CN116763917A - 一种稳定的抗人il15/pd-l1双功能融合蛋白制剂 - Google Patents
一种稳定的抗人il15/pd-l1双功能融合蛋白制剂 Download PDFInfo
- Publication number
- CN116763917A CN116763917A CN202310238467.9A CN202310238467A CN116763917A CN 116763917 A CN116763917 A CN 116763917A CN 202310238467 A CN202310238467 A CN 202310238467A CN 116763917 A CN116763917 A CN 116763917A
- Authority
- CN
- China
- Prior art keywords
- formulation
- fusion protein
- bifunctional fusion
- buffer
- human
- 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.)
- Pending
Links
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 97
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 97
- 102000008096 B7-H1 Antigen Human genes 0.000 title claims abstract description 92
- 102000003812 Interleukin-15 Human genes 0.000 title claims abstract description 89
- 108090000172 Interleukin-15 Proteins 0.000 title claims abstract description 89
- 108010074708 B7-H1 Antigen Proteins 0.000 title claims abstract description 87
- 230000001588 bifunctional effect Effects 0.000 title claims abstract description 84
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000000872 buffer Substances 0.000 claims abstract description 32
- 239000004094 surface-active agent Substances 0.000 claims abstract description 14
- 239000003381 stabilizer Substances 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims description 66
- 238000009472 formulation Methods 0.000 claims description 59
- 239000000427 antigen Substances 0.000 claims description 28
- 102000036639 antigens Human genes 0.000 claims description 28
- 108091007433 antigens Proteins 0.000 claims description 28
- 230000027455 binding Effects 0.000 claims description 19
- 239000012634 fragment Substances 0.000 claims description 19
- 206010028980 Neoplasm Diseases 0.000 claims description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- 201000010099 disease Diseases 0.000 claims description 14
- 238000004108 freeze drying Methods 0.000 claims description 11
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 11
- 229920000053 polysorbate 80 Polymers 0.000 claims description 11
- 239000008362 succinate buffer Substances 0.000 claims description 11
- 229930006000 Sucrose Natural products 0.000 claims description 10
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 claims description 10
- 229940068968 polysorbate 80 Drugs 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- 239000012931 lyophilized formulation Substances 0.000 claims description 9
- 210000004899 c-terminal region Anatomy 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
- 150000005846 sugar alcohols Chemical group 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 6
- 239000008351 acetate buffer Substances 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 101001055157 Homo sapiens Interleukin-15 Proteins 0.000 claims description 5
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 claims description 5
- 102000056003 human IL15 Human genes 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000001404 mediated effect Effects 0.000 claims description 5
- 229940068977 polysorbate 20 Drugs 0.000 claims description 5
- 230000004927 fusion Effects 0.000 claims description 4
- JISIBLCXFLGVJX-UHFFFAOYSA-M sodium;butanedioic acid;hydroxide Chemical group [OH-].[Na+].OC(=O)CCC(O)=O JISIBLCXFLGVJX-UHFFFAOYSA-M 0.000 claims description 4
- 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 claims description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 239000000600 sorbitol Substances 0.000 claims description 3
- 125000000185 sucrose group Chemical group 0.000 claims description 3
- 235000000346 sugar Nutrition 0.000 claims description 3
- CTKXFMQHOOWWEB-UHFFFAOYSA-N Ethylene oxide/propylene oxide copolymer Chemical compound CCCOC(C)COCCO CTKXFMQHOOWWEB-UHFFFAOYSA-N 0.000 claims description 2
- 229920001993 poloxamer 188 Polymers 0.000 claims description 2
- 229940044519 poloxamer 188 Drugs 0.000 claims description 2
- 239000007974 sodium acetate buffer Substances 0.000 claims description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical group [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 238000011321 prophylaxis Methods 0.000 claims 1
- 239000008194 pharmaceutical composition Substances 0.000 abstract description 5
- 238000003860 storage Methods 0.000 abstract description 5
- 239000000825 pharmaceutical preparation Substances 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 29
- 102000004169 proteins and genes Human genes 0.000 description 29
- 108090000623 proteins and genes Proteins 0.000 description 29
- 238000012216 screening Methods 0.000 description 16
- 239000000337 buffer salt Substances 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 239000003223 protective agent Substances 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 8
- 229940024606 amino acid Drugs 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- 150000001720 carbohydrates Chemical group 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 230000007774 longterm Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 230000004071 biological effect Effects 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 238000011835 investigation Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 4
- 206010039491 Sarcoma Diseases 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000013368 capillary electrophoresis sodium dodecyl sulfate analysis Methods 0.000 description 4
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 230000006240 deamidation Effects 0.000 description 4
- -1 fatty acid esters Chemical class 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 229920005862 polyol Polymers 0.000 description 4
- 150000003077 polyols Chemical class 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 201000009030 Carcinoma Diseases 0.000 description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 3
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 230000001133 acceleration Effects 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000007979 citrate buffer Substances 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000006317 isomerization reaction Methods 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000001542 size-exclusion chromatography Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 239000013598 vector Substances 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
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 206010008342 Cervix carcinoma Diseases 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 2
- 208000022072 Gallbladder Neoplasms Diseases 0.000 description 2
- 208000032612 Glial tumor Diseases 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 208000017604 Hodgkin disease Diseases 0.000 description 2
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 2
- 208000034578 Multiple myelomas Diseases 0.000 description 2
- 206010029260 Neuroblastoma Diseases 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 208000006265 Renal cell carcinoma Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 201000010881 cervical cancer Diseases 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 201000004101 esophageal cancer Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 201000010175 gallbladder cancer Diseases 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 229960002885 histidine Drugs 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 230000010494 opalescence Effects 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 229920001983 poloxamer Polymers 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 230000004845 protein aggregation Effects 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 210000003708 urethra Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- 206010000871 Acute monocytic leukaemia Diseases 0.000 description 1
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 206010000890 Acute myelomonocytic leukaemia Diseases 0.000 description 1
- 208000036762 Acute promyelocytic leukaemia Diseases 0.000 description 1
- 201000003076 Angiosarcoma Diseases 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 206010004146 Basal cell carcinoma Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101150091609 CD274 gene Proteins 0.000 description 1
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 1
- 208000005243 Chondrosarcoma Diseases 0.000 description 1
- 201000009047 Chordoma Diseases 0.000 description 1
- 208000006332 Choriocarcinoma Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 201000009051 Embryonal Carcinoma Diseases 0.000 description 1
- 206010014967 Ependymoma Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 208000006168 Ewing Sarcoma Diseases 0.000 description 1
- 108010008177 Fd immunoglobulins Proteins 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101100407305 Homo sapiens CD274 gene Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 102000004556 Interleukin-15 Receptors Human genes 0.000 description 1
- 108010017535 Interleukin-15 Receptors Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 208000018142 Leiomyosarcoma Diseases 0.000 description 1
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000007054 Medullary Carcinoma Diseases 0.000 description 1
- 208000000172 Medulloblastoma Diseases 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010027406 Mesothelioma Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 208000035489 Monocytic Acute Leukemia Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 208000033835 Myelomonocytic Acute Leukemia Diseases 0.000 description 1
- 208000005890 Neuroma Diseases 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- 201000010133 Oligodendroglioma Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000007641 Pinealoma Diseases 0.000 description 1
- 208000007452 Plasmacytoma Diseases 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 208000033826 Promyelocytic Acute Leukemia Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 102000001712 STAT5 Transcription Factor Human genes 0.000 description 1
- 108010029477 STAT5 Transcription Factor Proteins 0.000 description 1
- 201000010208 Seminoma Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- 206010057644 Testis cancer Diseases 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 208000017733 acquired polycythemia vera Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 208000011912 acute myelomonocytic leukemia M4 Diseases 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 150000005215 alkyl ethers Chemical class 0.000 description 1
- 238000013103 analytical ultracentrifugation Methods 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229960003121 arginine Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229960003589 arginine hydrochloride Drugs 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000012575 bio-layer interferometry Methods 0.000 description 1
- 230000008512 biological response Effects 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
- 230000037396 body weight Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000010983 breast ductal carcinoma Diseases 0.000 description 1
- 201000005200 bronchus cancer Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 238000000533 capillary isoelectric focusing Methods 0.000 description 1
- 238000005515 capillary zone electrophoresis Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000011970 concomitant therapy Methods 0.000 description 1
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 1
- 229940097362 cyclodextrins Drugs 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008355 dextrose injection Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000000612 dual polarization interferometry Methods 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 239000005350 fused silica glass Substances 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 208000025750 heavy chain disease Diseases 0.000 description 1
- 201000002222 hemangioblastoma Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000005746 immune checkpoint blockade Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000009851 immunogenic response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000000111 isothermal titration calorimetry Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 206010024627 liposarcoma Diseases 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 201000000564 macroglobulinemia Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 208000023356 medullary thyroid gland carcinoma Diseases 0.000 description 1
- 206010027191 meningioma Diseases 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 208000001611 myxosarcoma Diseases 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000009701 normal cell proliferation Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 208000004019 papillary adenocarcinoma Diseases 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000008180 pharmaceutical surfactant Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 208000024724 pineal body neoplasm Diseases 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 229960000502 poloxamer Drugs 0.000 description 1
- 208000037244 polycythemia vera Diseases 0.000 description 1
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229950008882 polysorbate Drugs 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 230000006318 protein oxidation Effects 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 208000015347 renal cell adenocarcinoma Diseases 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- 238000012954 risk control Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 201000008407 sebaceous adenocarcinoma Diseases 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000012475 sodium chloride buffer Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000001370 static light scattering Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 201000010965 sweat gland carcinoma Diseases 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical group [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical group [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 210000000626 ureter Anatomy 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- C07K16/2827—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 B7 molecules, e.g. CD80, CD86
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
Abstract
本发明涉及一种稳定的抗人IL15/PD‑L1双功能融合蛋白的药物制剂及其用途。所述药物制剂含有抗人IL15/PD‑L1双功能融合蛋白、稳定剂、缓冲剂和表面活性剂。本发明的抗人IL15/PD‑L1双功能融合蛋白制剂非常稳定,经长时间保存、强光、高温后仍能符合药学使用要求,有着广泛的应用前景。
Description
技术领域
本发明属于抗体制剂领域,具体的涉及一种抗人IL15/PD-L1的双功能融合蛋白的制剂及其用途。
背景技术
1994年,Grabstein等发现了一种分子量大小为12-14KD的细胞因子,白介素15(IL15)。白介素15作为T细胞和NK细胞生长因子,在免疫细胞的产生、复制和激活过程中起着重要的作用。美国国家癌症研究中心将白介素15列为癌症免疫治疗中最具有潜力的靶点之一。美国FDA已经批准另一种相关的细胞因子,白介素2,用来治疗肾细胞癌症和恶性黑色素瘤。
PD-L1,细胞程序性死亡配体1,是人类体内的一种蛋白质,由CD274基因编码,分子量大小为40KD。PD-L1一般表达于肿瘤细胞表面。PD-1,细胞程序性死亡受体-1(PD-1),为CD28超家族成员,表达于T细胞表面。当PD-L1与PD-1连接以后,T细胞就不能够发现肿瘤和向免疫系统发出攻击肿瘤的信号。通过设计PD-L1抗体,可以切断PD-L1与PD-1连接,使肿瘤细胞被免疫细胞识别并杀死肿瘤细胞。
IL15/PD-L1双功能融合蛋白为申请人自主研发的CHO细胞表达的创新靶点双功能融合蛋白药物。该双功能融合蛋白由IgG1型的抗PD-L1单抗通过其Fc段的C-末端与IL15RaSu及IL15连接而成,抗PD-L1单抗的每条重链的C-末端均通过G4S连接子(G4SLinker)连接1个IL15RαSu和1个IL15,IL15RαSu和IL15之间也是由G4S Linker连接。
上述IL15/PD-L1双功能融合蛋白通过抗PD-L1抗体与肿瘤细胞表面的PD-L1结合,可解除PD-L1/PD-1介导的免疫检查点抑制效应:同时可以通过激活人原代淋巴细胞表面IL15受体下游STAT5信号通路,促进T淋巴细胞和NK细胞的功能活性。
双功能融合蛋白虽然具有特异性好、靶向性强、起效剂量低等优点,但因其结构复杂,在生产和贮存过程中,已发生聚集、变性、沉淀等物理变化以及异构化、氧化和脱酰胺等化学变化。因此基于对人用抗体在安全性和有效性上的严格要求,需要针对抗人IL15/PD-L1的双功能融合蛋白单独进行最佳制剂配方的开发和优化。
发明内容
本发明在获得抗人IL15/PD-L1的双功能融合蛋白的基础上,进一步地对IL15/PD-L1双功能融合蛋白的制剂处方进行了大量摸索和研究,最终获得了一种针对该IL15/PD-L1双功能融合蛋白最为适用的、且能稳定保存所述双功能融合蛋白的制剂,该制剂能够充分防止双功能融合蛋白的蛋白聚集、降解、氧化或者变性等,从而保持其有效组分的生物学活性,适合于临床使用。
发明详述
1、术语
本说明书中提及的所有公布、专利和专利申请都以引用的方式并入本文,所述引用的程度就如同已特定地和个别地将各个别公布、专利或专利申请以引用的方式并入本文。
在下文详细描述本发明前,应理解本发明不限于本文中描述的特定方法学、方案和试剂,因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案,而并不意图限制本发明的范围。除非另外定义,本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
本文所公开的某些实施方案包含了数值范围,并且本发明的某些方面可采用范围的方式描述。除非另有说明,应当理解数值范围或者以范围描述的方式仅是出于简洁、便利的目的,并不应当认为是对本发明的范围的严格限定。因此,采用范围方式的描述应当被认为具体地公开了所有可能的子范围以及在该范围内的所有可能的具体数值点,正如这些子范围和数值点在本文中已经明确写出。不论所述数值的宽窄,上述原则均同等适用。当采用范围描述时,该范围包括范围的端点。
当涉及可测量值比如量、暂时持续时间等时,术语“约”是指包括指定值的±20%、或在某些情况下±10%、或在某些情况下±5%、或在某些情况下±1%、或在某些情况下±0.1%的变化。
本文所用氨基酸三字母代码和单字母代码如J.Biol.Chem,243,p3558(1968)中所述。
本文所用的术语“抗体”,典型的指包含通过共价二硫键和非共价相互作用保持在一起的两条重(H)多肽链和两条轻(L)多肽链的Y型四聚蛋白。天然IgG抗体即具有这样的结构。每条轻链由一个可变结构域(VL)和一个恒定结构域(CL)组成。每条重链包含一个可变结构域(VH)和恒定区。
本领域已知五个主要类别的抗体:IgA,IgD,IgE,IgG和IgM,对应的重链恒定结构域分别被称为α,δ,ε,γ和μ,IgG和IgA可以进一步分为不同的亚类,例如IgG可分为IgG1,IgG2,IgG3,IgG4,IgA可分为IgA1和IgA2。来自任何脊椎动物物种的抗体的轻链基于其恒定结构域的氨基酸序列可以被分配到两种明显相异的类型之一,称为K和λ。
在IgG、IgA和IgD抗体的情形中,该恒定区包含称为CH1、CH2和CH3的三个结构域(IgM和IgE具有第四个结构域CH4)。在IgG、IgA和IgD类别中,CH1和CH2结构域被柔性铰链区分离,该铰链区是可变长度的富含脯氨酸和半胱氨酸的区段。每类抗体进一步包含由配对半胱氨酸残基形成的链间和链内二硫键。
术语“可变区”或“可变结构域”显示出从一种抗体到另一种抗体的氨基酸组成的显著变化,并且主要负责抗原的识别和结合。每个轻链/重链对应的可变区形成抗体结合位点,使得完整的IgG抗体具有两个结合位点(即它是二价的)。重链的可变区(VH)和轻链的可变区(VL)结构域各包含具有极端变异性的三个区域,被称为高变区(HVR),或更通常地,被称为互补决定区(CDR),VH和VL各有4个骨架区FR,分别用FRI,FR2,FR3,FR4表示。因此,CDR和FR序列通常出现在重链可变结构域(或轻链可变结构域)的以下序列中:FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。
术语“Fc”用于定义免疫球蛋白重链的C端区域,所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。
术语“单克隆抗体”(或称“单抗”)指由单一细胞克隆产生的基本均质、仪针对某一特定抗原表位的抗体。单克隆抗体可以是用本领域中已知的多种技术制备,包括杂交瘤技术、重组技术、噬菌体展示技术、转基因动物、合成技术或上述技术的组合等。
需说明的是,本发明的单克隆抗体可变区的CDR和FR的划分是根据Kabat定义确定的。而其他命名和编号系统,例如Chothia、IMGT或AHo等,也是本领域技术人员已知的。因此,以本发明的单抗序列为基础,包含任何命名系统衍生的一种或多种CDR的人源化抗体均明确的保持在本发明的范围内。
术语“抗体片段”包含完整抗体的至少一部分。如在此所使用,抗体分于的“片段”包括抗体的“抗原结合片段”,并且术语“抗原结合片段”是指免疫球蛋白中或抗体中与所选抗原或其免疫原性决定部分特异性结合或反应的多肽片段,或由此片段进一步衍生的融合蛋白产物,例如单链抗体,嵌合抗原受体中的胞外结合区等。示例性的抗体片段或其抗原结合片段包括但不限于:可变轻链片段、可变重链片段、Fab片段、F(ab’)2片段、Fd片段、Fv片段、单结构域抗体、线性抗体、单链抗体(scFv)及由抗体片段形成的双功能融合蛋白或多特异性抗体等。
术语“抗原”是指被抗体或抗体结合片段识别并特异性结合的物质,广义上,抗原可以包括所选靶标的任何免疫原性片段或决定簇,包括单表位、多表位、单结构域、多结构域、完整的胞外结构域(ECD)或蛋白质。肽、蛋白质、糖蛋白、多糖和脂质,其部分及其组合均可构成抗原。非限制性示例性抗原包括肿瘤抗原或病原体抗原等。“抗原”也可以指引发免疫反应的分子。任何形式的抗原或含有该抗原的细胞或制剂都可以用于生成对抗原决定簇具有特异性的抗体。抗原可以是分离的全长蛋白质、细胞表面蛋白(例如,用在其表面上表达至少一部分抗原的细胞进行免疫的)、或可溶性蛋白质(例如,仅用该蛋白质的ECD部分进行免疫的)或蛋白质构建体(例如,Fc抗原)。该抗原可以在基因修饰的细胞中产生。前述任何抗原可以单独或与本领域已知的一种或多种免疫原性增强佐剂组合使用。编码该抗原的DNA可以是基因组或非基因组的(例如,cDNA),并且可以编码足以引起免疫原性应答的至少一部分ECD。可以使用任何载体来转化其中表达抗原的细胞,所述载体包括但不限于腺病毒载体、慢病毒载体、质粒以及非病毒载体如阳离子脂质。
术语“亲和力”或“结合亲和力”指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。术语“KD”是指特定的抗体-抗原相互作用的解离常数。可以使用本领域已知的各种技术来确定结合亲和力,例如表面等离子体共振、生物层干涉法、双极化干涉法、静态光散射、动态光散射、等温滴定量热法、ELISA、分析超速离心和流式细胞术等。
术语“生物学活性”指抗体结合抗原并导致可测量的生物学反应的能力,所述生物学反应可以在体外或体内进行测量。
术语“药物制剂”或“制剂处方”,表示这样的制品:其存在形式允许活性成分的生物学活性有效,并且不含有对所述制剂要施用的受试者有毒的其他组分,可用于皮下或静脉给药方式。
术语“溶液制剂”表示,在大气压下至少约2℃至约8℃的温度为液体的制剂。
术语“冻干剂”是指通过本领域所公知的冻干技术,制备的冻干剂。冻干剂施用于患者前,应使用水性重建成分重建。这个步骤允许冻干剂中的抗体和其他成分重新溶解,以得到适用于注射给患者的溶液。用于重建的水性物质体积决定了得到的药物组合物中抗体的浓度。用比冻干前体积小重建水性物质重建提供了比冻干前更加浓缩的组合物。重建系数(冻干后制剂的体积:冻干前制剂的体积)可以从1∶0.5至1∶6。本发明的冻干剂可重建,以得到具有抗人IL15和PD-L1的双特异性的抗体,浓度2mg/mL的水性组合物,并相应地选择重建成分的体积。如果需要,可在施用于患者前稀释重建的制剂,从而酌情递送预期剂量。
典型的用于冻干制剂的重建成分包括无菌水或缓冲液,任选地含有防腐剂。如果冻干剂包括缓冲剂,则重建成分可进一步包括缓冲剂(其可以是相同或不同的冻干剂的缓冲剂),或者其也可以不包括缓冲剂(例如:注射用水、生理盐水或葡萄糖注射液)。
术语“脱酰胺”表示,抗体或融合蛋白中的一个或多个天冬酰胺残基已经被衍生,例如,天冬氨酸或异-天冬氨酸。
术语“聚集”的抗体或融合蛋白指的是这样一种抗体或融合蛋白,其已经被发现与其他抗体或融合蛋白分子一起聚集,特别是在冷冻和/或搅动之后。
术语“稳定的”制剂是这样的制剂,其中的蛋白质在保存后基本上保持其物理稳定性和/或化学稳定性和/或生物学活性。优选地,该制剂在保存后基本上保持其物理和化学稳定性,以及其生物学活性。一般基于制剂保质期来选择贮存期。用来测量蛋白质稳定性的各种分析技术是本领域已有的。可以在选定的温度下测量稳定性持续选定的时间。稳定性能够以许多不同的方式进行定性和/或定量地评估,包括评估聚集物形成(例如利用大小排阻层析,通过测量浊度,和/或通过肉眼观察);通过利用阳离子交换层析或毛细管分区电泳评估电荷异质性;氨基末端或羧基末端序列分析;质谱分析;SDS-PAGE分析以比较减小的和完整的抗体;肽图谱分析;评估生物学活性或抗体的抗原结合功能;等等。不稳定性可以包括下列的任一种或多种:聚集,脱酰胺作用(例如Asn脱酰胺作用),氧化作用(例如Met氧化作用),异构化作用(例如Asp异构化作用),剪切/水解/片段化(例如铰链区片段化),琥珀酰亚胺形成,未配对的半胱氨酸,N-末端延伸,C-末端加工,糖基化作用差异,等等。
术语“缓冲剂”或“缓冲液”表示稳定药物制剂pH的药学上可接受的赋形剂。合适的缓冲剂是本领域公知的,且可以在文献中找到的。优选的药学上可接受的缓冲液包括但不限于:组氨酸缓冲液、枸橼酸盐缓冲液、琥珀酸盐缓冲液、醋酸盐缓冲液、精氨酸缓冲液、磷酸盐缓冲液或其混合物等等。缓冲液用本领域已知的酸或碱进行pH调节,可以将pH调至在4.0-8.0范围内的值,特别是调至在5.0-6.0范围内的值,最特别地是调至pH5.5。
语“稳定剂”表示药学可接受的赋形剂,其在制造,储存和应用过程中保护活性药物成分和/或制剂免受化学和/或物理降解。稳定剂包括但不限于糖,氨基酸,多元醇,环糊精,氯化钠等。
术语“表面活性剂”表示,用于保护蛋白制剂抵抗物理应力(如搅拌和剪切)的药学上可接受的赋形剂。药学上可接受的表面活性剂包括:聚氧乙烯脱水山梨糖醇脂肪酸酯(吐温)、聚氧乙烯烷基醚(例如在商标BrijTM下销售的那些)和聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆,Pluronic)。聚氧乙烯脱水山梨糖醇-脂肪酸酯包括聚山梨酯20(在商标吐温20TM下销售)和聚山梨酯80(在商标吐温80TM下销售)。
术语“有效量”指本发明的抗体或片段的药物制剂的剂量,其以单一或多次剂量施用患者后,在治疗的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定:诸如人种差异;体重、年龄和健康状况;涉及的具体疾病;疾病的严重程度;个体患者的应答;施用的具体抗体;施用模式;施用制剂的生物利用率特征;选择的给药方案;和任何伴随疗法的使用。
本发明工程化的抗体或其抗原结合片段可用常规方法制备和纯化。比如,编码重链和轻链的cDNA序列,可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术,哺乳动物类表达系统会导致抗体的糖基化,特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体,也可以用常规方法去除,比如分子筛、离子交换。
本文所用的术语“个体”或“受试者”是指任何动物,例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如食蟹猴或恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
本文所用的术语“肿瘤”指的是一种以细胞或组织的病理性增生为特征的疾病,及其随后的迁移或侵袭其他组织或器官。肿瘤生长通常是不受控制的和进行性的,不诱导或抑制正常细胞增殖。肿瘤可影响多种细胞、组织或器官,包括但不限于选自膀胱、骨、脑、乳腺、软骨、神经胶质细胞、食管、输卵管、胆囊、心脏、肠、肾、肝、肺、淋巴结、神经组织、卵巢、胰腺、前列腺、骨骼肌、皮肤、脊髓、脾、胃、睾丸、胸腺、甲状腺、气管、尿道、输尿管、尿道、子宫、阴道器官,或组织或相应的细胞。肿瘤包括癌症,如肉瘤,癌,或浆细胞瘤(浆细胞的恶性肿瘤)。本发明所述的肿瘤,可包括,但不限于白血病(如急性白血病、急性淋巴细胞白血病、急性髓细胞性白血病,急性粒细胞白血病,急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、慢性白血病、慢性粒细胞白血病、慢性淋巴细胞白血病、真性红细胞增多症),淋巴瘤(霍奇金病、非霍奇金病)、原发性巨球蛋白血症,重链病,实体瘤如肉瘤和癌症(如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、内皮肉瘤、淋巴管肉瘤、血管肉瘤、淋巴管内皮肉瘤,间皮瘤,尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、癌、支气管癌、髓样癌、肾细胞癌、肝癌,尼罗河管癌,绒癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、子宫癌、睾丸癌、肺癌、小细胞肺癌、膀胱癌、上皮癌、胶质瘤、星形细胞瘤、髓母细胞瘤,颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤,听神经瘤,少突胶质瘤、神经鞘瘤、脑膜瘤、黑色素瘤、神经母细胞瘤、视网膜母细胞瘤)、食管癌、胆囊癌、肾癌、多发性骨髓瘤。较佳地,所述的“肿瘤”包括但不限于:胰腺癌、肝癌、肺癌、胃癌、食管癌、头颈部鳞状细胞癌、前列腺癌、结肠癌、乳腺癌、淋巴瘤、胆囊癌、肾癌、白血病、多发性骨髓瘤、卵巢癌、宫颈癌和胶质瘤。
本文所用的术语“疾病”或“病症”或“紊乱”等是指任何损害或干扰细胞、组织或器官的正常功能的改变或失调。例如,所述的“疾病”包括但不限于:肿瘤、病原体感染、自身免疫性疾病、T细胞功能障碍性疾病、或免疫耐受能力缺陷(如移植排斥)。
本文所用的术语“治疗”是指在试图改变个人或处理细胞引起的疾病过程中的临床干预,既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于,防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
2、发明内容
本发明的目的在于提供一种稳定的适用于特定的抗人IL15/PD-L1的双功能融合蛋白的制剂及其用途。
本发明提供一种抗人IL15/PD-L1的双功能融合蛋白制剂,包含抗人IL15/PD-L1的双功能融合蛋白或其抗原结合片段和缓冲剂,所述抗人IL15/PD-L1双功能融合蛋白由IgG1型的抗PD-L1单克隆抗体通过其Fc段的C-末端与IL15RαSu及IL-15连接而成,所述双功能融合蛋白的融合重链序列如SEQ ID NO:1所示,所述双功能融合蛋白的轻链序列如SEQ IDNO:2所示。
优选的,所述缓冲剂选自醋酸盐缓冲液、琥珀酸盐缓冲液、枸橼酸盐缓冲液、组氨酸盐缓冲液、磷酸盐缓冲液。
优选的,醋酸盐缓冲剂为醋酸-醋酸钠缓冲液;琥珀酸盐缓冲剂为琥珀酸-氢氧化钠缓冲液、枸橼酸盐缓冲剂为枸橼酸-枸橼酸钠缓冲液、组氨酸盐缓冲剂为L-组氨酸-L-盐酸组氨酸、磷酸盐缓冲剂为磷酸二氢钠-磷酸氢二钠缓冲液;优选的所述缓冲剂为醋酸盐缓冲剂、琥珀酸盐缓冲剂;更为优选的为琥珀酸盐缓冲剂。
优选的,所述缓冲剂浓度为5mM至30mM,优选的为5mM,优选的为10mM,优选的为30mM,更为优选的为5mM。
优选的,所述制剂中还包含有稳定剂。
优选的,所述稳定剂为糖类、多元醇类、氨基酸类和盐类,其中糖类选自蔗糖和海藻糖,多元醇类选自甘露醇、山梨醇和甘油,氨酸类选自甲硫氨酸、精氨酸盐酸盐,盐类选自氯化钠;优选稳定剂为糖类和多元醇类;更为优选的为蔗糖、海藻糖。
优选的,所述稳定剂浓度为30mg/mL至110mg/mL,优选的为50mg/mL至100mg/mL,更有选的为90mg/mL。
优选的,制剂中还包含有表面活性剂。
优选的,所述表面活性剂选自聚山梨酯20、聚山梨酯80或泊洛沙姆188,优选为聚山梨酯80。
优选的,所述表面活性剂浓度0.2mg/mL至1mg/mL,优选的为0.3mg/mL至0.7mg/mL,更优选的为0.5mg/mL。
优选的,所述制剂的pH为4.0-7.0,优选为4.5-6.0,更优选的为5.3-5.7。
优选的,所述制剂包含0.5-30mg/ml的抗人IL15/PD-L1双功能融合蛋白,优选的为1-10mg/mL,更优选的为2-5mg/mL。
优选的,所述制剂包含1-10mg/ml的抗人IL15/PD-L1的双功能融合蛋白、50-100mg/ml的蔗糖、5-30mM的琥珀酸盐缓冲剂、0.3-0.7mg/ml的聚山梨酯80,溶液的pH为5.3-5.7。
本发明还提供一种含有抗人IL15/PD-L1双功能融合蛋白的冻干制剂,所述冻干制剂通过将本发明所述的制剂经冷冻干燥所得。
本发明还提供本发明还提供一种含有抗人IL15/PD-L1双功能融合蛋白的冻干制剂,所述冻干制剂经复溶后可形成本发明所述的制剂。
一种产品,其包括容器,所述容器中包含本发明所述的制剂,或本发明所述的冻干制剂。
本发明还提供本发明所述的制剂在制备用于治疗或预防IL15和PD-LI介导的疾病或者肿瘤的药物中的用途。
本发明还提供一种治疗方法,用于受试者中预防或治疗IL15和PD-L1介导的疾病或病症,所述的疾病优选为肿瘤;所述方法包括给予受试者施用本发明所述的制剂。
本发明所述的抗人IL15/PD-L1双功能融合蛋白的制剂,具有以下有益效果:该制剂非常稳定,经长时间保存、强光、高温或者低温、冻融循环后仍能够符合药学使用要求,有着广阔的应用前景。
附图说明
图1为抗人IL15/PD-L1双功能融合蛋白的结构示意图。
图2为抗人IL15/PD-L1双功能融合蛋白的等电点示意图。
图3为抗人IL15/PD-L1双功能融合蛋白不同pH和缓冲体系溶液中的蛋白含量变化。
图4为抗人IL15/PD-L1双功能融合蛋白不同保护剂筛选液体处方样品的稳定性考察中的聚集体变化。
图5为抗人IL15/PD-L1双功能融合蛋白保护剂筛选冻干样品稳定性考察中的聚集体变化。
图6为抗人IL15/PD-L1双功能融合蛋白优选pH和缓冲盐浓度的稳定性考察中的聚集体变化。
图7为抗人IL15/PD-L1双功能融合蛋白优选pH和缓冲盐浓度的稳定性考察中的碎片变化。
图8为抗人IL15/PD-L1双功能融合蛋白表面活性筛选中的聚集体变化。
图9为抗人IL15/PD-L1双功能融合蛋白不同蛋白浓度处方的聚集体变化。
图10为抗人IL15/PD-L1双功能融合蛋白优选处方的液体长期和加速稳定性的SEC纯度。
图11为抗人IL15/PD-L1双功能融合蛋白优选处方的液体长期和加速稳定性的CE-SDS纯度。
图12为抗人IL15/PD-L1双功能融合蛋白优选处方的液体长期和加速稳定性的iCIEF纯度。
图13为抗人IL15/PD-L1双功能融合蛋白优选冻干处方的长期稳定性。
图14为抗人IL15/PD-L1双功能融合蛋白优选冻干处方的加速稳定性。
图15为抗人IL15/PD-L1双功能融合蛋白优选冻干处方的光照稳定性。
具体实施方式
通过以下实施例进一步详细说明本发明。
以下列举具体实施例来阐明本发明,然而应当理解,列举这些实施例,只是为了对本发明起说明作用,而非限制本发明的范围。
电荷异质性(iCIEF)
采用全柱成像毛细管等电聚焦电泳法来检测本品样品的电荷异质性和等电点。毛细管为100μm内径FC涂层熔融石英毛细管,有效分离长度5cm;样品处理时分别加入终浓度为4.0%的GE pharmalyte 3-10、0.35%的甲基纤维素,2mol/L尿素,样品终浓度0.5mg/ml;聚焦分离电压和时间为1.5kV-1min,3kV-8min。以Marker pI值计算目标峰的pI值。
分子排阻层析(SEC)
使用分子大小排阻层析来量化聚合体、单体和片段。这种测定利用WatersXbridge BEH SEC,7.8×300mm柱并在WatersHPLC系统上运行。流动相为50mM磷酸盐,200mM氯化钠缓冲液,pH7.5±0.1。样品进样量为20μg蛋白,以0.5mL/min的流速将蛋白质等度洗脱30min,于280nm检测洗脱物的吸光度。利用Empower 3软件进行积分处理。
毛细管电泳(CE-SDS)
通过非还原CE-SDS(nrCE)和还原CE-SDS(rCE)分别测定主峰和(LC+HC)纯度,将这一测定在BECKMAN COULTER PA800 plus毛细管电泳系统上以50μm I.D.非涂层石英毛细管进行,毛细管有效分离长度20.2cm(全长30.2cm),PDA220nm带宽10nm检测。
以下列举具体实施例来阐明本发明,应理解这些例子仅仅是为了说明本发明,而非限制本发明的范围。
实施例1 IL15/PD-L1双功能融合蛋白的制备
IL15/PD-L1双功能融合蛋白是CHO细胞表达的双功能融合蛋白。该双功能融合蛋白由IgG1型的抗PD-L1单抗通过其Fc段的C-末端与IL15RαSu及IL-15连接而成,抗PD-L1单抗的每条重链的C-末端均通过G4S连接子(G4S Linker)连接1个IL15RαSu和1个IL-15,IL15RαSu和IL-15之间也是由G4S Linker连接。其结构如附图1所示。该蛋白完整分子含2条由658个氨基酸残基组成的融合重链(包含PD-L1重链、IL15RαSu/IL-15及G4S Linker)和2条由215个氨基酸残基组成的轻链,链间通过二硫键连接。所述融合重链的序列如SEQ IDNO:1所示,所述轻链的序列如SEQ ID NO:2所示。
在CHO细胞中表达上述IL15/PD-L1双功能融合蛋白,经纯化,以用于进一步实验。
实施例2最适pH范围和缓冲剂筛选
由于IL15/PD-L1双功能融合蛋白的等电点范围大约在6.7~7.5,见附图2。因此在pH4.0~pH8.0范围内,考察5种常见缓冲剂对IL15/PD-L1双功能融合蛋白稳定性影响,根据不同缓冲剂的缓冲能力,设计与其对应不同pH范围的处方,详细信息如表1所示。
表1 IL15/PD-L1双功能融合蛋白制剂pH范围和缓冲剂筛选考察处方
对上述处方进行了40℃高温考察,通过SEC检测ILl5/PD-L1双功能融合蛋白分子的化学稳定性,通过蛋白浓度检测IL15/PD-L1双功能融合蛋白分子的物理稳定性。结果显示,当pH大于7.0以上,要么蛋白浓度出现不同程度的降低,如附图3所示(R12),要么出现聚集体的增加,如表2所示(R13~R14),说明在此pH条件下,由于接近IL15/PD-L1双功能融合蛋白的等电点,出现蛋白沉淀或聚集。表2所示,IL15/PD-L1双功能融合蛋白聚集体在不同缓冲盐中表现不一致,其中在琥珀酸盐和醋酸盐中,在pH5.0-pH6.0之间,聚集体含量最低,且40℃高温6天稳定。
表2 IL15/PD-L1双功能融合蛋白pH和缓冲剂筛选实验结果
注:R11和R12聚集体含量较少,其原因为大部分聚集体随蛋白共沉淀。
实施例3保护剂种类筛选
在上述实验结果上,以pH5.0琥珀酸盐缓冲体系为基础缓冲体系,进行不同辅料种类的筛选。考察糖类、多元醇类、氨基酸类、NaCl共计10种保护剂,具体考察处方设计见表3。
表3 IL15/PD-L1双功能融合蛋白保护剂种类筛选制剂处方
/>
对上述处方进行了40℃高温考察,通过SEC检测IL15/PD-L1双功能融合蛋白分子在不同保护剂中的稳定性,结果显示,见附图4,IL15/PD-L1双功能融合蛋白在糖类和多元醇类保护剂中稳定性较好,其中糖类保护剂优于多元醇类保护剂。在氨基酸和NaCl保护剂中,聚集体增加明显,说明其对小分子盐类较为敏感。接下来将在上述优选的糖类和多元醇类保护剂中考察冻干处方,冻干处方设计见表4。
表4 IL15/PD-L1双功能融合蛋白保护剂种类筛选冻干制剂处方
采用平台冻干工艺,对上述处方进行冻干处理。将冻干后的样品进行高温考察,通过复溶后的外观考察物理稳定性,结果显示见表5,随着高温加速甘露醇和甘氨酸处方复溶后乳光和浊度增加趋势明显,海藻糖、蔗糖、山梨醇处方乳光和浊度无明显变化最为稳定。通过SEC检测化学稳定性结果如附图5所示,蔗糖和海藻糖的稳定性更好,从成本角度出发,优选蔗糖保护剂。
表5复溶后外观
/>
实施例4优选pH和缓冲盐浓度筛选
在上述实验结果上,以琥珀酸-氢氧化钠缓冲体系为基础缓冲体系,进行不同pH和缓冲盐浓度的筛选,具体考察处方设计见表6。
表6IL15/PD-L1双功能融合蛋白优选pH和缓冲盐浓度筛选考察处方
/>
对上述处方样品通过40℃高温条件进行稳定性检查,结果显示外观稳定。在高温稳定性考察过程中,如附图6和附图7所示,IL15/PD-L1双功能融合蛋白表现出随着pH增加,聚集体产生、碎片降解的变化呈现下降的趋势,说明蛋白越稳定。因pH6.0接近于IL15/PD-L1双功能融合蛋白等电点,而上述pH考察实验中显示出IL15/PD-L1双功能融合蛋白在等电点时易于产生沉淀,基于风险控制考虑,选择pH5.5为优选pH。缓冲盐浓度5mM-20mM范围内,IL15/PD-L1双功能融合蛋白表现为随着缓冲盐浓度增加,聚集体增加趋势。进一步稳定性考察中,IL15/PD-L1双功能融合蛋白表现为在低缓冲盐浓度下,碎片更加稳定。因此选择5mM缓冲盐浓度为优选缓冲盐浓度。
实施例5表面活性剂种类筛选
在确定最适的缓冲盐、pH和保护剂基础上,进行表面活性剂种类筛选。考察聚山梨酯80和聚山梨酯20两种表面活性剂处方设计见下表7。
表7 IL15/PD-L1双功能融合蛋白表面活性剂种类筛选考察的制剂处方
对上述处方进行40℃高温考察稳定性,如附图8所示,相较于聚山梨酯20,聚山梨酯80表面活性剂能跟好的抵抗聚集体的产生,其稳定性较好。因此选择聚山梨酯80为优选的表面活性剂。
实施例6蛋白浓度筛选
在确定上述最适缓冲盐、pH、保护剂和表面活性剂的基础上,考察2-10mg/ml的IL15/PD-L1双功能融合蛋白浓度进行蛋白浓度考察。表8显示,在2-10mg/mL的蛋白浓度下,随着蛋白浓度的增加,溶液外观乳光略有增加,但澄清度保持良好,因此乳光增加与蛋白浓度有关,不影响制剂溶液的稳定性。40℃高温加速过程中,见图9,处方蛋白浓度越高,聚体增加比例越明显,2mg/ml与3mg/ml聚集趋势未见明显差异,优于5mg/ml及以上浓度。结合临床试验需求,为方便7μg~1950μg剂量皮下用药,以2mg/ml为最终蛋白浓度。
表8 IL15/PD-L1双功能融合蛋白不同蛋白浓度下的外观
实施例7稳定性考察
根据上述实验结果,确定最优制剂组成为2mg/ml IL15/PD-L1双功能融合蛋白,90mg/ml蔗糖,0.5mg/ml聚山梨酯80,5mmol/L琥珀酸-氢氧化钠缓冲液,pH值5.5。对上述最优选冻干或液体制剂进行稳定性研究。包括:强制条件试验(高温试验、强光照射试验)、加速试验、长期试验。稳定性研究中样品放置方式为正置;考察了液体样品的长期、加速稳定性,考察了冻干样品的长期、高温和光照稳定性性,光照试验样品除去外包装及瓶签进行。各考察条件如下表9所述:
表9 IL15/PD-L1双功能融合蛋白各稳定性实验考察条件
所用的冻干循环见下表10所述:
表10 IL15/PD-L1双功能融合蛋白冻干循环参数
/>
图10-图15证明IL15/PD-L1双功能融合蛋白在优选处方条件下液体-40℃长期12个月稳定,2-8℃加速6个月稳定。冻干制剂在拟定条件下,2-8℃存放12个月稳定,40℃和光照条件下均稳定。
实施例8 IL15/PD-L1双功能融合蛋白的活性评价
根据上述结果,对最优制剂的双功能融合蛋白进行结合活性的评估。采用ELISA法,测定双功能融合蛋白与IL-15Rβ和PD-L1蛋白的结合能力。首先将人IL-15Rβ蛋白包被在96孔板上,然后加入梯度稀释的双功能融合蛋白,再加入固定浓度的生物素标记的PD-L1蛋白。本品Fc端与IL-15Rβ蛋白结合,Fab端与生物素标记的PD-L1蛋白结合。再向96孔板中加入辣根过氧化物酶标记的链霉亲和素,最后加入TMB读物溶液显色后读取450nm出吸光值。通过SoftMax Pro计算机软件,使用四参数拟合回归模型进行曲线绘制并获得EC50值,结果如表11所示。结果显示,IL15/PD-L1双功能融合蛋白与IL-15Rβ和PD-L1蛋白都有良好的结合活性。
表11 IL15/PD-L1双功能融合蛋白结合活性结果
样品名称 | EC50值(ng/mL) |
IL15/PD-L1双功能融合蛋白 | 0.0134 |
Claims (18)
1.一种抗人IL15/PD-L1的双功能融合蛋白制剂,其特征在于:包含抗人IL15/PD-L1的双功能融合蛋白或其抗原结合片段和缓冲剂,所述抗人IL15/PD-L1双功能融合蛋白由IgG1型的抗PD-L1单克隆抗体通过其Fc段的C-末端与IL15RαSu及IL15连接而成,所述双功能融合蛋白的融合重链序列如SEQ ID NO:1所示,所述双功能融合蛋白的轻链序列如SEQ IDNO:2所示。
2.如权利要求1所述的制剂,其特征在于:所述缓冲剂选自醋酸盐缓冲液、琥珀酸盐缓冲液。
3.如权利要求2所述的制剂,其特征在于:醋酸盐缓冲剂为醋酸-醋酸钠缓冲液;琥珀酸盐缓冲剂为琥珀酸-氢氧化钠缓冲液;优选的所述缓冲剂为琥珀酸盐缓冲剂。
4.如权利要求1-3任一项所述的制剂,其特征在于:所述缓冲剂浓度为5mM至30mM,优选的为5mM。
5.如权利要求1-4任一项所述的制剂,其特征在于:所述制剂中还包含有稳定剂。
6.如权利要求1-5任一项所述的制剂,其特征在于:所述稳定剂为糖类和多元醇类,糖类选自蔗糖和海藻糖,多元醇类选自甘露醇、山梨醇和甘油,优选稳定剂为蔗糖和或海藻糖。
7.如权利要求5-6任一项所述的制剂,其特征在于:所述稳定剂浓度为30mg/mL至110mg/mL,优选的为50mg/mL至100mg/mL,更有选的为90mg/mL。
8.如权利要求1-7任一项所述的制剂,其特征在于:制剂中还包含有表面活性剂。
9.如权利要求8所述的制剂,其特征在于:所述表面活性剂选自聚山梨酯20、聚山梨酯80或泊洛沙姆188,优选为聚山梨酯80。
10.如权利要求8-9任一项所述的制剂,其特征在于:所述表面活性剂浓度0.2mg/mL至1mg/mL,优选的为0.3mg/mL至0.7mg/mL,更优选的为0.5mg/mL。
11.如权利要求1-10任一项所述的制剂,其特征在于:所述制剂的pH为4.0-7.0,优选为4.5-6.0,更优选的为5.3-5.7。
12.如权利要求1-11任一项所述的制剂,其特征在于:所述制剂包含0.5-30mg/ml的抗人IL15/PD-L1双功能融合蛋白,优选的为1-10mg/mL,更优选的为2-5mg/mL。
13.如权利要求1-12任一项所述的制剂,其特征在于:所述制剂包含1-10mg/ml的抗人IL15/PD-L1的双功能融合蛋白、50-100mg/ml的蔗糖、5-30mM的琥珀酸盐缓冲剂、0.3-0.7mg/ml的聚山梨酯80,溶液的pH为5.3-5.7。
14.一种含有抗人IL15/PD-L1双功能融合蛋白的冻干制剂,其特征在于:所述冻干制剂通过将权利要求1-13中任一项所述的制剂经冷冻干燥所得。
15.一种含有抗人IL15/PD-L1双功能融合蛋白的冻干制剂,其特征在于:所述冻干制剂经复溶后可形成如权利要求1-15中任一项所述的制剂。
16.一种产品,其包括容器,所述容器中包含如权利要求1-15中任一所述的制剂,或如权利要求14-15任一项所述的含有抗人IL15/PD-L1双功能融合蛋白的冻干制剂。
17.如权利要求1-13任一所述的制剂在制备用于治疗或预防IL15和PD-L1介导的疾病或者肿瘤的药物中的用途。
18.一种治疗方法,其特征在于:用于受试者中预防或治疗IL15和PD-L1介导的疾病或病症,所述的疾病优选为肿瘤;所述方法包括给予受试者施用权利要求1-13中任一所述的制剂。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210262247 | 2022-03-16 | ||
CN2022102622475 | 2022-03-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116763917A true CN116763917A (zh) | 2023-09-19 |
Family
ID=87986729
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310238467.9A Pending CN116763917A (zh) | 2022-03-16 | 2023-03-13 | 一种稳定的抗人il15/pd-l1双功能融合蛋白制剂 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116763917A (zh) |
-
2023
- 2023-03-13 CN CN202310238467.9A patent/CN116763917A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111971062B (zh) | 一种抗人pd-1的单克隆抗体制剂、联合用药物及其用途 | |
CN103608071B (zh) | 抗α4β7抗体的制剂 | |
AU2007238677B2 (en) | Use of IL-I antibodies for treating ophthalmic disorders | |
KR20160034307A (ko) | 안정화 항체 조성물 | |
CN110559435A (zh) | 包含离子性液体的液体蛋白质制剂 | |
TW201036650A (en) | Lyophilised antibody formulation | |
JP7475335B2 (ja) | Csf-1r抗体製剤 | |
TW201945031A (zh) | 低ph藥物製劑 | |
KR102106914B1 (ko) | Gm-csf를 중화하는 화합물을 포함하는 액체 제제 | |
US20220073616A1 (en) | Methods of administering anti-tim-3 antibodies | |
WO2020259605A1 (zh) | 包含抗cd47/pd-l1双特异性抗体的制剂及其制备方法和用途 | |
CN115803057A (zh) | 治疗多发性骨髓瘤的方法 | |
EP3714901A1 (en) | Lag-3 antibody pharmaceutical composition and use thereof | |
CN110960490A (zh) | 一种抗egfr抗体偶联药物组合物及其用途 | |
CN104780940B (zh) | 用于双特异性t细胞衔接体(bites)的制剂 | |
CN114129723A (zh) | 一种高浓度抗her2的抗体制剂及其用途 | |
US20200129633A1 (en) | Pharmaceutical composition comprising c-met antibody-drug conjugate and use thereof | |
CN116323657B (zh) | 同时靶向PD-L1和TGFβ的双功能分子及其医药用途 | |
WO2022068894A1 (zh) | 同时靶向pd-l1和vegf的双功能分子及其医药用途 | |
BR112020021082A2 (pt) | regimes de dosagem para inibição de tgf-b direcionada para uso no tratamento de câncer em indivíduos sem tratamento prévio | |
WO2019246595A1 (en) | Dosing regimens for targeted tgf-b inhibition for use in treating biliary tract cancer | |
CN116763917A (zh) | 一种稳定的抗人il15/pd-l1双功能融合蛋白制剂 | |
KR20180069906A (ko) | 항-인자 d 항체 제제 | |
CN116688115B (zh) | 一种PD-L1/TGF-β双功能融合蛋白制剂及其用途 | |
JP2023511356A (ja) | 組換え完全ヒト抗tigitモノクローナル抗体製剤及びその調製方法と使用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |