression sequence. The immunogenic polypeptide can be isolated from the cell or the culture medium using standard protein purification methods.
- The skilled artisan has many suitable techniques for using the binding agents (e.g., antibodies) described herein to identify biological samples containing proteins that bind thereto. For instance, antibodies may be utilized to isolate PD-1 using, for example, immunoprecipitation or other capture-type assay. This well-known technique is performed by attaching the antibody to a solid support or chromatographic material (e.g., a bead coated with Protein A, Protein G and/or Protein L). The bound antibody is then introduced into a solution either containing or believed to contain the PD-1 (e.g., an HIV-infected T cell lysate). PD-1 may then bind to the antibody and non-binding materials are washed away under conditions in which the PD-1 remains bound to the antibody. The bound protein may then be separated from the antibody and analyzed as desired. Similar methods for isolating a protein using an antibody are well-known in the art. The binding agents (e.g., antibodies) may also be utilized to detect PD-1 within a biological sample. For instance, the antibodies may be used in assays such as, for example, flow cytometric analysis, ELISA, immunoblotting (e.g., western blot), in situ detection, immunocytochemistry, and/or immunhistochemistry. Methods of carrying out such assays are well-known in the art.
- The binding agents described herein may be also be used to determine the presence of a disease state in a patient, to predict prognosis, or to determine the effectiveness of a chemotherapeutic or other treatment regimen. Expression profile assays, performed as described herein or as is otherwise known in the art, may be used to determine the relative level of expression of PD-1. The level of expression may then be correlated with base (e.g., control) levels to determine whether a particular disease is present within the patient, the patient's prognosis, or whether a particular treatment regimen is effective. For example, if the patient is being treated with a particular anti-infective regimen, an increased or decreased level of expression of PD-1 in the patient's tissues (e.g., in peripheral blood, breast tissue biopsy) may indicate the regimen is worsening or improving the load of the infectious agent in that host. The increase or decrease in expression may indicate the regimen is having or not having the desired effect and another therapeutic modality may therefore be selected.
- It is also possible to use the binding agents described herein as reagents in drug screening assays to test, for example, new drug candidates. The reagents may be used to ascertain the effect of a drug candidate on the expression of the immunogenic target in a cell line, or a cell or tissue of a patient. The expression profiling technique may be combined with high throughput screening techniques to allow rapid identification of useful compounds and monitor the effectiveness of treatment with a drug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8 (1998)). Drug candidates may be chemical compounds, nucleic acids, proteins, antibodies, or derivatives therefrom, whether naturally occurring or synthetically derived. Drug candidates thus identified may be utilized, among other uses, as pharmaceutical compositions for administration to patients or for use in further screening assays.
- In some embodiments, the binding agents are in purified form. A “purified” binding agent (e.g., antibody) may be one that is separated from at least about 50% of the proteins and/or other components with which it is initially found (e.g., as part of a hybridoma supernatant or ascites preparation in the case of a monoclonal antibody). A purified binding agent (e.g., antibody) may be one that is separated from at least about 50%, 60%, 75%, 90%, or 95% of the proteins and/or other components with which it is initially found.
- The polypeptides and nucleic acids described herein may be combined with one or more pharmaceutically acceptable carriers prior to administration to a host. A pharmaceutically acceptable carrier is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Suitable pharmaceutical carriers and their formulations are described in, for example, Remington's: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally from about 5 to about 8 or from about 7 to about 7.5. Other carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing polypeptides or fragments thereof. Matrices may be in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of polypeptides and/or fragments thereof to humans or other subjects. Pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives, surface active agents, adjuvants, immunostimulants, in addition to the immunogenic polypeptide. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents and anesthetics. The pharmaceutical composition may be administered orally, parentally, by inhalation spray, rectally, intranodally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a nucleic acid, polypeptide, or peptide as a pharmaceutical composition. A “pharmaceutical composition” is a composition comprising a therapeutically effective amount of a nucleic acid or polypeptide. The terms “effective amount” and “therapeutically effective amount” each refer to the amount of a binding agent, nucleic acid or the like used to observe the desired therapeutic effect (e.g., restore T cell function).
- Methods for treating one or more disease conditions (e.g., HIV or cancer) in a mammalian host comprising administering to the mammal at least one or more effective doses of one or more binding agents (and/or derivative(s) thereof) described herein are also provided. In some embodiments, the binding agent is a monoclonal antibody or fragment or derivative thereof comprising one or more of SEQ ID NOS. 1-138 and/or shown in Tables 1A and 1B. The one or more binding agents may be administered in a dosage amount of about 1 to about 50 mg/kg, about 1 to about 30 mg/kg, or about 5 to about 30 mg/kg (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 mg/kg). In certain embodiments, the one or more binding agents may be administered to the mammal (e.g., intradermally, intravenously, orally, rectally) at about 10 mg/kg one or more times. When multiple doses are administered, the doses may comprise about the same or different amount of binding agent in each dose. The doses may also be separated in time from one another by the same or different intervals. For instance, the doses may be separated by about any of 6, 12, 24, 36, 48, 60, 72, 84, or 96 hours, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 3 years, 4 years, 5 years, or any time period before, after, and/or between any of these time periods. In some embodiments, the binding agents may be administered in conjunction with other agents (e.g., anti-infective agents and/or chemotherapeutic agent). Such other agents may be administered about simultaneously with the binding agents, or at a different time and/or frequency. Other embodiments of such methods may also be appropriate as could be readily determined by one of ordinary skill in the art.
- To assist the skilled artisan in using the antibodies described herein, the same may be provided in kit format. A kit including such antibodies and optionally other components necessary for using the antibodies to detect cells expressing PD-1 is provided. The antibodies of the kit may be provided in any suitable form, including frozen, lyophilized, or in a pharmaceutically acceptable buffer such as TBS or PBS. The kit may also include other reagents required for utilization of the antibodies in vitro or in vivo such as buffers (e.g., TBS, PBS), blocking agents (solutions including nonfat dry milk, normal sera, Tween-20 Detergent, BSA, or casein), and/or detection reagents (e.g., goat anti-mouse IgG biotin, streptavidin-HRP conjugates, allophycocyanin, B-phycoerythrin, R-phycoerythrin, peroxidase, detectable labels, and other labels and/or staining kits (e.g., ABC Staining Kit, Pierce)). The kits may also include other reagents and/or instructions for using the antibodies in commonly utilized assays described above such as, for example, flow cytometric analysis, ELISA, immunoblotting (e.g., western blot), in situ detection, immunocytochemistry, immunhistochemistry. In one embodiment, the kit provides a binding agent in purified form. In another embodiment, the binding agent may be provided in biotinylated form either alone or along with an avidin-conjugated detection reagent (e.g., antibody). In another embodiment, the kit includes a binding agents comprising one or more detectable labels that may be used to directly detect PD-1. Buffers and the like required for using any of these systems are well-known in the art and/or may be prepared by the end-user or provided as a component of the kit. The kit may also include a solid support containing positive- and negative-control protein and/or tissue samples. For example, kits for performing spotting or western blot-type assays may include control cell or tissue lysates for use in SDS-PAGE or nylon or other membranes containing pre-fixed control samples with additional space for experimental samples. Kits for visualization of PD-1 in cells on slides may include pre-formatted slides containing control cell or tissue samples with additional space for experimental samples. Other embodiments of kits are also contemplated herein as would be understood by those of ordinary skill in the art.
- Thus, this disclosure provides a binding agent that binds PD-1 agonistically or antagonistically. In some embodiments, the binding agent is a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOS. 1-138 and/or shown in Tables 1A and 1B. In some embodiments, the binding agent is a polypeptide comprising one or more combinations of SEQ ID NOS. 1-138 (e.g., as shown in Tables 1A and/or 1B). In some embodiments, the binding agent is an antibody. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR1 amino acid sequence selected from the group consisting of SEQ ID NOS. 1-23. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR2 amino acid sequence selected from the group consisting of SEQ ID NOS. 24-46. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR3 amino acid sequence selected from the group consisting of 47-69. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a light chain CDR1 amino acid sequence selected from the group consisting of SEQ ID NOS. 70-92. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR2 amino acid sequence selected from the group consisting of SEQ ID NOS. 93-115. In some embodiments, the binding agent is a polypeptide such as an antibody comprising a heavy chain CDR3 amino acid sequence selected from the group consisting of 116-138. In some embodiments, the binding agent comprises the combinations of CDRs shown in Tables 1A and/or 1B and/or has the properties described in Table 2. In some embodiments, the binding agent is derived from or related to (e.g., by sequence or derivation) a human antibody, human IgG, human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgA1, human IgA2, human IgD, human IgE, canine antibody, canine IgGA, canine IgGB, canine IgGC, canine IgGD, chicken antibody, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, goat antibody, goat IgG, mouse antibody, mouse IgG, pig antibody, and/or rat antibody, and/or a derivative thereof. In some embodiments, the derivative may be selected from the group consisting of an Fab, Fab2, Fab′ single chain antibody, Fv, single chain, mono-specific antibody, bispecific antibody, trimeric antibody, multi-specific antibody, multivalent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized antibody, CDR-grafted antibody, shark antibody, nanobody, and/or canelid antibody. In some embodiments, the binding agent comprises at least a least a first and second specificity, the first being against PD-1 and the second being against a different antigen (e.g., an antigen of an infectious agent such as HIV (e.g., env) and/or a tumor antigen). In some embodiments, the binding agent and/or derivative thereof may comprise a detectable label fixably attached thereto. In some embodiments, the binding agent of any one and/or derivative thereof comprises an effector moiety (e.g., a cytotoxic drug, toxin, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and radiochemical) fixably attached thereto. In some embodiments, polynucleotides encoding one or more binding agents are also provided (e.g., as an expression vector). Host cells comprising and/or expressing the polypeptide products of such polynucleotides are also provided. In some embodiments, compositions comprising at least one binding agent or derivative; at least one isolated polynucleotide; at least one expression vector; and/or, at least one host cell; or a combination thereof; and, a pharmaceutically acceptable carrier are also provided.
- This disclosure also provides methods for detecting PD-1 on a cell, the method comprising contacting a test biological sample with a binding agent or derivative described herein and detecting the binding agent bound to the biological sample or components thereof. Such methods may be an in vivo method or an in vitro method. In some embodiments, the method may comprise comparing the amount of binding to the test biological sample or components thereof to the amount of binding to a control biological sample or components thereof, wherein increased binding to the test biological sample or components thereof relative to the control biological sample or components thereof indicates the presence of a cell expressing PD-1 in the test biological sample (e.g., mammalian blood). In some embodiments, a system for identifying a PD-1 antibody binding agent by assaying the candidate binding agent by the exhaustion functional recovery assay (EFRA); determining the affinity of the candidate binding agent for PD-1; and, determining the nucleotide sequence of the CDR of the candidate binding agent is provided.
- In some embodiments, a kit for detecting the expression of PD-1 in or on a cell, the kit comprising a binding agent or derivative thereof and instructions for use. In some embodiments, the binding agent and/or derivative thereof is in lyophilized form.
- In some embodiments, this disclosure provides methods for treating, preventing and/or ameliorating an infectious disease, cancer and/or autoimmunity in a mammal comprising administering to the mammal at least one effective dose of a pharmaceutical composition comprising a binding agent or derivative thereof. In some embodiments, the infectious disease is human immunodeficiency virus (HIV). In some embodiments, the binding agent and/or derivative thereof used to treat infectious disease and/or cancer is a PD-1 antagonist. In some embodiments, the binding agent and/or derivative thereof used to treat an autoimmune condition is a PD-1 agonist. In some embodiments, multiple doses are administered to the animal. In some embodiments, the binding agent and/or derivative thereof may be administered in a dosage amount of about 1 to 50 mg/kg.
- This disclosure also provides combinations of PD-1 binding agents. In some embodiments, the combination comprises a first binding agent that blocks the interaction of PD-1 and PD-L1 and a second binding agent that does not block the interaction of PD-1 and PD-L1. Such combinations may be used for any use described herein or as may be otherwise ascertained by those of ordinary skill in the art. For instance, such combinations may be used in the methods for treating, preventing and/or ameliorating an infectious disease, cancer and/or autoimmunity in a mammal described herein.
- This disclosure also provides methods for producing the binding agents described herein by expressing the binding agent in a cell and isolating the binding agent from the cell or a culture supernatant of the cell. In some embodiments, such methods may further comprise expressing a nucleic acid encoding such binding agent(s). In some embodiments, such methods may also include combining the binding agent(s) following isolation with one or more pharmaceutically acceptable excipients.
- Methods for producing a combination(s) of binding agents, such as a first binding agent that blocks the interaction of PD-1 and PD-L1 and a second binding agent that does not block the interaction of PD-1 and PD-L1, are also provided by this disclosure. In some embodiments, the second binding agent binds PD-1. In some embodiments, the first and/or second binding agents are antibodies such monoclonal antibodies or fragments or derivatives thereof. In some embodiments, the second binding agent comprises the amino acid sequences SEQ ID NOS. 2, 25, 48, 71, 94 and 117; or SEQ ID NOS. 17, 40, 63, 86, 109, and 132. In some embodiments, these methods may further include the addition of a pharmaceutically acceptable excipient.
- The terms “about”, “approximately”, and the like, when preceding a list of numerical values or range, refer to each individual value in the list or range independently as if each individual value in the list or range was immediately preceded by that term. The terms mean that the values to which the same refer are exactly, close to, or similar thereto.
- As used herein, a subject or a host is meant to be an individual. The subject can include domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs) and birds. In one aspect, the subject is a mammal such as a primate or a human.
- Optional or optionally means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase optionally the composition can comprise a combination means that the composition may comprise a combination of different molecules or may not include a combination such that the description includes both the combination and the absence of the combination (i.e., individual members of the combination).
- Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximately, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Ranges (e.g., 90-100%) are meant to include the range per se as well as each independent value within the range as if each value was individually listed.
- The term “combined” or “in combination” or “in conjunction” may refer to a physical combination of agents that are administered together or the use of two or more agents in a regimen (e.g., administered separately, physically and/or in time) for treating, preventing and/or ameliorating a particular disease.
- When the terms treat, prevent, and/or ameliorate or derivatives thereof are used herein in connection with a given treatment for a given condition (e.g., preventing cancer infection by HIV), it is meant to convey that the treated patient either does not develop a clinically observable level of the condition at all, or develops it more slowly and/or to a lesser degree than he/she would have absent the treatment. These terms are not limited solely to a situation in which the patient experiences no aspect of the condition whatsoever. For example, a treatment will be said to have prevented the condition if it is given during exposure of a patient to a stimulus that would have been expected to produce a given manifestation of the condition, and results in the patient's experiencing fewer and/or milder symptoms of the condition than otherwise expected. For instance, a treatment can “prevent” infection by resulting in the patient's displaying only mild overt symptoms of the infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism.
- Similarly, reduce, reducing, and reduction as used herein in connection with prevention, treatment and/or amelioration of a given condition by a particular treatment typically refers to a subject developing an infection more slowly or to a lesser degree as compared to a control or basal level of developing an infection in the absence of a treatment (e.g., administration of one or more PD-1 binding agents). A reduction in the risk of infection may result in the patient's displaying only mild overt symptoms of the infection or delayed symptoms of infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism.
- All references cited within this disclosure are hereby incorporated by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.
- Four mice strains (total of 16 mice) have been immunized with two PD-1 proteins, i.e. human PD-1 Fc fusion protein and a human PD-1 monomeric protein. Mice showing serum reactivity to PD-1 expressed on activated human T lymphocytes have been selected for generation of anti-PD-1 hybridoma cell lines. A total of 240 PD-1 hybridoma cell lines were selected for producing antibodies that bind to recombinant PD-1 protein. The primary criteria for the first round of antibodies selection were: i) staining of PD-1 on activated human T lymphocytes by flow cytometry; ii) diversity of CDR VH and VL sequences as compared to those of the existing anti-PD-1 antibodies; and, iii) epitope mapping performed by competitive binding studies with PD-1 conjugated Luminex beads with two commercially available anti-PD-1 antibodies binding to different epitopes on PD-1. A second round of selection was then carried out by: iv) affinity binding assays (not a primary criteria as it does not correlate with the stimulatory potential of anti-PD-1 antibodies); v) evaluation of anti-PD-1 antibodies that bind PD-1 and are either competitive, partially competitive or non-competitive with the binding of PD-L1 in a Luminex biochemical assay; and, vi) functional characterization of antibodies as agonist (not able to restore T-cells from functional exhaustion) or antagonist (able to restore T-cells from functional exhaustion). In these studies, the antibodies were tested and differentiated based on their ability to rescue proliferation in HIV-specific exhausted CD8 T-cells.
- In a primary screen of antibody supernatants from individual cell cultured hybridoma cell clones, the EFRA assay was carried out to evaluate the functional effect of anti-PD1 antibodies on the proliferation of HIV-specific CD8 T cells (
FIG. 2 ). Antibody clones in the upper box (E8-3, C2-3, E1-3, F3-3, H8-3, C10-2, G2-1, G3-2, H2-1, and H4-2) act as PD-1 antagonists and stimulate proliferation while antibody clones in the lower box (C8-1 and G10-2) are agonistic and promote the PD-1 negative regulatory effect. The level of proliferation induced by the peptide control (Pep 8) is indicated by the lower horizontal line (just below 1%) and the induced proliferation by the Merck MK-3475 anti-PD1 antibody is shown in the upper horizontal line (just above 2%). Antibodies of interest identified by these processes underwent a second round of subcloning and the resulting hybridoma clones were used for the production and purification of the antibodies in Table 2. Binding assays were carried out with the purified anti-PD-1 antibodies to ensure that the subclones retained their affinity for PD-1. The concentration response binding of anti-PD-1 antibodies to cell surface PD-1 was evaluated on activated CD4 T-cells (FIG. 3 ). - The EFRA provides for the selection of binding agents that restore T-cells from functional exhaustion but are not necessarily antagonistic, meaning those binding agents that do not necessarily interfere with the interaction between PD-1 and its biological ligand(s) (e.g., PD-L1 or PD-L2). An embodiment of the EFRA was to identify such binding agents (antibodies) that bind PD-1. Epitope mapping of antibody binding to PD-1 was performed with two separate biochemical assays. In one assay, competitive binding to PD-1 Fc fusion protein labeled beads was evaluated between one of two commercial anti-PD-1 antibodies (BMS-5C4 and MK3475) and the anti-PD-1 antibodies listed (also described in Table 2). Four classes of monoclonal antibodies binding to distinct epitopes were identified based on this assay. These are: class 1 (competitive with the EH12.2H7 commercial monoclonal antibody clone that blocks the interaction of PD-1 with PD-L1), class 2 (competitive with the J116 commercial monoclonal antibody clone that binds PD-1 but does not effectively block the interaction of PD-1 with PD-L1), class 3 (competitive with both the EH12.2H7 and J116 commercial monoclonal antibodies), and class 4 (non-competitive with either the EH12.2H7 or J116 commercial antibodies). The relative binding of these antibodies for cell surface PD-1 is represented by the mean fluorescence intensity (MFI) relative to a control anti-PD-1 antibody in
FIG. 4 . These results show that tight binding antibodies were identified from all four binding classes. A second Luminex binding assay was used to directly evaluate if an anti-PD-1 antibody blocks the interaction between PD-1 and PD-L1. This assay was carried out using PD-1 Fc fusion protein coated beads that were incubated in the absence or presence of different concentrations of the anti-PD-1 antibodies listed in Table 2. A fixed concentration of biotinylated PD-L1, approximately equivalent to the IC50 of the PD-1/PD-L1 interaction, was then incubated with the PD-1/antibody complex and PD-L1 binding was detected using fluorescently labeled streptavidin (representative binding curves shown inFIG. 5a-c ). Using this biochemical assay, antibodies were defined as being competitive, partially competitive or non-competitive with the PD-1/PD-L1 interaction. The competitive, partially competitive and non-competitive antibodies were identified which bind distinct sites on PD-1 and exhibit high binding affinity (Table 2). Further evaluation of antibodies that bind PD-1 and are non-competitive or partially competitive with PD-L1 were performed by incubating PD-1 Fc fusion protein coated Luminex beads with 20 nM of an anti-PD-1 antibody from Table 2 followed by an incubation of the PD-1/antibody complex with different concentrations of biotinylated PD-L1.FIG. 5d-f shows that a completive anti-PD-1 antibody completely blocks the interaction between PD-1 and PD-L1. Partially competitive and non-competitive anti-PD-1 antibodies listed in Table 2 results in a shift in the binding affinity of PD-L1 for PD-1, but does not block the interaction when concentration of PD-L1 are increased. Some of these antibodies were also shown to exhibit statistically significant increases in proliferation relative to controls (peptide 8 alone control or the peptide and IgG1 isotype control antibody inFIGS. 6a and 6b ).Class 1 antibodies (competitive with the EH12.2H7 commercial monoclonal antibody that blocks the interaction of PD-1 with PD-L1) or competitive in the PD-1/PD-L1 interaction assay were generally determined to provide improved proliferative restoration. In combining the data from multiple EFRA experiments and using MK-3475 as the common comparator,FIG. 7 shows that select antibodies described in Table 2 exhibited equivalent or statistically improved activity (p<0.007) compared to the benchmark MK-3475 antibody. - Combinations of antibodies binding to different PD-1 epitopes were found to enhance restoration of HIV peptide specific CD8 T-cell proliferation in a functional exhaustion recovery assay (
FIG. 8 ). Synergy between antibody types was also observed. For instance, it was determined that class 1 (MK-3475 inFIG. 8 ) and class 2 (139D6 inFIG. 8 ) antibodies are able to simultaneously bind PD-1. While the maximum stimulation observed for MK-3475 is consistently about 200% relative to the HIV peptide, combinations of MK-3475 and 139D6 monoclonal antibodies at 5 pg/ml exhibited synergy with a 288% increase in HIV-specific CD8 T cell proliferation relative to the HIV peptide control alone or a 144% increased proliferation relative to MK-3475 or 139D6 added alone (FIG. 8 ). This synergistic increase in proliferation was observed in several experimental tests with a statistically significant p value of 0.007. As a comparison, addition of 10 pg/ml of either MK-3475 or 139D6 alone did not result in an increased proliferation in the EFRA. Thus, the combination of a first binding agent that blocks the interaction of PD-1 and PD-L1 with a second binding agent that does not block the interaction of PD-1 and PD-L1 has been determined to act synergistically to rescue T cells from exhaustion. - While certain embodiments have been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the following claims.
-
- 1. Chun T W et al. Nature 387: 183-188, 1997
- 2. Chun T W et al. Proc Natl Acad Sci USA 94: 13193-13197, 1997
- 3. Finzi D et al. Science 278: 1295-1300, 1997
- 4. Chomont N et al. Nat Med 15:893-900, 2009
- 5. Siliciano J D et al. Nat Med 9:727-728, 203
- 6. Perreau M et al. J Exp Med 210: 143-156, 2013
- 7. Rong L and Perelson A J Theoret Biol 260:308-331, 2009
- 8. Sigal A et al. Nature 477:95-98, 2011
- 9. Katlama C et al. Lancet 381:2109-2117, 2013
- 10. Hansen S G et al. Nature 503:100-106, 2013
- 11. Klein F et al. Nature 492: 518.522, 2012
- 12. Barouch D H et al. Nature 503:224-228, 2013
- 13. Trautmann L et al. Nat Med 12:1198-1202, 2006
- 14. Day C L et al. Nature 443:350-354, 2006
- 15. Archin N M et al. Nature 487:482-485, 2012
- 16. Sievers E L and Senter P D Annu Rev Med. 64:15-29, 2013
- 17. Zolot R S et al. Nat Rev Drug Discov 4:259-260, 2013
Claims (3)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/485,752 US20220169733A1 (en) | 2014-08-05 | 2021-09-27 | Immunological Reagents |
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201462033177P | 2014-08-05 | 2014-08-05 | |
US201462053366P | 2014-09-22 | 2014-09-22 | |
US201462093368P | 2014-12-17 | 2014-12-17 | |
PCT/IB2015/055943 WO2016020856A2 (en) | 2014-08-05 | 2015-08-05 | Immunological reagents |
US201715329760A | 2017-01-27 | 2017-01-27 | |
US15/981,977 US11130807B2 (en) | 2014-08-05 | 2018-05-17 | Immunological reagents |
US17/485,752 US20220169733A1 (en) | 2014-08-05 | 2021-09-27 | Immunological Reagents |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/981,977 Continuation US11130807B2 (en) | 2014-08-05 | 2018-05-17 | Immunological reagents |
Publications (1)
Publication Number | Publication Date |
---|---|
US20220169733A1 true US20220169733A1 (en) | 2022-06-02 |
Family
ID=53836144
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/329,760 Active 2035-08-18 US9982053B2 (en) | 2014-08-05 | 2015-08-05 | Immunological reagents |
US15/981,977 Active 2036-03-22 US11130807B2 (en) | 2014-08-05 | 2018-05-17 | Immunological reagents |
US17/485,752 Abandoned US20220169733A1 (en) | 2014-08-05 | 2021-09-27 | Immunological Reagents |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/329,760 Active 2035-08-18 US9982053B2 (en) | 2014-08-05 | 2015-08-05 | Immunological reagents |
US15/981,977 Active 2036-03-22 US11130807B2 (en) | 2014-08-05 | 2018-05-17 | Immunological reagents |
Country Status (13)
Country | Link |
---|---|
US (3) | US9982053B2 (en) |
EP (1) | EP3177644B1 (en) |
JP (1) | JP6629321B2 (en) |
KR (1) | KR102357893B1 (en) |
CN (1) | CN107074947B (en) |
AU (1) | AU2015298356B2 (en) |
CA (1) | CA2957258C (en) |
DK (1) | DK3177644T3 (en) |
ES (1) | ES2847311T3 (en) |
PL (1) | PL3177644T3 (en) |
PT (1) | PT3177644T (en) |
SG (1) | SG11201700672YA (en) |
WO (1) | WO2016020856A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20220259315A1 (en) * | 2016-01-22 | 2022-08-18 | MabQuest SA | Immunological Reagents |
Families Citing this family (103)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4169951A1 (en) | 2008-12-09 | 2023-04-26 | F. Hoffmann-La Roche AG | Anti-pd-l1 antibodies and their use to enhance t-cell function |
DK2992017T3 (en) | 2013-05-02 | 2021-01-25 | Anaptysbio Inc | ANTIBODIES AGAINST PROGRAMMED DEATH-1 (PD-1) |
TWI693232B (en) | 2014-06-26 | 2020-05-11 | 美商宏觀基因股份有限公司 | Covalently bonded diabodies having immunoreactivity with pd-1 and lag-3, and methods of use thereof |
US9982052B2 (en) * | 2014-08-05 | 2018-05-29 | MabQuest, SA | Immunological reagents |
DK3177644T3 (en) * | 2014-08-05 | 2021-01-11 | MabQuest SA | Immunological reagents that bind to PD-1 |
CN113603784A (en) | 2015-05-29 | 2021-11-05 | 艾吉纳斯公司 | anti-CTLA-4 antibodies and methods of use thereof |
TWI773646B (en) | 2015-06-08 | 2022-08-11 | 美商宏觀基因股份有限公司 | Lag-3-binding molecules and methods of use thereof |
CN108368170B (en) | 2015-07-13 | 2022-04-15 | 西托姆克斯治疗公司 | anti-PD-1 antibodies, activatable anti-PD-1 antibodies, and methods of use thereof |
SI3456346T1 (en) | 2015-07-30 | 2021-11-30 | Macrogenics, Inc. | Pd-1 and lag-3 binding molecules and methods of use thereof |
EP3355920A4 (en) | 2015-09-29 | 2019-05-15 | Celgene Corporation | Pd-1 binding proteins and methods of use thereof |
JP2019500893A (en) | 2015-11-03 | 2019-01-17 | ヤンセン バイオテツク,インコーポレーテツド | Antibodies that specifically bind to PD-1 and TIM-3 and uses thereof |
GEP20217328B (en) | 2015-12-14 | 2021-12-10 | Macrogenics Inc | Bispecific molecules having immunoreactivity with pd-1 and ctla-4, and methods of use thereof |
WO2017125815A2 (en) | 2016-01-22 | 2017-07-27 | MabQuest SA | Immunological reagents |
CN106008714B (en) * | 2016-05-24 | 2019-03-15 | 瑞阳(苏州)生物科技有限公司 | Anti-human PD-1 Humanized monoclonal antibodies and its application |
BR112018074463A2 (en) | 2016-05-27 | 2019-03-06 | Agenus Inc. | anti-tim-3 antibodies and methods of use. |
MX2018015592A (en) * | 2016-06-14 | 2019-04-24 | Xencor Inc | Bispecific checkpoint inhibitor antibodies. |
CN109952317A (en) | 2016-09-19 | 2019-06-28 | 细胞基因公司 | Use the method for PD-1 binding protein treatment immune disorders |
US10766958B2 (en) | 2016-09-19 | 2020-09-08 | Celgene Corporation | Methods of treating vitiligo using PD-1 binding antibodies |
CN110023337B (en) | 2016-10-11 | 2024-01-05 | 艾吉纳斯公司 | anti-LAG-3 antibodies and methods of use thereof |
BR112019008859A2 (en) | 2016-11-01 | 2019-07-09 | Anaptysbio Inc | antibodies directed against programmed death 1 (pd-1) |
HRP20211703T1 (en) * | 2016-11-02 | 2022-02-04 | Jounce Therapeutics, Inc. | Antibodies to pd-1 and uses thereof |
US11013802B2 (en) | 2016-12-07 | 2021-05-25 | Agenus Inc. | Anti-CTLA-4 antibodies and methods of use thereof |
KR102603681B1 (en) | 2016-12-07 | 2023-11-17 | 아게누스 인코포레이티드 | Antibodies and methods of using them |
CN110167966A (en) | 2017-01-06 | 2019-08-23 | 克雷森多生物制剂有限公司 | The single domain antibody of apoptosis (PD-1) |
CN110382545A (en) | 2017-01-09 | 2019-10-25 | 泰萨罗公司 | With the method for anti-PD-1 antibodies for treating cancer |
WO2018132739A2 (en) | 2017-01-13 | 2018-07-19 | Agenus Inc. | T cell receptors that bind to ny-eso-1 and methods of use thereof |
CN107474135B (en) * | 2017-02-17 | 2020-08-18 | 广西医科大学 | anti-PD-1 nano antibody PD-1/Nb20 and preparation method and application thereof |
US11161905B2 (en) | 2017-03-04 | 2021-11-02 | Xiangtan Tenghua Bioscience | Recombinant antibodies to programmed death 1 (PD-1) and uses thereof |
BR112019018915A2 (en) | 2017-03-15 | 2020-04-14 | Pandion Therapeutics Inc | targeted immunotolerance |
BR112019017241A2 (en) | 2017-04-13 | 2020-04-14 | Agenus Inc | anti-cd137 antibodies and methods of using them |
CN106939049B (en) * | 2017-04-20 | 2019-10-01 | 苏州思坦维生物技术股份有限公司 | The monoclonal antibody and the preparation method and application thereof of antagonism inhibition people PD-1 antigen and its ligand binding |
DK3618863T3 (en) | 2017-05-01 | 2023-08-21 | Agenus Inc | ANTI-TIGIT ANTIBODIES AND METHODS OF USING THEREOF |
MX2019013517A (en) | 2017-05-24 | 2020-08-17 | Pandion Operations Inc | Targeted immunotolerance. |
AU2018282094B2 (en) | 2017-06-05 | 2024-06-27 | Janssen Biotech, Inc. | Antibodies that specifically bind PD-1 and methods of use |
EP3679062A1 (en) | 2017-09-04 | 2020-07-15 | Agenus Inc. | T cell receptors that bind to mixed lineage leukemia (mll)-specific phosphopeptides and methods of use thereof |
MX2020002612A (en) | 2017-09-07 | 2020-07-13 | Univ Res Inst Inc Augusta | Antibodies to programmed cell death protein 1. |
CN108218988B (en) * | 2017-11-29 | 2019-10-11 | 广西医科大学 | Nano antibody PD-1/Nb52 of anti-PD-1 and the preparation method and application thereof |
US10946068B2 (en) | 2017-12-06 | 2021-03-16 | Pandion Operations, Inc. | IL-2 muteins and uses thereof |
US10174092B1 (en) | 2017-12-06 | 2019-01-08 | Pandion Therapeutics, Inc. | IL-2 muteins |
GB201802573D0 (en) | 2018-02-16 | 2018-04-04 | Crescendo Biologics Ltd | Therapeutic molecules that bind to LAG3 |
AR114127A1 (en) * | 2018-03-02 | 2020-07-22 | Lilly Co Eli | AGONIST ANTIBODIES AGAINST PD-1 AND USES OF THEM |
EP3768713A2 (en) * | 2018-03-22 | 2021-01-27 | Keires AG | Antagonistic pd-1, pd-l1 and lag-3 binding proteins |
CN112218883A (en) | 2018-04-26 | 2021-01-12 | 艾吉纳斯公司 | Heat shock protein-binding peptide compositions and methods of use thereof |
CN110404066B (en) * | 2018-04-28 | 2022-06-17 | 齐鲁制药有限公司 | Monoclonal antibody preparation for resisting human PD-1, combined medicament and application thereof |
CN108840932B (en) * | 2018-04-28 | 2022-03-29 | 中国科学院微生物研究所 | PD-1 specific antibody and anti-tumor application thereof |
BR112021013157A8 (en) | 2019-01-03 | 2022-12-06 | Inst Nat Sante Rech Med | USES OF AN NRP-1 INHIBITOR, USE OF A COMBINATION, USE OF A MULTIESPECIFIC ANTIBODY, EX VIVO METHOD TO PREDICT, USE OF AN INHIBITOR, MULTIESPECIFIC ANTIBODY, MODIFIED CELL POPULATION, EX VIVO METHOD OF PRODUCTION AND USE OF A POPULATION OF T CELLS |
TW202110885A (en) | 2019-05-20 | 2021-03-16 | 美商潘迪恩治療公司 | Madcam targeted immunotolerance |
US11629190B2 (en) | 2019-08-15 | 2023-04-18 | Oregon State University | Canine antibody therapeutic for treating cancer |
KR102231974B1 (en) * | 2019-08-23 | 2021-03-25 | 한국과학기술원 | Novel polypeptide selective binding with human programmed cell death protein 1 protein and uses thereof |
MX2022002315A (en) | 2019-08-30 | 2022-03-25 | Agenus Inc | Anti-cd96 antibodies and methods of use thereof. |
WO2021092130A1 (en) | 2019-11-05 | 2021-05-14 | Pairwise Plants Services, Inc. | Compositions and methods for rna-encoded dna-replacement of alleles |
WO2021099576A1 (en) | 2019-11-21 | 2021-05-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Novel immunotherapies targeting pd-1 with anti-pd-1/il-15 immunocytokines |
WO2021113788A1 (en) | 2019-12-06 | 2021-06-10 | Pairwise Plants Services, Inc. | Recruitment methods and compounds, compositions and systems for recruitment |
AU2021212189A1 (en) | 2020-01-30 | 2022-08-04 | Pairwise Plants Services, Inc. | Compositions, systems, and methods for base diversification |
WO2021155084A1 (en) | 2020-01-31 | 2021-08-05 | Pairwise Plants Services, Inc. | Suppression of shade avoidance response in plants |
US20230063560A1 (en) | 2020-02-04 | 2023-03-02 | Pairwise Plans Services, Inc. | Thornless and/or prickleless rubus plants |
WO2021168288A1 (en) | 2020-02-21 | 2021-08-26 | Pairwise Plants Services, Inc. | Improved resistance to soybean cyst nematode through gene editing |
EP4107187A4 (en) | 2020-02-21 | 2024-07-03 | Pandion Operations Inc | Tissue targeted immunotolerance with a cd39 effector |
US11999946B2 (en) | 2020-03-26 | 2024-06-04 | Pairwise Plants Services, Inc. | Methods for controlling meristem size for crop improvement |
US11882808B2 (en) | 2020-03-27 | 2024-01-30 | Pairwise Plants Services, Inc. | Methods for improving resistance to soybean rust |
WO2021207148A1 (en) | 2020-04-06 | 2021-10-14 | Pairwise Plants Services, Inc. | Methods and compositions for increasing resistance to ear rot and stem rot disease in maize |
CN115697045A (en) | 2020-04-16 | 2023-02-03 | 成对植物服务股份有限公司 | Method for controlling meristem size to improve crop plants |
AU2021266688A1 (en) * | 2020-05-04 | 2022-12-08 | Inhibrx Biosciences, Inc. | Canine PD-1-binding polypeptides and uses thereof |
CA3185017A1 (en) | 2020-06-02 | 2021-12-09 | Pairwise Plants Services, Inc. | Methods for controlling meristem size for crop improvement |
AR122661A1 (en) | 2020-06-17 | 2022-09-28 | Pairwise Plants Services Inc | MERISTEM SIZE CONTROL METHODS FOR CROP IMPROVEMENT |
CN116113694A (en) | 2020-06-30 | 2023-05-12 | 成对植物服务股份有限公司 | Compositions, systems, and methods for base diversity |
IL300823A (en) | 2020-08-28 | 2023-04-01 | Pairwise Plants Services Inc | Engineered crispr-cas proteins and methods of use thereof |
WO2022098993A2 (en) | 2020-11-06 | 2022-05-12 | Pairwise Plants Services, Inc. | Compositions and methods for rna-encoded dna-replacement of alleles |
BR112023015909A2 (en) | 2021-02-11 | 2023-11-21 | Monsanto Technology Llc | METHODS AND COMPOSITIONS FOR MODIFYING CYTOKININ OXIDASE LEVELS IN PLANTS |
MX2023009799A (en) | 2021-02-25 | 2023-10-30 | Pairwise Plants Services Inc | Methods and compositions for modifying root architecture in plants. |
CN113189078B (en) * | 2021-03-04 | 2024-04-16 | 吉林大学 | High-throughput screening method of targeted drugs |
WO2022265905A2 (en) | 2021-06-14 | 2022-12-22 | Pairwise Plants Services, Inc. | Reporter constructs, compositions comprising the same, and methods of use thereof |
CA3223995A1 (en) | 2021-06-17 | 2022-12-22 | Pairwise Plants Services, Inc. | Modification of growth regulating factor family transcription factors in soybean |
UY39827A (en) | 2021-06-24 | 2023-01-31 | Pairwise Plants Services Inc | MODIFICATION OF UBIQUITIN LIGASE E3 HECT GENES TO IMPROVE PERFORMANCE TRAITS |
MX2023014883A (en) | 2021-07-01 | 2024-02-12 | Pairwise Plants Services Inc | Methods and compositions for enhancing root system development. |
CN113702351A (en) * | 2021-07-14 | 2021-11-26 | 吉林大学 | Method for screening and fishing natural drug products |
CN118076742A (en) | 2021-08-12 | 2024-05-24 | 成对植物服务股份有限公司 | Modification of brassinolide receptor genes to improve yield traits |
WO2023023496A1 (en) | 2021-08-17 | 2023-02-23 | Pairwise Plants Services, Inc. | Methods and compositions for modifying cytokinin receptor histidine kinase genes in plants |
CN118265446A (en) | 2021-08-30 | 2024-06-28 | 成对植物服务股份有限公司 | Modification of ubiquitin-binding peptidase genes in plants to improve yield traits |
AR126938A1 (en) | 2021-09-02 | 2023-11-29 | Pairwise Plants Services Inc | METHODS AND COMPOSITIONS TO IMPROVE PLANT ARCHITECTURE AND PERFORMANCE TRAITS |
WO2023049720A1 (en) | 2021-09-21 | 2023-03-30 | Pairwise Plants Services, Inc. | Methods and compositions for reducing pod shatter in canola |
US20230266293A1 (en) | 2021-09-21 | 2023-08-24 | Pairwise Plants Services, Inc. | Color-based and/or visual methods for identifying the presence of a transgene and compositions and constructs relating to the same |
CA3237641A1 (en) | 2021-10-04 | 2023-04-13 | Pairwise Plants Services, Inc. | Methods for improving floret fertility and seed yield |
CA3234455A1 (en) | 2021-10-07 | 2023-04-13 | Pairwise Plants Services, Inc. | Methods for improving floret fertility and seed yield |
AR127904A1 (en) | 2021-12-09 | 2024-03-06 | Pairwise Plants Services Inc | METHODS TO IMPROVE FLOWER FERTILITY AND SEED YIELD |
US20230295646A1 (en) | 2021-12-13 | 2023-09-21 | Pairwise Plants Services, Inc. | Model editing systems and methods relating to the same |
WO2023133440A1 (en) | 2022-01-06 | 2023-07-13 | Pairwise Plants Services, Inc. | Methods and compositions for trichome removal |
AR128372A1 (en) | 2022-01-31 | 2024-04-24 | Pairwise Plants Services Inc | SUPPRESSION OF THE SHADE AVOIDANCE RESPONSE IN PLANTS |
WO2023164722A1 (en) | 2022-02-28 | 2023-08-31 | Pairwise Plants Services, Inc. | Engineered crispr-cas effector proteins and methods of use thereof |
WO2023168217A1 (en) | 2022-03-02 | 2023-09-07 | Pairwise Plants Services, Inc. | Modification of brassinosteroid receptor genes to improve yield traits |
WO2023192838A1 (en) | 2022-03-31 | 2023-10-05 | Pairwise Plants Services, Inc. | Early flowering rosaceae plants with improved characteristics |
US20230357789A1 (en) | 2022-04-07 | 2023-11-09 | Pairwise Plants Services, Inc. | Methods and compositions for improving resistance to fusarium head blight |
WO2023205714A1 (en) | 2022-04-21 | 2023-10-26 | Pairwise Plants Services, Inc. | Methods and compositions for improving yield traits |
WO2023215704A1 (en) | 2022-05-02 | 2023-11-09 | Pairwise Plants Services, Inc. | Methods and compositions for enhancing yield and disease resistance |
WO2023215809A1 (en) | 2022-05-05 | 2023-11-09 | Pairwise Plants Services, Inc. | Methods and compositions for modifying root architecture and/or improving plant yield traits |
US20230416771A1 (en) | 2022-06-27 | 2023-12-28 | Pairwise Plants Services, Inc. | Methods and compositions for modifying shade avoidance in plants |
WO2024006791A1 (en) | 2022-06-29 | 2024-01-04 | Pairwise Plants Services, Inc. | Methods and compositions for controlling meristem size for crop improvement |
WO2024006792A1 (en) | 2022-06-29 | 2024-01-04 | Pairwise Plants Services, Inc. | Methods and compositions for controlling meristem size for crop improvement |
WO2024030984A1 (en) | 2022-08-04 | 2024-02-08 | Pairwise Plants Services, Inc. | Methods and compositions for improving yield traits |
US20240060081A1 (en) | 2022-08-11 | 2024-02-22 | Pairwise Plants Services, Inc. | Methods and compositions for controlling meristem size for crop improvement |
US20240090466A1 (en) | 2022-09-08 | 2024-03-21 | Pairwise Plants Services, Inc. | Methods and compositions for improving yield characteristics in plants |
US20240200102A1 (en) | 2022-12-16 | 2024-06-20 | Pairwise Plants Services, Inc. | Fusion proteins comprising an intein polypeptide and methods of use thereof |
WO2024137911A1 (en) | 2022-12-21 | 2024-06-27 | Pairwise Plants Services, Inc. | Engineered proteins and methods of use thereof |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9402899B2 (en) * | 2002-07-03 | 2016-08-02 | Ono Pharmaceutical Co., Ltd. | Immunopotentiative composition |
US9982053B2 (en) * | 2014-08-05 | 2018-05-29 | MabQuest, SA | Immunological reagents |
US9982052B2 (en) * | 2014-08-05 | 2018-05-29 | MabQuest, SA | Immunological reagents |
US10294299B2 (en) * | 2016-01-22 | 2019-05-21 | MabQuest SA | Immunological reagents |
US11214617B2 (en) * | 2016-01-22 | 2022-01-04 | MabQuest SA | Immunological reagents |
Family Cites Families (53)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US5504005A (en) | 1987-03-02 | 1996-04-02 | Albert Einstein College Of Medicine Of Yeshiva University | Recombinant mycobacterial vaccine |
JP3011939B2 (en) | 1987-03-02 | 2000-02-21 | ホワイトヘツド・インスチチユート・フオー・バイオメデイカル・リサーチ | Recombinant mycobacterial vaccine |
GB8823869D0 (en) | 1988-10-12 | 1988-11-16 | Medical Res Council | Production of antibodies |
US5530101A (en) | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
AU648505B2 (en) | 1989-05-19 | 1994-04-28 | Amgen, Inc. | Metalloproteinase inhibitor |
JPH05504067A (en) | 1990-02-26 | 1993-07-01 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガナイゼイション | Shuttle plasmid for E. coli and mycobacteria |
GB9015888D0 (en) | 1990-07-19 | 1990-09-05 | Smithkline Biolog | Vectors |
US5545806A (en) | 1990-08-29 | 1996-08-13 | Genpharm International, Inc. | Ransgenic non-human animals for producing heterologous antibodies |
US5625126A (en) | 1990-08-29 | 1997-04-29 | Genpharm International, Inc. | Transgenic non-human animals for producing heterologous antibodies |
DE69127627T2 (en) | 1990-08-29 | 1998-02-19 | Genpharm Int | Production and Use Non-human transgene heterologous antibodies for production |
US5661016A (en) | 1990-08-29 | 1997-08-26 | Genpharm International Inc. | Transgenic non-human animals capable of producing heterologous antibodies of various isotypes |
US5633425A (en) | 1990-08-29 | 1997-05-27 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
CA2110682A1 (en) | 1991-06-06 | 1992-12-10 | Charles K. Stover | Induction of ctl responses to foreign antigens expressed in mycobacteria |
DK0590058T3 (en) | 1991-06-14 | 2004-03-29 | Genentech Inc | Humanized heregulin antibody |
JP3454275B2 (en) | 1992-06-05 | 2003-10-06 | 佑 本庶 | Novel polypeptide associated with programmed cell death and DNA encoding the same |
AU7203194A (en) | 1993-06-04 | 1995-01-03 | New York University | Bispecific human monoclonal antibodies specific for human immunodeficiency virus |
JPH07291996A (en) | 1994-03-01 | 1995-11-07 | Yuu Honshiyo | Polypeptide related to programmed cell death in human, dna coding the same, vector consisting of the same dna, host cell transformed with the same vector, antibody of the same polypeptide and pharmaceutical composition containing the same polypeptide or the same antibody |
PT1210428E (en) | 1999-08-23 | 2015-07-21 | Genetics Inst Llc | Pd-1, a receptor for b7-4, and uses therefor |
JP4249013B2 (en) | 2001-07-31 | 2009-04-02 | 佑 本庶 | Substance with specificity for PD-1 |
JP4511943B2 (en) * | 2002-12-23 | 2010-07-28 | ワイス エルエルシー | Antibody against PD-1 and use thereof |
DE602004031589D1 (en) | 2003-01-07 | 2011-04-14 | Dyax Corp | Kunitz DOMAIN LIBRARY |
US7563869B2 (en) | 2003-01-23 | 2009-07-21 | Ono Pharmaceutical Co., Ltd. | Substance specific to human PD-1 |
PL2439273T3 (en) | 2005-05-09 | 2019-08-30 | Ono Pharmaceutical Co., Ltd. | Human monoclonal antibodies to programmed death 1(PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics |
KR101514463B1 (en) | 2005-06-08 | 2015-04-28 | 다나-파버 캔서 인스티튜트 인크. | Methods and compositions for the treatment of persistent infections and cancer by inhibiting the programmed cell death 1 (pd-1) pathway |
CA3045637A1 (en) | 2006-12-27 | 2008-07-10 | Emory University | Compositions and methods for the treatment of infections and tumors |
CA2691357C (en) | 2007-06-18 | 2014-09-23 | N.V. Organon | Antibodies to human programmed death receptor pd-1 |
WO2009014708A2 (en) | 2007-07-23 | 2009-01-29 | Cell Genesys, Inc. | Pd-1 antibodies in combination with a cytokine-secreting cell and methods of use thereof |
JOP20080381B1 (en) | 2007-08-23 | 2023-03-28 | Amgen Inc | Antigen Binding Proteins to Proprotein Convertase subtillisin Kexin type 9 (pcsk9) |
WO2009114335A2 (en) * | 2008-03-12 | 2009-09-17 | Merck & Co., Inc. | Pd-1 binding proteins |
TW201007669A (en) | 2008-08-01 | 2010-02-16 | Integrated Solutions Technology Inc | A display driving device and the driving method thereof |
EP2342228B1 (en) * | 2008-09-12 | 2017-09-06 | Oxford University Innovation Limited | Pd-1 specific antibodies and uses thereof |
US8927697B2 (en) | 2008-09-12 | 2015-01-06 | Isis Innovation Limited | PD-1 specific antibodies and uses thereof |
CA2998281C (en) | 2008-09-26 | 2022-08-16 | Dana-Farber Cancer Institute, Inc. | Human anti-pd-1 antobodies and uses therefor |
JP5844159B2 (en) | 2009-02-09 | 2016-01-13 | ユニヴェルシテ デクス−マルセイユUniversite D’Aix−Marseille | PD-1 antibody and PD-L1 antibody and use thereof |
WO2011100841A1 (en) | 2010-02-16 | 2011-08-25 | Valorisation-Recherche, Limited Partnership | Pd-1 modulation and uses thereof for modulating hiv replication |
TW201134488A (en) * | 2010-03-11 | 2011-10-16 | Ucb Pharma Sa | PD-1 antibodies |
US8993731B2 (en) | 2010-03-11 | 2015-03-31 | Ucb Biopharma Sprl | PD-1 antibody |
WO2011159877A2 (en) | 2010-06-18 | 2011-12-22 | The Brigham And Women's Hospital, Inc. | Bi-specific antibodies against tim-3 and pd-1 for immunotherapy in chronic immune conditions |
US9029315B2 (en) | 2010-11-11 | 2015-05-12 | The University Of Hong Kong | Soluble PD-1 variants, fusion constructs, and uses thereof |
KR102289394B1 (en) | 2011-03-31 | 2021-08-13 | 머크 샤프 앤드 돔 코포레이션 | Stable formulations of antibodies to human programmed death receptor pd-1 and related treatments |
WO2012145493A1 (en) | 2011-04-20 | 2012-10-26 | Amplimmune, Inc. | Antibodies and other molecules that bind b7-h1 and pd-1 |
US9090994B2 (en) | 2011-06-08 | 2015-07-28 | Nanjingjinsirui Science & Technology Biology Corp. | Antibody humanization by framework assembly |
KR20190133790A (en) * | 2011-08-01 | 2019-12-03 | 제넨테크, 인크. | Methods of treating cancer using pd-1 axis binding antagonists and mek inhibitors |
US20130071403A1 (en) | 2011-09-20 | 2013-03-21 | Vical Incorporated | Synergistic anti-tumor efficacy using alloantigen combination immunotherapy |
EP2817619B1 (en) * | 2012-02-23 | 2018-07-25 | Sloan Kettering Institute For Cancer Research | Prediction of responsiveness to treatment with immunomodulatory therapeutics and method of monitoring abscopal effects during such treatment |
WO2013169693A1 (en) * | 2012-05-09 | 2013-11-14 | Bristol-Myers Squibb Company | Methods of treating cancer using an il-21 polypeptide and an anti-pd-1 antibody |
ES2643887T3 (en) | 2012-10-02 | 2017-11-27 | Bristol-Myers Squibb Company | Combination of anti-KIR antibodies and anti-PD-1 antibodies to treat cancer |
DK2992017T3 (en) | 2013-05-02 | 2021-01-25 | Anaptysbio Inc | ANTIBODIES AGAINST PROGRAMMED DEATH-1 (PD-1) |
CN104250302B (en) | 2013-06-26 | 2017-11-14 | 上海君实生物医药科技股份有限公司 | The anti-antibody of PD 1 and its application |
JP6623353B2 (en) | 2013-09-13 | 2019-12-25 | ベイジーン スウィッツァーランド ゲーエムベーハー | Anti-PD-1 antibodies and their use for therapy and diagnosis |
TWI681969B (en) | 2014-01-23 | 2020-01-11 | 美商再生元醫藥公司 | Human antibodies to pd-1 |
CA2971734A1 (en) | 2014-12-22 | 2016-06-30 | Enumeral Biomedical Holdings, Inc. | Anti-pd-1 antibodies |
-
2015
- 2015-08-05 DK DK15750136.2T patent/DK3177644T3/en active
- 2015-08-05 KR KR1020177004575A patent/KR102357893B1/en active IP Right Grant
- 2015-08-05 PT PT157501362T patent/PT3177644T/en unknown
- 2015-08-05 CN CN201580042199.7A patent/CN107074947B/en active Active
- 2015-08-05 WO PCT/IB2015/055943 patent/WO2016020856A2/en active Application Filing
- 2015-08-05 AU AU2015298356A patent/AU2015298356B2/en active Active
- 2015-08-05 JP JP2017526774A patent/JP6629321B2/en active Active
- 2015-08-05 SG SG11201700672YA patent/SG11201700672YA/en unknown
- 2015-08-05 US US15/329,760 patent/US9982053B2/en active Active
- 2015-08-05 CA CA2957258A patent/CA2957258C/en active Active
- 2015-08-05 ES ES15750136T patent/ES2847311T3/en active Active
- 2015-08-05 EP EP15750136.2A patent/EP3177644B1/en active Active
- 2015-08-05 PL PL15750136T patent/PL3177644T3/en unknown
-
2018
- 2018-05-17 US US15/981,977 patent/US11130807B2/en active Active
-
2021
- 2021-09-27 US US17/485,752 patent/US20220169733A1/en not_active Abandoned
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9402899B2 (en) * | 2002-07-03 | 2016-08-02 | Ono Pharmaceutical Co., Ltd. | Immunopotentiative composition |
US9982053B2 (en) * | 2014-08-05 | 2018-05-29 | MabQuest, SA | Immunological reagents |
US9982052B2 (en) * | 2014-08-05 | 2018-05-29 | MabQuest, SA | Immunological reagents |
US11130807B2 (en) * | 2014-08-05 | 2021-09-28 | MabQuest, S.A. | Immunological reagents |
US10294299B2 (en) * | 2016-01-22 | 2019-05-21 | MabQuest SA | Immunological reagents |
US11214617B2 (en) * | 2016-01-22 | 2022-01-04 | MabQuest SA | Immunological reagents |
Non-Patent Citations (8)
Title |
---|
Barthelemy et al, Journal of Biological Chemistry, 2008, 283:3639-3654. (Year: 2008) * |
Beiboer et al, Journal of Molecular Biology, 2000, 296:833-849. (Year: 2000) * |
Choi et al, 2011, Molecular Biosystems, 2011, 7:3327-3334. (Year: 2011) * |
De Genst et al, Developmental and Comparative Immunology, 2006, 30:187-98. (Year: 2006) * |
Griffiths et al, The EMBO Journal, 1993, 12:725-734. (Year: 1993) * |
Klimka et al, British Journal of Cancer, 2000, 83:252-260. (Year: 2000) * |
Malia et al, Proteins, 2016, 84:427-434. (Year: 2016) * |
Ward et al, Nature, 1989, 341:544-546. (Year: 1989) * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20220259315A1 (en) * | 2016-01-22 | 2022-08-18 | MabQuest SA | Immunological Reagents |
Also Published As
Publication number | Publication date |
---|---|
PL3177644T3 (en) | 2021-06-14 |
AU2015298356A1 (en) | 2017-02-16 |
ES2847311T3 (en) | 2021-08-02 |
CA2957258A1 (en) | 2016-02-11 |
WO2016020856A2 (en) | 2016-02-11 |
WO2016020856A3 (en) | 2016-03-31 |
US20170226210A1 (en) | 2017-08-10 |
EP3177644B1 (en) | 2020-10-07 |
DK3177644T3 (en) | 2021-01-11 |
US9982053B2 (en) | 2018-05-29 |
JP2017531028A (en) | 2017-10-19 |
CN107074947A (en) | 2017-08-18 |
AU2015298356B2 (en) | 2020-11-19 |
CN107074947B (en) | 2021-04-09 |
US11130807B2 (en) | 2021-09-28 |
KR102357893B1 (en) | 2022-02-04 |
EP3177644A2 (en) | 2017-06-14 |
JP6629321B2 (en) | 2020-01-15 |
KR20170069996A (en) | 2017-06-21 |
PT3177644T (en) | 2021-01-13 |
CA2957258C (en) | 2023-11-07 |
SG11201700672YA (en) | 2017-02-27 |
US20190106493A1 (en) | 2019-04-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220169733A1 (en) | Immunological Reagents | |
AU2017208819B2 (en) | PD1 specific antibodies | |
US9982052B2 (en) | Immunological reagents | |
US20210009661A1 (en) | HIV Binding Agents | |
US20220259315A1 (en) | Immunological Reagents | |
WO2016077720A1 (en) | Binding agents and uses thereof | |
WO2020012435A1 (en) | Hiv binding agents | |
US20220267416A1 (en) | Hiv binding agents |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
AS | Assignment |
Owner name: MABQUEST SA, SWITZERLAND Free format text: NUNC PRO TUNC ASSIGNMENT;ASSIGNORS:PANTALEO, GIUSEPPE;FENWICK, CRAIG;REEL/FRAME:064055/0545 Effective date: 20170510 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE AFTER FINAL ACTION FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS |
|
ZAAB | Notice of allowance mailed |
Free format text: ORIGINAL CODE: MN/=. |