WO2023011662A1 - 抗Her-2抗体-粒细胞调节因子融合蛋白及其制法和应用 - Google Patents
抗Her-2抗体-粒细胞调节因子融合蛋白及其制法和应用 Download PDFInfo
- Publication number
- WO2023011662A1 WO2023011662A1 PCT/CN2022/110926 CN2022110926W WO2023011662A1 WO 2023011662 A1 WO2023011662 A1 WO 2023011662A1 CN 2022110926 W CN2022110926 W CN 2022110926W WO 2023011662 A1 WO2023011662 A1 WO 2023011662A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fusion protein
- antibody
- protein
- chain
- csf
- Prior art date
Links
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 169
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 165
- 230000001105 regulatory effect Effects 0.000 title abstract description 11
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 61
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims abstract description 59
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims abstract description 59
- 239000012634 fragment Substances 0.000 claims abstract description 48
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 45
- 239000000427 antigen Substances 0.000 claims abstract description 8
- 102000036639 antigens Human genes 0.000 claims abstract description 8
- 108091007433 antigens Proteins 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 96
- 206010028980 Neoplasm Diseases 0.000 claims description 88
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 49
- 210000002865 immune cell Anatomy 0.000 claims description 22
- 239000013598 vector Substances 0.000 claims description 22
- 108091033319 polynucleotide Proteins 0.000 claims description 20
- 102000040430 polynucleotide Human genes 0.000 claims description 20
- 239000002157 polynucleotide Substances 0.000 claims description 20
- 150000001413 amino acids Chemical class 0.000 claims description 19
- 206010006187 Breast cancer Diseases 0.000 claims description 14
- 208000026310 Breast neoplasm Diseases 0.000 claims description 14
- 239000008194 pharmaceutical composition Substances 0.000 claims description 11
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 10
- 206010017758 gastric cancer Diseases 0.000 claims description 10
- 230000014509 gene expression Effects 0.000 claims description 10
- 201000011549 stomach cancer Diseases 0.000 claims description 10
- 201000001441 melanoma Diseases 0.000 claims description 8
- 239000003937 drug carrier Substances 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 210000004899 c-terminal region Anatomy 0.000 claims description 5
- 206010005003 Bladder cancer Diseases 0.000 claims description 4
- 206010009944 Colon cancer Diseases 0.000 claims description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 4
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 201000007270 liver cancer Diseases 0.000 claims description 4
- 208000014018 liver neoplasm Diseases 0.000 claims description 4
- 201000005202 lung cancer Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 201000002528 pancreatic cancer Diseases 0.000 claims description 4
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 4
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 4
- 239000000833 heterodimer Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 28
- 210000004881 tumor cell Anatomy 0.000 abstract description 16
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 abstract description 9
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000009169 immunotherapy Methods 0.000 abstract description 3
- 231100001159 controllable toxicity Toxicity 0.000 abstract description 2
- 230000002195 synergetic effect Effects 0.000 abstract description 2
- 230000022534 cell killing Effects 0.000 abstract 1
- 231100000053 low toxicity Toxicity 0.000 abstract 1
- 229960000575 trastuzumab Drugs 0.000 description 68
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 39
- 238000000034 method Methods 0.000 description 31
- 229920001184 polypeptide Polymers 0.000 description 30
- 102000004196 processed proteins & peptides Human genes 0.000 description 30
- 210000000440 neutrophil Anatomy 0.000 description 28
- 241000699670 Mus sp. Species 0.000 description 25
- 125000003275 alpha amino acid group Chemical group 0.000 description 25
- 102000004127 Cytokines Human genes 0.000 description 17
- 108090000695 Cytokines Proteins 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 14
- 108060003951 Immunoglobulin Proteins 0.000 description 14
- 230000006870 function Effects 0.000 description 14
- 230000012010 growth Effects 0.000 description 14
- 102000018358 immunoglobulin Human genes 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 238000003752 polymerase chain reaction Methods 0.000 description 13
- 238000000338 in vitro Methods 0.000 description 12
- 239000002773 nucleotide Substances 0.000 description 12
- 125000003729 nucleotide group Chemical group 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 238000002965 ELISA Methods 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 10
- 239000013604 expression vector Substances 0.000 description 10
- 230000004614 tumor growth Effects 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 9
- 230000000295 complement effect Effects 0.000 description 9
- 239000013613 expression plasmid Substances 0.000 description 9
- 239000012091 fetal bovine serum Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 210000004962 mammalian cell Anatomy 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 7
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 108020004511 Recombinant DNA Proteins 0.000 description 7
- 230000027455 binding Effects 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 238000007796 conventional method Methods 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 230000001988 toxicity Effects 0.000 description 7
- 231100000419 toxicity Toxicity 0.000 description 7
- 238000010276 construction Methods 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 241000282412 Homo Species 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 230000006907 apoptotic process Effects 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 210000003527 eukaryotic cell Anatomy 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000001976 improved effect Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 239000012224 working solution Substances 0.000 description 5
- 210000005253 yeast cell Anatomy 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 4
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 230000000259 anti-tumor effect Effects 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108010075254 C-Peptide Proteins 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 description 3
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 239000000611 antibody drug conjugate Substances 0.000 description 3
- 229940049595 antibody-drug conjugate Drugs 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229960004927 neomycin Drugs 0.000 description 3
- 210000001236 prokaryotic cell Anatomy 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102000001398 Granzyme Human genes 0.000 description 2
- 108060005986 Granzyme Proteins 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 101000686031 Homo sapiens Proto-oncogene tyrosine-protein kinase ROS Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 241000235649 Kluyveromyces Species 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 102000003896 Myeloperoxidases Human genes 0.000 description 2
- 108090000235 Myeloperoxidases Proteins 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 102100023347 Proto-oncogene tyrosine-protein kinase ROS Human genes 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 101000996372 Rattus norvegicus Glutamine synthetase Proteins 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 229940126547 T-cell immunoglobulin mucin-3 Drugs 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000007123 defense Effects 0.000 description 2
- 229940127276 delta-like ligand 3 Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000004520 electroporation 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
- 230000004927 fusion Effects 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 208000025113 myeloid leukemia Diseases 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- -1 small molecule compounds Chemical class 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 208000037816 tissue injury Diseases 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- WGYHKLTYYQEAPK-BYPYZUCNSA-N (2S)-4-methylsulfanyl-2-(sulfoamino)butanoic acid Chemical compound S(=O)(=O)(O)N[C@@H](CCSC)C(=O)O WGYHKLTYYQEAPK-BYPYZUCNSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-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
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- XZWXFWBHYRFLEF-FSPLSTOPSA-N Ala-His Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 XZWXFWBHYRFLEF-FSPLSTOPSA-N 0.000 description 1
- PTVGLOCPAVYPFG-CIUDSAMLSA-N Arg-Gln-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PTVGLOCPAVYPFG-CIUDSAMLSA-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
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 102000001301 EGF receptor Human genes 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
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 208000017891 HER2 positive breast carcinoma Diseases 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 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
- 229940126528 Immuno-Oncology Drug Drugs 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 241001138401 Kluyveromyces lactis Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 239000002136 L01XE07 - Lapatinib Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 101710118186 Neomycin resistance protein Proteins 0.000 description 1
- BZQFBWGGLXLEPQ-UHFFFAOYSA-N O-phosphoryl-L-serine Natural products OC(=O)C(N)COP(O)(O)=O BZQFBWGGLXLEPQ-UHFFFAOYSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700026244 Open Reading Frames 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
- YTILBRIUASDGBL-BZSNNMDCSA-N Phe-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 YTILBRIUASDGBL-BZSNNMDCSA-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
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 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
- FZXOPYUEQGDGMS-ACZMJKKPSA-N Ser-Ser-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O FZXOPYUEQGDGMS-ACZMJKKPSA-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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 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
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 229940047120 colony stimulating factors Drugs 0.000 description 1
- 230000022811 deglycosylation Effects 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000014726 immortalization of host cell Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 229960004891 lapatinib Drugs 0.000 description 1
- BCFGMOOMADDAQU-UHFFFAOYSA-N lapatinib Chemical compound O1C(CNCCS(=O)(=O)C)=CC=C1C1=CC=C(N=CN=C2NC=3C=C(Cl)C(OCC=4C=C(F)C=CC=4)=CC=3)C2=C1 BCFGMOOMADDAQU-UHFFFAOYSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- USRGIUJOYOXOQJ-GBXIJSLDSA-N phosphothreonine Chemical compound OP(=O)(O)O[C@H](C)[C@H](N)C(O)=O USRGIUJOYOXOQJ-GBXIJSLDSA-N 0.000 description 1
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical compound OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 108020001775 protein parts Proteins 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000013605 shuttle vector Substances 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
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960003989 tocilizumab Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
-
- 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
- A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
-
- 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
- A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
-
- 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
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
Definitions
- the invention belongs to the fields of biotechnology and medicine, and in particular relates to an anti-Her-2 antibody-granulocyte regulatory factor fusion protein and its preparation method and application.
- HER-2 is a proto-oncogene, which belongs to the human epidermal growth factor receptor family. It inhibits cancer cell apoptosis and promotes its proliferation and invasion by regulating downstream signaling pathways. HER2 amplification or overexpression accounts for about 20% of breast cancer patients. %-30%. In addition, HER2 is often detected in gastric cancer.
- Trastuzumab the representative drug of HER-2 target, has achieved good curative effect in HER-2+ breast cancer and gastric cancer, but the resistance rate and recurrence rate of breast cancer to trastuzumab are increasing year by year High, the final drug resistance rate is as high as 65%, including 70% of patients who are sensitive to trastuzumab at the beginning of treatment, and eventually develop drug resistance. Therefore, new combinations are urgently needed to achieve effective control of HER-2+ tumors.
- Neutrophils are one of the most important defense systems in tissue injury, accounting for about 50%-70% of circulating leukocytes. Recent studies have shown that tumor-associated neutrophils (TANs) also play an important role in tumor immunity. Neutrophils can not only directly kill tumor cells through mechanisms such as granzyme B, but also induce apoptosis through antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, by enhancing the ADCC activity of anti-tumor antibody drugs, the function of neutrophils to kill tumor cells can be improved.
- ADCC antibody-dependent cell-mediated cytotoxicity
- the purpose of the present invention is to provide a fusion protein of anti-Her-2 antibody-granulocyte regulatory factor that is safer, more effective and precisely targets tumors.
- the purpose of the present invention is to provide an anti-Her-2 antibody-granulocyte regulatory factor fusion protein and its application.
- a fusion protein single chain is provided, and the fusion protein single chain includes the following elements fused together:
- the first protein element is an antigen recognition module
- the second protein element is granulocyte colony stimulating factor.
- the antigen recognition module includes an antibody or an active fragment thereof, and the antibody is selected from the group consisting of anti-CD20 antibody, anti-TIM-3 antibody, anti-LAG-3 antibody, anti-CD73 antibody, anti-CD47 antibody , anti-DLL3 antibody, anti-FRmAb antibody, anti-CTLA-4 antibody, anti-OX40 antibody, anti-CD137 antibody, anti-PD-1 antibody.
- the antibody is a monoclonal antibody.
- the active fragment is an active fragment containing antibody F(ab), F(ab')2, scFv, VH, CH, VL or VHH.
- the antigen recognition module is an anti-Her-2 antibody or an active fragment thereof.
- the anti-Her-2 antibody or its active fragment is an active fragment containing F(ab), F(ab')2, scFv, VH, CH, VL or VHH.
- the anti-Her-2 antibody or its active fragment is selected from the active fragment of trastuzumab.
- the linker element is a peptide bond or a peptide linker.
- the granulocyte colony-stimulating factor is derived from humans or non-human mammals, more preferably from rodents (such as mice, rats), primates and humans.
- the G-CSF includes wild type and mutant type.
- the G-CSF includes full-length, mature G-CSF, or an active fragment thereof.
- the G-CSF also includes derivatives of G-CSF.
- the derivatives of G-CSF include modified G-CSF, protein molecules whose amino acid sequence is homologous to natural G-CSF and have natural G-CSF activity, and dimers of G-CSF Or multimer, fusion protein containing G-CSF amino acid sequence.
- the "protein molecule whose amino acid sequence is homologous to natural G-CSF and has natural G-CSF activity” means that its amino acid sequence has a homology of ⁇ 85% compared with G-CSF, Preferably ⁇ 90% homology, more preferably ⁇ 95% homology, most preferably ⁇ 98% homology; and a protein molecule with G-CSF activity.
- a fusion protein consisting of a single chain of the fusion protein according to the first aspect of the present invention, the fusion protein comprising two single chains, wherein each single chain is from the N-terminus to the C-terminus Has the structure shown in following formula I:
- M1, M2, M3, M4 are each independently none or granulocyte colony-stimulating factor G-CSF, and at least one is not none;
- L1, L2, L3, L4 are each independently none or a bond or a peptide linker
- H-Chain is the heavy chain of anti-Her-2 antibody or its active fragment
- V-Chain is the anti-Her-2 antibody light chain or its active fragment
- the is one or more interchain disulfide bonds between heavy or light chains.
- said M1, M4, L1, L2, L3 and L4 are none.
- said M2, M3, L2 and L3 are none.
- the fusion proteins each have a structure selected from the following formulas II, III, IV or V:
- H-Chain is the heavy chain of anti-Her-2 antibody or its active fragment
- V-Chain is the anti-Her-2 antibody light chain or its active fragment
- M is granulocyte colony-stimulating factor
- the fusion protein is a dimer.
- the fusion protein is a homodimer or a heterodimer.
- the active heavy chain fragment includes or contains the heavy chain, VH, CH, VHH, Fc region or HCDR of the anti-Her-2 antibody.
- the active fragment of the light chain includes or contains the light chain, VL, CL or LCDR of the anti-Her-2 antibody.
- the anti-Her-2 antibody heavy chain or active fragment thereof includes a heavy chain variable region, a heavy chain constant region, and an Fc segment.
- the Fc fragment is derived from human or non-human mammal, more preferably from rodent (such as mouse, rat), primate and human.
- the Fc fragment is the Fc fragment of immunoglobulin IgG, preferably the Fc portion of IgG1.
- the anti-Her-2 antibody light chain or active fragment thereof includes a light chain variable region and a light chain constant region.
- the H-Chain is the heavy chain of trastuzumab.
- the V-Chain is the light chain of trastuzumab.
- M is granulocyte colony-stimulating factor G-CSF.
- the H-Chain or V-Chain is connected to G-CSF in a head-to-head, head-to-tail, or tail-to-tail manner.
- the "head” refers to the N-terminal of the polypeptide or its fragments, especially the N-terminal of the wild-type polypeptide or its fragments.
- the "tail” refers to the C-terminal of the polypeptide or its fragments, especially the C-terminal of the wild-type polypeptide or its fragments.
- the length of the peptide linker is 0-20 amino acids, preferably 1-15 amino acids.
- the H-Chain contains or has amino acids 1-449 in SEQ ID NO:11
- the V-Chain contains or has amino acids 1-214 in SEQ ID NO:14 amino acid
- the G-CSF contains or has amino acids 450-624 in SEQ ID NO:11, or amino acids 222-396 in SEQ ID NO:14.
- sequence of the peptide linker is 215-221 in SEQ ID NO:14.
- sequence of the fusion protein is selected from the following group:
- the H-chain-M is a fusion protein heavy chain, the sequence of which is shown in SEQ ID NO: 11.
- V-chain-M is a fusion protein light chain, the sequence of which is shown in SEQ ID NO:14.
- the polynucleotide additionally contains auxiliary elements selected from the following group at the flanks of the ORF of the mutein or fusion protein: signal peptide, secretory peptide, tag sequence (such as 6His), or its combination.
- the polynucleotide is selected from the group consisting of DNA sequence, RNA sequence, or a combination thereof.
- a vector comprising the polynucleotide described in the third aspect of the present invention.
- the vectors include: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, or other vectors.
- the vector contains one or more promoters, which are operably associated with the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, replication origin, selectable marker , nucleic acid restriction sites, and/or homologous recombination site connections.
- the vectors include expression vectors, shuttle vectors, and integration vectors.
- a host cell containing the vector according to the fourth aspect of the present invention or the polynucleotide according to the third aspect of the present invention integrated in the genome.
- the host cells include prokaryotic cells and eukaryotic cells.
- the host cells include mammalian cells.
- the host cells are eukaryotic cells, such as yeast cells, plant cells or mammalian cells (including human and non-human mammals).
- the host cell is a prokaryotic cell, such as Escherichia coli.
- the yeast cells are selected from one or more sources of yeast from the following group: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cells include: Luveromyces, more preferably Kluyveromyces marx, and/or Kluyveromyces lactis.
- the host cell is selected from the group consisting of Escherichia coli, wheat germ cells, insect cells, SF9, SP2/0, Hela, HEK293, CHO (such as CHOKS), yeast cells, or combinations thereof.
- a method for producing a fusion protein as described in the second aspect of the present invention comprising the steps of:
- composition comprising:
- a fusion protein according to the second aspect of the present invention is a fusion protein according to the second aspect of the present invention.
- the pharmaceutical composition also contains: additional active ingredients, preferably the active ingredients include: small molecule compounds, cytokines, antibodies (such as anti-PD-1 antibody, anti-OX40 antibody , anti-CD137 antibody, anti-CD47 antibody, ADC, CAR-immune cells).
- additional active ingredients include: small molecule compounds, cytokines, antibodies (such as anti-PD-1 antibody, anti-OX40 antibody , anti-CD137 antibody, anti-CD47 antibody, ADC, CAR-immune cells).
- the pharmaceutical composition is in the form of injection.
- an immune cell carrying the fusion protein according to the second aspect of the present invention there is provided an immune cell carrying the fusion protein according to the second aspect of the present invention.
- the immune cells include T cells.
- composition comprising:
- the fusion protein according to the second aspect of the present invention or the immune cell according to the eighth aspect of the present invention for preparing a drug for treating tumors.
- the tumors include: breast cancer tumors, gastric cancer tumors, bladder cancer tumors, pancreatic cancer tumors, colorectal cancer tumors, lung cancer tumors, liver cancer tumors, and melanoma tumors.
- the drug for treating tumors can be used in combination with another tumor immunotherapy, including but not limited to: chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb , anti-CD47 mAb, anti-DLL3 mAb, anti-FRmAb mAb, anti-CTLA-4 antibody, anti-OX40 antibody, anti-CD137 antibody, anti-PD-1 antibody, PD-1/PD-L1 therapy, other immuno-oncology drugs, anti-angiogenic agents , radiation therapy, antibody-drug conjugate (ADC), targeted therapy, or other anticancer drugs.
- another tumor immunotherapy including but not limited to: chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb , anti-CD47 mAb, anti-DLL3 mAb, anti-FRmAb mAb, anti-CTLA-4 antibody, anti-OX40 antibody, anti-CD137 antibody,
- the eleventh aspect of the present invention provides a method for preventing and/or treating tumors, comprising the step of: administering the fusion protein described in the second aspect of the present invention to a subject in need.
- the fusion protein is administered in the form of monomer and/or dimer.
- the subject is human.
- the tumors include: breast cancer tumors, gastric cancer tumors, bladder cancer tumors, pancreatic cancer tumors, colorectal cancer tumors, lung cancer tumors, liver cancer tumors, and melanoma tumors.
- Fig. 1 shows four structural schematic diagrams of the embodiment of the anti-Her-2 antibody-granulocyte colony stimulating factor fusion protein of the present invention (Fig. 1A, Fig. 1B, Fig. 1C, Fig. 1D).
- Figure 2 shows the SDS-PAGE electrophoresis analysis of the heavy chain-G-CSF trastuzumab fusion protein.
- Figure 2A Non-reducing 6% SDS-PAGE electrophoresis analysis.
- Figure 2B Analysis by reducing 10% SDS-PAGE electrophoresis.
- Lane 1 is trastuzumab;
- lane 2 is heavy chain-G-CSF trastuzumab fusion protein;
- MW is protein molecular weight standard (kDa).
- Figure 3 shows the ELISA study of the heavy chain-G-CSF trastuzumab fusion protein binding to recombinant human Her-2 ECD in vitro ( Figure 3A), and the flow cytometric analysis of the binding to membrane Her-2 ( Figure 3A). 3B).
- Figure 4 shows the study of the heavy chain-G-CSF trastuzumab fusion protein inhibiting the growth of Her-2 positive breast cancer cell BT-474 in vitro.
- Figure 5 shows the study of heavy chain-G-CSF trastuzumab fusion protein stimulating the growth of mouse myeloid leukemia lymphocyte NFS-60 in vitro
- Figure 6 shows that the heavy chain-G-CSF trastuzumab fusion protein promotes the proliferation of neutrophils in mouse blood in vivo.
- Figure 7 shows the study of the heavy chain-G-CSF trastuzumab fusion protein inhibiting the growth of mouse melanoma cell B16 expressing human Her-2 in mice.
- Fig. 7A curves of average tumor volume changes in mice in each group;
- Fig. 7B curves of average weight changes in mice in each group.
- the present invention relates to a novel fusion protein consisting of tumor-associated targeting elements, preferably monoclonal antibodies or fragments thereof, and colony-stimulating factors. It specifically recognizes molecules expressed on human tumors, such as human epidermal growth factor receptor (HER-2) and carries granulocyte colony stimulating factor (G-CSF).
- the resulting fusion protein can specifically bind to HER-2 expressed in tumor tissue to inhibit tumor growth, and at the same time deliver G-CSF to targeted tumor tissue to enhance the tumor-killing effect of neutrophils.
- the new fusion protein can be used for the treatment of HER2+ tumors. The present invention has been accomplished on this basis.
- the fusion protein involved in the present invention consists of the following two parts: (1) a full-length monoclonal antibody that recognizes the tumor-specific antigen Her-2 or a part that recognizes the minimum antigen; (2) regulates the proliferation, differentiation and activation of neutrophils Cytokines such as granulocyte colony-stimulating factor G-CSF.
- the fusion protein contains the heavy chain of anti-Her-2 antibody, which contains or does not contain CH1 or CH2 or CH3 of the heavy chain constant region, and its C-terminus is connected with active cytokines such as G-CSF fusion.
- an anti-Her-2 antibody-cytokine (such as G-CSF) fusion protein can be produced, and this fusion protein can bind to the expression of Her-2 tumor cells and can deliver cytokines to tumor sites.
- a biologically active neutrophil regulatory cytokine can also be fused with an anti-Her-2 single-chain antibody.
- the complete fusion protein is a polypeptide chain, and each functional region is connected by a connecting peptide to ensure that the fusion protein has the correct The spatial structure maintains its biological activity.
- the fusion protein of the present invention is a class of brand-new molecules with two biological functions: first, they can target tumor tissues expressing Her-2, and inhibit the growth of tumors by blocking the function of Her-2; ADCC or CDC function to kill tumor cells. Second, they can specifically deliver biologically active cytokines to tumor sites. These cytokines have the function of regulating the activity of immune cells. Therefore, they can increase the infiltration of immune cells in tumor tissues and enhance the activity of immune cells, so that the growth of tumors, such as breast cancer and gastric cancer, can be inhibited. Since cytokines are mainly confined to the tumor tissue site, the toxicity to patients is relatively small. Therefore, the object of the present invention is to provide an antibody-cytokine fusion protein containing a monoclonal antibody or antibody fragment targeting Her-2-expressing tumors and fused with a biologically active cytokine.
- the antibody in the fusion protein of the present invention can be a full-length antibody, or a key fragment of the antibody, such as scFv, F(ab)2, etc.
- all antibodies that can bind to the Her-2 receptor on the tumor cell membrane are suitable for constructing the antibody-granulocyte colony-stimulating factor fusion protein of the present invention.
- trastuzumab is the preferred antibody.
- the object of the present invention is to provide an antibody-granulocyte colony-stimulating factor fusion protein, the antibody part of which is a full-length antibody or an antibody fragment containing the necessary variable region sequence, such as F(ab) or F(ab)2 or scFv .
- the cytokine part of the fusion protein of the present invention is selected from biologically active G-CSF, and is connected with the antibody part directly or through a connecting peptide chain.
- the content of the present invention also includes a method for producing and preparing an antibody-cytokine fusion protein, by directly or indirectly fusing the nucleotide sequence encoding the antibody with the nucleotide sequence of the cytokine, cloning it into an expression vector, and then transfecting the vector into cells , culture the transfected cells in a suitable medium to obtain the antibody-cytokine fusion protein.
- the antibody-cytokine fusion protein of the present invention can be used for clinical tumor treatment. Therefore, the content of the present invention includes the composition of a clinical therapeutic drug, which contains at least one of the fusion proteins of the present invention and a physiologically acceptable carrier.
- fusion protein of the present invention Her-2 antibody-granulocyte colony-stimulating factor fusion protein of the present invention
- Her-2 antibody-G-CSF fusion protein refers to the fusion protein mentioned in the second aspect of the present invention.
- the term "about” when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value.
- the expression “about 100” includes all values between 99 and 101 and in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).
- Fc refers to the Fc fragment of a human immunoglobulin.
- immunoglobulin Fc region refers to the constant region of the immunoglobulin chain, especially the carboxyl terminal of the constant region of the heavy chain of the immunoglobulin or a part thereof.
- the Fc region of the immunoglobulin used comprises at least one immunoglobulin hinge region, a CH2 domain and a CH3 domain, preferably lacking CH1 domain.
- the globulin Fc region is within the scope of those skilled in the art.
- the immunoglobulin Fc region can be selected to include the coding sequence of the Fc region of the human immunoglobulin IgG4 subclass, in which an immunoglobulin Fc region is deleted.
- Globulin heavy chain 1 domain (CH1) but includes the coding sequence of the hinge region and CH2, CH3, two domains.
- the words “comprising”, “having” or “comprising” include “comprising”, “consisting essentially of”, “consisting essentially of”, and “consisting of”;” “Mainly consist of”, “essentially consist of” and “consist of” belong to the sub-concepts of "contain", “have” or “include”.
- Neutrophils are one of the most important defense systems in tissue injury, accounting for about 50%-70% of circulating leukocytes. Recent studies have shown that tumor-associated neutrophils (TANs) also play an important role in tumor immunity. Under the induction of some cytokines, neutrophils can effectively kill tumor cells, such as under the induction of TNF ⁇ , neutrophils kill tumor cells through the ROS pathway, and under the induction of IFN- ⁇ and IL-2 Under these conditions, neutrophils exert direct toxicity on tumor cells by expressing granzyme B. Neutrophils can also induce tumor cell apoptosis through antibody-dependent cell-mediated cytotoxicity (ADCC).
- ADCC antibody-dependent cell-mediated cytotoxicity
- neutrophils Under the induction of radiotherapy, it will cause a large number of neutrophils to infiltrate the tumor tissue, and finally pass through The ROS pathway induces tumor cell apoptosis.
- neutrophils can express tumor necrosis factor-related apoptosis ligand (TRAIL) and myeloperoxidase (MPO) with direct killing activity to exert anti-tumor effects.
- TRAIL tumor necrosis factor-related apoptosis ligand
- MPO myeloperoxidase
- neutrophils can also indirectly enhance the anti-tumor effect by stimulating the proliferation of T cells, promoting the release of IFN- ⁇ , and activating dendritic cells.
- G-CSF granulocyte colony-stimulating factor
- neutrophils involved in anti-tumor have a short half-life, and after a certain period of time, they will After neutrophil depletion occurs, significant tumor growth is observed. If G-CSF is added to the treatment at the same time to promote the activation and migration of neutrophils in the tumor microenvironment, neutrophils The number of granulocytes increased significantly, the tumor immune response was significantly enhanced, and the growth of tumors was effectively inhibited.
- the fusion protein is an isolated protein, not associated with other proteins, polypeptides or molecules, expressed by recombinant host cells, or an isolated or purified product.
- the fusion protein constructed by the present invention consists of the following two parts:
- a biologically active granulocyte colony-stimulating factor in the granulocyte colony-stimulating factor family such as G-CSF or GM-CSF.
- the fusion protein contains the heavy chain of anti-Her-2 antibody, which contains or does not contain CH1 or CH2 or CH3 of the heavy chain constant region, and its C-terminus is connected to the active cell granulocyte Colony-stimulating factor fusion.
- an anti-Her-2 antibody-granulocyte colony-stimulating factor (such as G-CSF) fusion protein can be produced, and this fusion protein can bind and express Her -2 tumor cells, and can deliver granulocyte colony-stimulating factor to the tumor site.
- G-CSF anti-Her-2 antibody-granulocyte colony-stimulating factor
- the biologically active granulocyte colony-stimulating factor is fused with an anti-Her-2 single-chain antibody.
- the complete fusion protein is a polypeptide chain, and each functional region is connected by a connecting peptide to ensure that the fusion protein has the correct spatial structure and maintains its biological structure. active.
- the fusion proteins of the present invention are a class of brand-new molecules with two biological functions: first, they can target tumor tissues expressing Her-2, and second, they can specifically deliver biologically active cytokines to tumors parts. These cytokines have the function of attracting immune cells and regulating the activity of immune cells. Therefore, they can increase the tumor tissue infiltration of immune cells and enhance the activity of immune cells, so that the growth of tumors, such as breast cancer and gastric cancer, can be inhibited. Since granulocyte colony-stimulating factor is mainly confined to the tumor tissue site, the toxicity to patients is relatively small.
- the antibody in the fusion protein of the present invention can be a full-length antibody, or a key fragment of the antibody, such as scFv, F(ab)2 or VHH.
- all antibodies that can bind to the Her-2 receptor on the tumor cell membrane are suitable for constructing the antibody-granulocyte colony-stimulating factor fusion protein of the present invention (trastuzumab, lapatinib, and Tocilizumab).
- trastuzumab is preferred.
- the granulocyte colony-stimulating factor part of the fusion protein of the present invention is linked to the antibody part directly or through a peptide linker.
- the invention provides a fusion protein comprising the following elements:
- G-CSF granulocyte colony stimulating factor
- linker element there may or may not be a linker between the elements (such as between element a and element b).
- the fusion protein of the present invention not only has a longer half-life in vivo, but also can more effectively inhibit the concentration of antibodies (especially IgE) related to immune diseases in serum.
- amino acid sequence provided by the present invention, those skilled in the art can conveniently use various known methods to prepare the fusion protein of the present invention. These methods are for example but not limited to: recombinant DNA method, artificial synthesis, etc.
- a preferred fusion protein is Trastuzumab HC-G-CSF fusion protein, its heavy chain nucleotide sequence is as shown in SEQ ID NO: 6, and the heavy chain amino acid sequence is as shown in SEQ ID NO: 11; wherein , the 1-449th position in the heavy chain amino acid sequence (SEQ ID NO: 11) is the amino acid sequence of trastuzumab; the 450th-624th position is the G-CSF amino acid sequence.
- a preferred fusion protein is Trastuzumab LC-linker-G-CSF fusion protein, its light chain nucleotide sequence is as shown in SEQ ID NO:9, and the light chain amino acid sequence is as shown in SEQ ID NO:14 ; Wherein, 1-214 in the light chain amino acid sequence (SEQ ID NO: 14) is the light chain amino acid sequence of trastuzumab; 222-396 is the G-CSF amino acid sequence.
- the light chain nucleotide sequence of the trastuzumab HC-G-CSF fusion protein of the present invention is shown in SEQ ID NO: 7, and the light chain amino acid sequence is shown in SEQ ID NO: 12 .
- the heavy chain nucleotide sequence of Trastuzumab LC-G-CSF or LC-linker-G-CSF fusion protein of the present invention is shown in SEQ ID NO: 8, and the heavy chain amino acid sequence As shown in SEQ ID NO:13.
- isolated means that the material is separated from its original environment (if the material is native, the original environment is the natural environment).
- polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state.
- isolated recombinant fusion protein means that the recombinant fusion protein is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Essentially pure proteins yield a single major band on non-reducing polyacrylamide gels.
- fusion protein also includes variant forms of the fusion protein (such as the sequence shown in SEQ ID NO.: 1 or 2) having the above-mentioned activity. These variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, and additions or substitutions at the C-terminal and/or N-terminal Deletion of one or several (usually within 3, preferably within 2, more preferably within 1) amino acids. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. Furthermore, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (eg, cyclic peptides).
- the present invention also includes active fragments, derivatives and analogs of the above fusion proteins.
- fragment refers to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention.
- polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A polypeptide with substituent groups in amino acid residues, or (iii) a polypeptide formed by fusing an antigenic peptide to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a fusion protein fused to a leader sequence, a secretory sequence, or a tag sequence such as 6 ⁇ His).
- polypeptide fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.
- One class of preferred active derivatives refers to the formation of at most 3, preferably at most 2, and more preferably at most 1 amino acid replaced by amino acids with similar or similar properties compared with the amino acid sequence of formula I or formula II peptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
- the invention also provides analogs of the fusion proteins of the invention.
- the difference between these analogues and the polypeptide shown in SEQ ID NO.: 11, 12, 13 or 14 may be a difference in amino acid sequence, or a modification that does not affect the sequence, or both.
- Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, ⁇ , ⁇ -amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
- Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.
- glycosylation such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme
- a polynucleotide of the invention may be in the form of DNA or RNA.
- Forms of DNA include cDNA, genomic DNA or synthetic DNA.
- DNA can be single-stranded or double-stranded.
- DNA can be either the coding strand or the non-coding strand.
- the present invention also relates to variants of the above polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention.
- Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants.
- an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the encoded polypeptide.
- primer refers to a general term for oligonucleotides that can be used as a starting point to synthesize a DNA chain complementary to a template under the action of a DNA polymerase when paired with a template.
- Primers can be natural RNA, DNA, or any form of natural nucleotides. Primers can even be non-natural nucleotides such as LNA or ZNA, etc.
- a primer is “substantially” (or “essentially”) complementary to a particular sequence on one strand of the template. A primer must be sufficiently complementary to one strand of the template to initiate extension, but the sequence of the primer does not have to be perfectly complementary to that of the template.
- a non-complementary sequence to the 5' end of a primer whose 3' end is complementary to the template, such a primer is still substantially complementary to the template.
- non-completely complementary primers can also form a primer-template complex with the template, thereby performing amplification.
- amino acid sequence provided by the present invention, those skilled in the art can conveniently use various known methods to prepare the fusion protein of the present invention. These methods are for example but not limited to: recombinant DNA method, artificial synthesis, etc.
- the full-length nucleotide sequence or fragments of the elements of the fusion protein of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis.
- primers can be designed according to the published relevant nucleotide sequences, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as Template, amplified to obtain related sequences.
- a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as Template, amplified to obtain related sequences.
- the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then the fragments amplified each time are spliced together in the correct order.
- recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
- related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
- the method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention.
- Primers for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods.
- Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
- the present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering with the vector or fusion protein coding sequence of the present invention, and a method for producing the protein of the present invention through recombinant technology.
- polynucleotide sequences of the present invention can be used to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally speaking, there are the following steps:
- expression vectors containing the coding DNA sequence of the protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis.
- the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
- the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
- selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
- Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.
- the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
- a prokaryotic cell such as a bacterial cell
- a lower eukaryotic cell such as a yeast cell
- a higher eukaryotic cell such as a mammalian cell.
- Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS, or 293 cells, etc.
- a particularly preferred cell is human and non-human mammalian cells, especially immune cells, including T cells, NK cells.
- Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
- the host is a prokaryotic organism such as Escherichia coli
- competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with CaCl2 using procedures well known in the art.
- Another method is to use MgCl2 . Transformation can also be performed by electroporation, if desired.
- the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
- the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
- the medium used in the culture can be selected from various conventional media according to the host cells used.
- the culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
- the protein in the above method may be expressed inside the cell, or on the cell membrane, or secreted outside the cell. Proteins can be isolated and purified by various separation methods by taking advantage of their physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
- the bifunctional fusion proteins of the invention may optionally contain a peptide linker or not.
- Peptide linker size and complexity may affect protein activity.
- the peptide linker should be of sufficient length and flexibility to ensure that the two proteins being linked have sufficient degrees of freedom in space to function. At the same time, the influence of the formation of ⁇ -helix or ⁇ -sheet in the peptide linker on the stability of the fusion protein is avoided.
- the length of the peptide linker is generally 0-20 amino acids, preferably 1-15 amino acids.
- Examples of preferred peptide linkers include (but are not limited to): GSGGGGS, (GGGGS) n , wherein n is an integer of 1-8, preferably n is 1, 2 or 3.
- the amino acid sequence of the peptide linker is: 215-221 in the trastuzumab LC-linker-G-CSF amino acid sequence (SEQ ID NO: 14).
- the present invention also provides a composition, which contains (a) an effective amount of the fusion protein of the present invention and/or an effective amount of the immune cell of the present invention, and a pharmaceutically acceptable carrier.
- the fusion protein of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably, the pH is about 6-8.
- the term "effective amount” or “effective dose” refers to the amount that can produce functions or activities on humans and/or animals and can be accepted by humans and/or animals, such as 0.001-99wt%; preferably 0.01-95wt%; more preferably, 0.1-90wt%.
- an effective amount or “effective dose” refers to 1 ⁇ 10 3 -1 ⁇ 10 7 immune cells/ml.
- a "pharmaceutically acceptable” ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation and allergic reactions), ie, has a reasonable benefit/risk ratio.
- pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
- the pharmaceutical composition of the present invention contains a safe and effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier.
- Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
- the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants.
- the pharmaceutical composition is preferably produced under sterile conditions.
- the amount of active ingredient administered is a therapeutically effective amount.
- the pharmaceutical preparations of the present invention can also be made into sustained-release preparations.
- the effective amount of the fusion protein of the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. The selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the fusion protein of the present invention such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the body weight of the patient, the immune status of the patient, the dose way etc. Usually, when the fusion protein of the present invention is administered at a dose of about 5 mg-20 mg/kg animal body weight (preferably 5 mg-10 mg/kg animal body weight) per day, satisfactory effects can be obtained. For example, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.
- the fusion protein of the present invention is particularly suitable for treating diseases such as tumors.
- Representative tumors include (but are not limited to): breast cancer tumors, gastric cancer tumors, bladder cancer tumors, pancreatic cancer tumors, colorectal cancer tumors, lung cancer tumors, liver cancer tumors, and melanoma tumors.
- the present invention constructs a new combination of anti-Her-2 antibody and G-CSF, the fusion protein targets the tumor tissue expressing Her-2, and inhibits the growth of the tumor by blocking the function of Her-2; Or kill tumor cells through ADCC or CDC function. It has the advantages of precise identification, immunotherapy, controllable toxicity, and enhanced ADCC.
- the fusion protein of the present invention can specifically deliver the biologically active cytokine to the tumor site.
- These cytokines have the function of regulating the activity of immune cells. Therefore, they can increase the infiltration of immune cells in tumor tissues and enhance the activity of immune cells, so that the growth of tumors, such as breast cancer and gastric cancer, can be inhibited. Since cytokines are mainly confined to the tumor tissue site, the toxicity to patients is relatively small.
- trastuzumab is an example of a Her-2 antibody.
- the complete cDNAs encoding the heavy and light chains of trastuzumab were synthesized by GenScrip (USA) and cloned in the pUC57 vector, respectively.
- the cDNA of human G-CSF was purchased from OpenBiosystems (USA).
- the gene encoding the heavy chain of trastuzumab and the gene encoding G-CSF were linked by two-step polymerase chain reaction (PCR) method.
- PCR polymerase chain reaction
- the first step use the PCR method (high-fidelity polymerase Pfx, Invitrogen) to amplify the heavy chain gene using artificially synthesized antibody heavy chain DNA as a substrate:
- 5' primer M13-F (SEQ ID NO: 1): 5'-TGTAAAACGACGGCCAGT-3', located on the pUC57 vector.
- the 3' end primer KDP004 (SEQ ID NO: 2): 5'-TCCTGGGGACAGTGACAGTG-3' is a specific primer for antibody heavy chain genes.
- the first 20 nucleotide sequences of the primer KDP047 are complementary to the nucleotide sequence of the primer KDP004, so that the two PCR fragments can be connected in the second step of overlapping extension PCR.
- the 5' end primer M13-F (SEQ ID NO: 1), the 3' end primer KDP048 (SEQ ID NO: 5), 5'-TGGTGGTGTCTAGAGACTCAGGGCTGGGCAAGGTGG-3', contains the Xba I restriction sequence for cloning.
- Not I enzyme cutting site before the transcription initiation site of the heavy chain gene of trastuzumab, so that Not I/Xba I double enzyme cutting (Takara) is performed after gel purification of the fragment obtained by overlapping PCR.
- the digested PCR fragment is then cloned into a similarly digested mammalian cell expression vector.
- This mammalian cell expression vector is an improved pcDNA3.1 (Invitrogen).
- the anti-neomycin (neomycin) gene in pcDNA3.1 is replaced by the rat glutamine synthetase gene.
- the improved vector is suitable for screening stable transfection Mammalian cells with high protein expression.
- the recombinant plasmid was transfected into DH5a competent bacteria, the positive colony containing the correct recombinant plasmid was identified by colony PCR method, and the recombinant plasmid was purified. It was identified by enzyme digestion and sequencing that the trastuzumab heavy chain-G-CSF recombinant gene had the correct sequence.
- trastuzumab light chain-linker-G-CSF was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (China), and cloned into the above-mentioned improved pcDNA3.1 vector.
- trastuzumab heavy chain and light chain expression genes in the pUC57 vector were respectively cloned into the improved pcDNA3.1 vector by subcloning.
- Cloning enzymes are Not I and Xba I.
- CHO-KS is a CHO-K1 cell grown in a medium containing fetal bovine serum (FBS) through gradually reducing the FBS content in the medium until cultured in a FBS-free medium, and finally acclimated to an OptiCHO medium without FBS ( Cells grown in suspension in Invitrogen).
- FBS fetal bovine serum
- the anti-neomycin gene in the pcDNA3.1 vector containing the antibody-cytokine fusion protein gene was replaced with the rat glutamine synthetase gene, and the heavy chain and The light chain expression plasmid was co-transfected into CHO-KS cells, and after the transfected cells were cultured for 24-48 hours, the transfected cells were screened and cultured on a 96-well culture plate by the limited dilution method.
- the selection medium was OptiCHO, 5 ⁇ g/ml recombinant human insulin and 10 M sulfomethionine (MSX). Incubate the cells in an incubator at 37°C with 8% CO 2 .
- ELISA method alkaline phosphatase-coupled goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab
- the positively expressed cell population is further amplified, detected by ELISA, amplified again, and finally a stable cell line expressing the antibody-cytokine fusion protein is obtained.
- the heavy chain-G-CSF trastuzumab and light chain-G-CSF trastuzumab highly expressed cell lines obtained in Example 2 were cultured and expanded. Centrifuge the cell culture fluid, collect the supernatant, and purify the fusion protein from the supernatant with Protein-A affinity chromatography column.
- the non-reducing SDS-PAGE gel of Figure 2A shows that the molecular weight of the heavy chain-G-CSF trastuzumab fusion protein is larger than that of trastuzumab, which is very close to its theoretical value of 183 kDa.
- the reducing SDS-PAGE electrophoresis gel of Figure 2B shows that the heavy chain molecular weight of the heavy chain-G-CSF trastuzumab fusion protein is larger than that of the trastuzumab heavy chain, which is consistent with its theoretical molecular weight (68kDa).
- the light chain of the heavy chain-G-CSF trastuzumab fusion protein is identical to that of trastuzumab.
- the heavy chain-G-CSF trastuzumab and trastuzumab were diluted in PBST-conjugated solution containing 1% BSA, respectively, to prepare 3-fold serially diluted solutions. Pour off the blocking solution, add diluted antibody, 50 ⁇ L/well, and react in a 37°C incubator for 1 hour. Pour off the solution, wash the ELISA plate three times with PBST, add 50 ⁇ L/well of secondary antibody (alkaline phosphatase-coupled goat anti-human IgG Fab antibody, Jackson ImmunoResearch Lab), and react in a 37°C incubator for 1 hour.
- secondary antibody alkaline phosphatase-coupled goat anti-human IgG Fab antibody, Jackson ImmunoResearch Lab
- Human breast cancer cell line BT-474 (purchased from the Cell Bank of Chinese Academy of Sciences) is a cell line with high expression of Her-2. Take an appropriate amount of BT-474 cells, adjust the cell density to 2 ⁇ 10 6 cells/ml with pre-cooled FACS working solution (0.1% FBS in PBS), aliquot 100 ⁇ L/tube, and block on ice for 1 hour.
- FIG. 3A the heavy chain-G-CSF trastuzumab fusion protein could specifically bind to Her-2 ECD, with an EC 50 of 72 ng/mL.
- the flow cytometry test showed that the heavy chain-G-CSF trastuzumab fusion protein can bind to Her-2 on the cell membrane, and the binding ability is equivalent to that of trastuzumab, as shown in Figure 3B.
- the fusion protein of the present invention fully retains the characteristics of the combination of trastuzumab and Her-2.
- trastuzumab inhibits the growth of Her-2-positive human tumor cells, such as human breast cancer BT-474, in vitro.
- BT-474 cells were cultured in RPMI1660 medium containing 10% FBS. After BT-474 cells were cultured in 96-well culture plates for 1 day, serial dilutions of trastuzumab-G-CSF fusion protein were added, and the culture was continued for 5 days. Then add the cell viability detection reagent CCK-8, and read the plate with a microplate reader at a dual wavelength of 450nm/655nm.
- NFS-60 mouse myeloid leukemia lymphocytes
- G-CSF G-CSF
- trastuzumab-G-CSF fusion protein The effect of trastuzumab-G-CSF fusion protein on the growth of NFS-60 cells in vitro was studied.
- NFS-60 cells come from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, and the cells are cultured in RPMI1640/10% FBS (Gibco) medium. After NFS-60 cells were cultured in a 96-well culture plate for 1 day, serially diluted trastuzumab-G-CSF fusion protein or recombinant human G-CSF (rhG-CSF) was added, and the culture was continued for 3 days. Then add the cell viability detection reagent CCK-8, and read the plate with a microplate reader at a dual wavelength of 450nm/655nm.
- mice Eight 10-12-week-old C57BL/6 mice were divided into two groups, and were given PBS and 2.5mg/kg dose of heavy chain-G-CSF trastuzumab fusion protein intravenously, and blood was collected after 72 hours, and put into In a test tube containing anticoagulant. Then FITC fluorescently labeled anti-mouse CD45 antibody and PE fluorescently labeled anti-mouse Gr-1 antibody were added, and the blood cells bound to the fluorescent antibodies were analyzed by flow cytometry.
- the content of neutrophils (Gr-1 positive) in the blood of mice in the PBS group was about 28%, while the content of neutrophils in the blood of mice in the heavy chain-G-CSF trastuzumab fusion protein group was about 50%. , indicating that the heavy chain-G-CSF trastuzumab fusion protein can promote the proliferation of neutrophils in mouse blood (results shown in Figure 6).
- Example 8 Construction of mouse tumor cells stably expressing human Her-2
- the mouse melanoma cell B16 comes from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, and the cells are cultured in RPMI1640/10% FBS (Gibco) medium.
- the human Her-2 expression gene was cloned in the expression vector pcDNA3.1 (Invitrogen), and the recombinant plasmid was transfected into the mouse melanoma cell B16 with Lipofectmaine 3000 (Invitrogen), and the transfected cells were prepared in RPMI/ Cultured in 10% FBS medium to obtain a stable cell pool. From the stable cell pool, the monoclonal stable cell line B16/Her-2 expressing human Her-2 was screened by flow cytometry (Influx, BD Biosciences).
- C57BL/6 mice were from Shanghai Slack Co., Ltd. and were raised in an SPF environment.
- mice Thirty-two C57BL/6 mice aged 6-7 weeks were divided into 4 groups, 8 mice in each group, half male and half male. By subcutaneous inoculation, 1 x 10 6 cells/mouse of B16/Her-2 cells were injected into the underarm of mice.
- mice Eight days after cell inoculation (D8), mice were given PBS (control group) or Trastuzumab-G-CSF fusion protein 10 mg/kg or Trastuzumab 10 mg/kg or combined Trastuzumab 8mg/kg and rhG-CSF 2mg/kg (the mass ratio of trastuzumab to G-CSF in the trastuzumab-G-CSF fusion protein molecule is 4:1), administered twice a week for a total of Dosing 4 times. The tumor volume was measured at each administration, and the mice were weighed.
- Figure 7 shows that compared with the average tumor volume of mice in the control group, at D20, the relative proliferation rate of the tumors in the heavy chain-G-CSF trastuzumab fusion protein group was 30%, significantly inhibiting the B16/Her- 2 Tumor growth in mice. Trastuzumab had little effect on tumor growth at a dose of 10 mg/kg. Experiments have proved that the heavy chain-G-CSF trastuzumab fusion protein is much better than trastuzumab in inhibiting the growth of B16/Her-2 tumors in vivo.
- Nude mice come from Shanghai Slack Co., Ltd. and are kept in an SPF environment.
- mice aged 6-7 weeks were divided into 3 groups, 8 in each group, half male and half male.
- 1 x 10 6 cells/mouse of NCI-N87 cells were injected into the underarm of mice.
- mice Eight days after cell inoculation (D8), mice were given PBS (control group) or Trastuzumab-G-CSF fusion protein 10 mg/kg or Trastuzumab 10 mg/kg or combined Trastuzumab 8mg/kg and rhG-CSF 2mg/kg (the mass ratio of trastuzumab to G-CSF in the trastuzumab-G-CSF fusion protein molecule is 4:1), administered twice a week for a total of Dosing 4 times. The tumor volume was measured at each administration, and the mice were weighed.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Peptides Or Proteins (AREA)
Abstract
提供一种抗Her-2抗体-粒细胞调节因子融合蛋白及其制法和应用。具体地,涉及一种融合蛋白,所述融合蛋白的单链包括融合在一起的以下元件:(a)第一蛋白元件;(b)第二蛋白元件;以及(c)任选的位于第一蛋白元件和第二蛋白之间的接头元件;其中,所述第一蛋白元件抗原识别模块,例如可以为抗Her-2抗体或其活性片段的蛋白元件;第二蛋白元件为粒细胞集落刺激因子。该融合蛋白具有精确识别、免疫治疗、毒性可控、ADCC增强的优势,并具有高效杀伤肿瘤细胞活性、毒副作用小的协同作用。
Description
本发明属于生物技术和医学领域,具体地涉及一种抗Her-2抗体-粒细胞调节因子融合蛋白及其制法和应用。
HER-2为原癌基因,属于人表皮生长因子受体家族,通过调节下游信号通路来抑制癌细胞凋亡,促进其增殖、侵袭,HER2扩增或过表达在乳腺癌患者中占比约20%-30%,此外,胃癌中也常检测到HER2阳性。HER-2靶点的代表药物曲妥珠单抗目前在HER-2+的乳腺癌和胃癌中取得了不错的疗效,但是目前乳腺癌对曲妥珠单抗的耐药率及复发率逐年升高,其最终耐药率高达65%,这其中包括70%的患者在治疗初期对曲妥珠单抗治疗敏感,最终也出现耐药性。因此亟需新的组合来实现对HER-2+肿瘤的有效控制。
嗜中性粒细胞是组织损伤中最重要的防御体系之一,约占循环白细胞的50%-70%。近期研究显示,肿瘤相关的中性粒细胞(TANs)在肿瘤免疫中也有着重要的作用。嗜中性粒细胞除了可以通过颗粒酶B等机制对肿瘤细胞产生直接杀伤外,还可以通过抗体依赖的细胞介导的细胞毒作用(ADCC)来诱导细胞凋亡。因此,通过增强抗肿瘤抗体药物的ADCC活性,能提高嗜中性粒细胞杀伤肿瘤细胞的功能。
综上所述,本领域迫切需要开发一种更安全、有效、精准靶向Her-2高表达肿瘤的抗Her-2抗体-粒细胞调节因子的融合蛋白。
发明内容
本发明的目的在于提供一种更安全、有效、精准靶向肿瘤的抗Her-2抗体-粒细胞调节因子的融合蛋白。
本发明的目的在于提供一种抗Her-2抗体-粒细胞调节因子融合蛋白及其用途。
在本发明的第一方面,提供了一种融合蛋白单链,所述融合蛋白单链包括融合在一起的以下元件:
(a)第一蛋白元件;
(b)第二蛋白元件;以及
(c)任选的位于第一蛋白元件和第二蛋白元件之间的接头元件;
其中,所述第一蛋白元件为抗原识别模块;
第二蛋白元件为粒细胞集落刺激因子。
在另一优选例中,所述抗原识别模块包括抗体或其活性片段,所述抗体选自下组:抗CD20抗体、抗TIM-3抗体、抗LAG-3抗体、抗CD73抗体、抗CD47抗体、抗DLL3抗体、抗FRmAb抗体、抗CTLA-4抗体、抗OX40抗体、抗CD137抗体、抗PD-1抗体。
在另一优选例中,所述抗体为单克隆抗体。
在另一优选例中,所述活性片段为含有抗体的F(ab)、F(ab')2、scFv、VH、CH、VL或VHH的活性片段。
在另一优选例中,所述抗原识别模块为抗Her-2抗体或其活性片段。
在另一优选例中,所述抗Her-2抗体或其活性片段为含有F(ab)、F(ab')2、scFv、VH、CH、VL或VHH的活性片段。
在另一优选例中,所述抗Her-2抗体或其活性片段选自曲妥珠单抗的活性片段。
在另一优选例中,所述接头元件为肽键或肽接头。
在另一优选例中,所述粒细胞集落刺激因子(G-CSF)来源于人或非人哺乳动物,更佳地来源于啮齿动物(如小鼠、大鼠)、灵长动物和人。
在另一优选例中,所述G-CSF包括野生型和突变型。
在另一优选例中,所述G-CSF包括全长的、成熟形式的G-CSF,或其活性片段。
在另一优选例中,所述G-CSF还包括G-CSF的衍生物。
在另一优选例中,所述G-CSF的衍生物包括经修饰的G-CSF、氨基酸序列与天然G-CSF同源且具有天然G-CSF活性的蛋白分子、G-CSF的二聚体或多聚体、含有G-CSF氨基酸序列的融合蛋白。
在另一优选例中,所述“氨基酸序列与天然G-CSF同源且具有天然G-CSF活性的蛋白分子”是指其氨基酸序列与G-CSF相比具有≥85%的同源性,较佳地≥90%的同源性,更佳地≥95%的同源性,最佳地≥98%同源性;并且具有G-CSF活性的蛋白分子。
在本发明的第二方面,提供了一种由本发明第一方面所述的融合蛋白单链组 成的融合蛋白,所述融合蛋白包含两条单链,其中每个单链从N端到C端具有如下式I所示的结构:
其中,M1、M2、M3、M4各自独立地为无或粒细胞集落刺激因子G-CSF,且至少一个不为无;
L1、L2、L3、L4各自独立地为无或键或肽接头;
式中,
H-Chain为抗Her-2抗体重链或其活性片段;
V-Chain为抗Her-2抗体轻链或其活性片段;
“-”代表肽键。
在另一优选例中,所述的M1、M4、L1、L2、L3和L4为无。
在另一优选例中,所述的M2、M3、L2和L3为无。
在另一优选例中,所述融合蛋白各自地具有选自如下式II、III、IV或V所示的结构:
式中,
H-Chain为抗Her-2抗体重链或其活性片段;
V-Chain为抗Her-2抗体轻链或其活性片段;
M为粒细胞集落刺激因子;
“-”代表肽键或肽接头。
在另一优选例中,所述融合蛋白为二聚体。
在另一优选例中,所述融合蛋白为同源或异源二聚体。
在另一优选例中,所述的重链活性片段包括或含有抗Her-2抗体中的重链、VH、CH、VHH、Fc区或HCDR。
在另一优选例中,所述的轻链活性片段包括或含有抗Her-2抗体中的轻链、VL、CL或LCDR。
在另一优选例中,所述的抗Her-2抗体重链或其活性片段包括重链可变区、重链恒定区、Fc段。
在另一优选例中,所述Fc片段来源于人或非人哺乳动物,更佳地来源于啮齿动物(如小鼠、大鼠)、灵长动物和人。
在另一优选例中,所述Fc片段为免疫球蛋白IgG的Fc片段,较佳地为IgG1的Fc部分。
在另一优选例中,所述的抗Her-2抗体轻链或其活性片段包括轻链可变区、轻链恒定区。
在另一优选例中,H-Chain为曲妥珠单抗的重链。
在另一优选例中,V-Chain为曲妥珠单抗的轻链。
在另一优选例中,M为粒细胞集落刺激因子G-CSF。
在另一优选例中,在所述的融合蛋白中,所述的H-Chain或V-Chain与G-CSF以头-头、头-尾、或尾-尾方式相连。
在另一优选例中,所述的“头部”指多肽或其片段的N端,尤其是野生型多肽的或其片段的N端。
在另一优选例中,所述的“尾部”指多肽或其片段的C端,尤其是野生型多肽的 或其片段的C端。
在另一优选例中,所述的肽接头的长度为0-20个氨基酸,较佳地1-15个氨基酸。
在另一优选例中,所述的H-Chain含有或具有SEQ ID NO:11中的第1-449位氨基酸,所述的V-Chain含有或具有SEQ ID NO:14中的第1-214位氨基酸,所述的G-CSF含有或具有SEQ ID NO:11中的第450-624位氨基酸,或SEQ ID NO:14中的第222-396位氨基酸。
在另一优选例中,所述肽接头的序列为SEQ ID NO:14中的第215-221位。
在另一优选例中,所述融合蛋白的序列选自下组:
(1)如SEQ ID NO:11所示的H-chain-M的序列;和如SEQ ID NO:12所示的V-chain的序列;
(2)如SEQ ID NO:13所示的H-chain的序列;和如SEQ ID NO:14所示的V-chain-M的序列;和
(3)与上述序列≥80%同源性(优选地,≥90%的同源性;等优选地≥95%的同源性;最优选地,≥97%的同源性,如98%以上,99%以上)的衍生序列。
在另一优选例中,所述H-chain-M为融合蛋白重链,其序列如SEQ ID NO:11所示。
在另一优选例中,所述V-chain-M为融合蛋白轻链,其序列如SEQ ID NO:14所示。
在本发明的第三方面,提供了一种分离的多核苷酸,所述的多核苷酸编码如本发明的第二方面所述的融合蛋白。
在另一优选例中,所述的多核苷酸在所述突变蛋白或融合蛋白的ORF的侧翼还额外含有选自下组的辅助元件:信号肽、分泌肽、标签序列(如6His)、或其组合。
在另一优选例中,所述的多核苷酸选自下组:DNA序列、RNA序列、或其组合。
在本发明的第四方面,提供了一种载体,它含有本发明的第三方面所述的多核苷酸。
在另一优选例中,所述的载体包括:细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在另一优选例中,所述载体包含一个或多个启动子,所述启动子可操作地与所述核酸序列、增强子、转录终止信号、多腺苷酸化序列、复制起点、选择性标记、 核酸限制性位点、和/或同源重组位点连接。
在另一优选例中,所述载体包括表达载体、穿梭载体、整合载体。
在本发明的第五方面,提供了一种宿主细胞,它含有如本发明的第四方面所述的载体或基因组中整合有本发明的第三方面所述的多核苷酸。
在另一优选例中,所述的宿主细胞包括原核细胞和真核细胞。
在另一优选例中,所述的宿主细胞包括哺乳动物细胞。
在另一优选例中,所述的宿主细胞为真核细胞,如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。
在另一优选例中,所述的宿主细胞为原核细胞,如大肠杆菌。
在另一优选例中,所述酵母细胞选自下组的一种或多种来源的酵母:毕氏酵母、克鲁维酵母、或其组合;较佳地,所述的酵母细胞包括:克鲁维酵母,更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母。
在另一优选例中,所述宿主细胞选自下组:大肠杆菌、麦胚细胞,昆虫细胞,SF9、SP2/0、Hela、HEK293、CHO(比如CHOKS)、酵母细胞、或其组合。
在本发明的第六方面,提供了一种产生如本发明的第二方面所述融合蛋白的方法,它包括步骤:
(1)在适合表达的条件下,培养如本发明的第五方面所述的宿主细胞,从而表达如本发明的第二方面所述的融合蛋白;和
(2)任选地分离所述融合蛋白。
在本发明的第七方面,提供了一种药物组合物,所述组合物包含:
如本发明的第二方面所述的融合蛋白,以及
药学上可接受的载体。
在另一优选例中,所述的药物组合物还含有:额外的活性成分,较佳地所述的活性成分包括:小分子化合物、细胞因子、抗体(如抗PD-1抗体、抗OX40抗体、抗CD137抗体、抗CD47抗体、ADC、CAR-免疫细胞)。
在另一优选例中,所述的药物组合物为注射剂型。
在本发明的第八方面,提供了一种免疫细胞,所述的免疫细胞携带如本发明的第二方面所述的融合蛋白。
在另一优选例中,所述的免疫细胞包括T细胞。
在本发明的第九方面,提供了一种药物组合物,所述的组合物包含:
如本发明的第八方面所述的免疫细胞,以及
药学上可接受的载体。
在本发明的第十方面,提供了一种如本发明的第二方面所述的融合蛋白或如本发明的第八方面所述的免疫细胞的用途,用于制备治疗肿瘤的药物。
在另一优选例中,所述的肿瘤包括:乳腺癌肿瘤、胃癌肿瘤、膀胱癌肿瘤、胰腺癌肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、黑色素肿瘤。
在另一优选例中,所述治疗肿瘤的药物可与另一种肿瘤免疫治疗联用,包括但不限于:化疗、抗CD20 mAb、抗TIM-3 mAb、抗LAG-3 mAb、抗CD73 mAb、抗CD47mAb、抗DLL3 mAb、抗FRmAb mAb、抗CTLA-4抗体、抗OX40抗体、抗CD137抗体、抗PD-1抗体、PD-1/PD-L1治疗、其他免疫肿瘤药物、抗血管生成剂、放射治疗、抗体-药物偶联物(ADC)、靶向治疗或其他抗癌药物。
本发明第十一方面提供了一种预防和/或治疗肿瘤的方法,包括步骤:给需要的对象施用本发明第二方面所述的融合蛋白。
在另一优选例中,所述的融合蛋白以单体和/或二聚体形式施用。
在另一优选例中,所述的对象是人。
在另一优选例中,所述的肿瘤包括:乳腺癌肿瘤、胃癌肿瘤、膀胱癌肿瘤、胰腺癌肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、黑色素肿瘤。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
图1显示了本发明抗Her-2抗体-粒细胞集落刺激因子融合蛋白实施方式的其中四种结构示意图(图1A、图1B、图1C、图1D)。
图2显示重链-G-CSF曲妥珠单抗融合蛋白的SDS-PAGE电泳分析研究。图2A:非还原6%SDS-PAGE电泳分析。图2B:还原10%SDS-PAGE电泳分析。泳道1为曲妥珠单抗;泳道2为重链-G-CSF曲妥珠单抗融合蛋白;MW为蛋白分子量标准(kDa)。
图3显示了重链-G-CSF曲妥珠单抗融合蛋白体外与重组人Her-2 ECD结合的ELISA研究(图3A),以及与膜Her-2结合的流式细胞技术分析研究(图3B)。
图4显示了重链-G-CSF曲妥珠单抗融合蛋白体外抑制Her-2阳性乳腺癌细胞BT-474生长的研究。
图5显示了重链-G-CSF曲妥珠单抗融合蛋白体外刺激小鼠髓性白血病淋巴细胞NFS-60生长的研究
图6显示了重链-G-CSF曲妥珠单抗融合蛋白体内促进小鼠血液中中性粒细胞增殖。
图7显示了重链-G-CSF曲妥珠单抗融合蛋白抑制表达人Her-2的小鼠黑色素瘤细胞B16在小鼠体内生长的研究。图7A:各组小鼠肿瘤平均体积变化曲线;图7B:各组小鼠平均重量变化曲线。
本发明人经过广泛而深入地研究,首次意外发现,将(a)抗Her-2抗体或其活性片段、(b)粒细胞集落刺激因子G-CSF相融合,获得的融合蛋白具有高效杀伤肿瘤细胞活性、毒副作用小的协同作用。具体地,本发明涉及一种新的融合蛋白,其由肿瘤相关的靶向原件、优选单克隆抗体或其片段、集落刺激因子组成。其特异性识别人肿瘤上表达的分子,例如人表皮生长因子受体(HER-2)并携带粒细胞集落刺激因子(G-CSF)。所得的融合蛋白可以在特异性结合肿瘤组织表达的HER-2,抑制肿瘤生长的同时,将G-CSF传递到靶向的肿瘤组织,增强嗜中性粒细胞杀伤肿瘤的作用。新的融合蛋白可用于HER2+肿瘤的治疗。在此基础上完成了本发明。
本发明涉及的融合蛋白,由以下二部分组成:(1)识别肿瘤特异性抗原Her-2的全长单克隆抗体或最小识别抗原的部分;(2)调控中性粒细胞增殖、分化和激活的细胞因子,比如粒细胞集落刺激因子G-CSF。利用重组DNA技术构建融合蛋白编码核糖核苷酸,融合蛋白含有抗Her-2抗体重链,其含或不含重链恒定区的CH1或CH2或CH3,其C末端与有活性的细胞因子如G-CSF融合。当融合蛋白重链表达质粒与抗Her-2抗体轻链表达质粒共转染,可以产生抗Her-2抗体-细胞因子(比如G-CSF)融合蛋白,此融合蛋白能结合表达Her-2的肿瘤细胞,并能把细胞因子递送到肿瘤部位。类似的,也可以把有生物活性的中性粒细胞调控细胞因子与抗Her-2的单链抗体融合,完整的融合蛋白是一条多肽链,各功能区域由连接肽连接,保证融合蛋白具有正确的空间结构,保持其生物活性。
本发明的融合蛋白是一类全新的分子,具有二种生物功能:第一,它们能靶向表达Her-2的肿瘤组织,通过阻断Her-2的功能,从而抑制肿瘤的生长;或 通过ADCC或CDC功能杀伤肿瘤细胞。第二,它们能把具有生物活性的细胞因子特异性递送到肿瘤部位。这些细胞因子具有调节免疫细胞活性的功能,因此,能增加免疫细胞肿瘤组织浸润以及增强免疫细胞活性,使肿瘤,例如乳腺癌、胃癌等,生长得到抑制。由于细胞因子主要局限在肿瘤组织部位,给患者造成的毒性相对较小。因此,本发明的目的就是提供一种含有靶向Her-2表达的肿瘤的单克隆抗体或抗体片段,并与有生物活性的细胞因子融合的抗体-细胞因子融合蛋白。
本发明中融合蛋白里的抗体可以是全长抗体,也可以是抗体的某一关键片段,比如,scFv、F(ab)2等。从理论上来说,所有能结合肿瘤细胞膜上Her-2受体的抗体都适用于构建本发明的抗体-粒细胞集落刺激因子融合蛋白。在本发明中,曲妥珠单抗是优选抗体。
本发明的目的是提供一种抗体-粒细胞集落刺激因子融合蛋白,其抗体部分是全长抗体或者是含有必要可变区序列的抗体片段,比如F(ab)或F(ab)2或scFv。本发明的融合蛋白细胞因子部分,选自有生物活性的G-CSF,直接或通过连接肽链与抗体部分连接。
本发明内容还包括产生和制备抗体-细胞因子融合蛋白的方法,通过直接或间接融合编码抗体的核苷酸序列与细胞因子的核苷酸序列,克隆到表达载体上,然后转染载体进入细胞,在合适的培养基里培养转染的细胞,获得抗体-细胞因子融合蛋白。
本发明的抗体-细胞因子融合蛋白可用于临床肿瘤治疗。因此,本发明内容包含临床治疗药物的组成,其至少含有本发明所述融合蛋白的其中1个,以及生理上可接受的载体。
术语
除非另外定义,否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
在本发明中,术语“本发明的融合蛋白”、“本发明的Her-2抗体-粒细胞集落刺激因子融合蛋白”、“Her-2抗体-G-CSF融合蛋白”可互换使用,皆指本发明中第二方面所提及的融合蛋白。
如本文所用,在提到具体列举的数值中使用时,术语“约”意指该值可以从列举的值变动不多于1%。例如,如本文所用,表述“约100”包括99和101和之间的全部值(例如,99.1、99.2、99.3、99.4等)。
如本文所用,除非另外说明,Fc是指人免疫球蛋白的Fc片段。术语“免疫球蛋白Fc区”指免疫球蛋白链恒定区,特别是免疫球蛋白重链恒定区的羧基端或 其中的一部分,例如免疫球蛋白Fc区可包括重链CH1、CH2、CH3的两个或更多结构域与免疫球蛋白铰链区的组合,在优选例中,所用的免疫球蛋白的Fc区包括至少一个免疫球蛋白绞链区,一个CH2结构域和一个CH3结构域,优选缺少CH1结构域。
已知人免疫球蛋白有多种类别,如IgA、IgD、IgE、IgM及IgG(包括IgG1、IgG2、IgG3、IgG4四个亚类),从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区是在本领域技术人员所掌握的范围之内的,在一个优选的实例中,免疫球蛋白Fc区可选择包含有人免疫球蛋白IgG4亚类Fc区的编码序列,其中缺失一个免疫球蛋白重链1结构域(CH1),但包括了铰链区以及CH2、CH3、二个结构域的编码序列。
如本文所用,所述的“含有”,“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”;“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
嗜中性粒细胞
嗜中性粒细胞是组织损伤中最重要的防御体系之一,约占循环白细胞的50%-70%。近期研究显示,肿瘤相关的中性粒细胞(TANs)在肿瘤免疫中也有着重要的作用。在一些细胞因子的诱导下,中性粒细胞可以对肿瘤细胞实现有效的杀伤,如在TNFα的诱导下,嗜中性粒细胞通过ROS途径杀伤肿瘤细胞,在IFN-γ和IL-2的诱导下,嗜中性粒细胞通过表达颗粒酶B对肿瘤细胞产生直接毒性。嗜中性粒细胞还可以通过抗体依赖的细胞介导的细胞毒作用(ADCC)来诱导肿瘤细胞凋亡(。在放疗的诱导下,会引起大量中性粒细胞在肿瘤组织浸润,并最终通过ROS途径诱导肿瘤细胞凋亡。此外,中性粒细胞可以表达具有直接杀伤活性的肿瘤坏死因子相关凋亡配体(TRAIL)、髓过氧化酶(MPO)等发挥抗肿瘤作用。除了上述直接杀伤作用外,嗜中性粒细胞还可以通过刺激T细胞增殖、促IFN-γ释放、激活树突状细胞等途径来间接增强抗肿瘤作用。嗜中性粒细胞主要由粒细胞集落刺激因子(G-CSF)调控,G-CSF可诱导嗜中性粒细胞增殖、分化、并向外周血释放。一项研究结果显示,参与抗肿瘤的嗜中性粒细胞由于半衰期较短,在一定时间后会发生衰竭,而嗜中性粒细胞耗竭后,观察到了肿瘤显著性的生长。若在治疗的同时,加用G-CSF干预,促进肿瘤微环境嗜中性粒细胞的激活和迁移后,嗜中性粒细胞的数量明显增加,肿瘤免疫反应显著增强,有效的抑制了肿瘤的生长。
融合蛋白
如本文所用,除非另外说明,所述的融合蛋白是一种分离的蛋白,与其它蛋白、多肽或分子无联系,是重组宿主细胞所表达的,或经分离或纯化的产物。
本发明构建的融合蛋白,由以下二部分组成:
(1)识别肿瘤特异性抗原Her-2的全长单克隆抗体或最小识别抗原的部分;
(2)粒细胞集落刺激因子家族中的一个具有生物活性的粒细胞集落刺激因子,比如G-CSF或GM-CSF。
利用重组DNA技术构建融合蛋白编码核糖核苷酸,融合蛋白含有抗Her-2抗体重链,其含或不含重链恒定区的CH1或CH2或CH3,其C末端与有活性的细胞粒细胞集落刺激因子融合。
当融合蛋白重链表达质粒与抗Her-2抗体轻链表达质粒共转染,可以产生抗Her-2抗体-粒细胞集落刺激因子(如G-CSF)融合蛋白,此融合蛋白能结合表达Her-2的肿瘤细胞,并能把粒细胞集落刺激因子递送到肿瘤部位。
将有生物活性的粒细胞集落刺激因子与抗Her-2的单链抗体融合,完整的融合蛋白是一条多肽链,各功能区域由连接肽连接,保证融合蛋白具有正确的空间结构,保持其生物活性。
本发明的融合蛋白是一类全新的分子,具有二种生物功能:第一,它们能靶向表达Her-2的肿瘤组织,第二,它们能把具有生物活性的细胞因子特异性递送到肿瘤部位。这些细胞因子具有吸引免疫细胞并调节免疫细胞活性的功能,因此,能增加免疫细胞肿瘤组织浸润以及增强免疫细胞活性,使肿瘤,例如乳腺癌、胃癌等,生长得到抑制。由于粒细胞集落刺激因子主要局限在肿瘤组织部位,给患者造成的毒性相对较小。
本发明中融合蛋白里的抗体可以是全长抗体,也可以是抗体的某一关键片段,比如,scFv、F(ab)2或VHH。从理论上来说,所有能结合肿瘤细胞膜上Her-2受体的抗体都适用于构建本发明的抗体-粒细胞集落刺激因子融合蛋白(曲妥珠单抗、拉帕替尼单抗、和帕妥珠单抗)。在本发明中,优选曲妥珠单抗。
本发明的融合蛋白粒细胞集落刺激因子部分,直接或通过肽接头与抗体部分连接。
本发明提供了一种融合蛋白,包含以下元件:
(a)抗Her-2抗体或其活性片段的蛋白元件、(b)粒细胞集落刺激因子(G-CSF)的蛋白元件、和(c)接头元件。本发明所述的融合蛋白中,所述的各元件之间(如元件a与元件b之间),可以含有或不含有接头。
本发明的融合蛋白,不仅具有更长的体内半衰期,可以更有效地抑制血清中免疫疾病相关的抗体(尤其是IgE)的浓度。
根据本发明提供的氨基酸序列,本技术领域人员可方便地用各种已知方法 制得本发明的融合蛋白。这些方法例如但不限于:重组DNA法,人工合成,等。
在得知了本发明的融合蛋白的氨基酸序列后,本领域人员可以方便地根据所述的氨基酸序列获得编码本发明的融合蛋白的基因序列。
一种优选的融合蛋白为曲妥珠单抗HC-G-CSF融合蛋白,其重链核苷酸序列如SEQ ID NO:6所示,重链氨基酸序列如SEQ ID NO:11所示;其中,重链氨基酸(SEQ ID NO:11)序列中的1-449位为曲妥珠单抗的氨基酸序列;第450-624位为G-CSF氨基酸序列。
一种优选的融合蛋白为曲妥珠单抗LC-linker-G-CSF融合蛋白,其轻链核苷酸序列如SEQ ID NO:9所示,轻链氨基酸序列如SEQ ID NO:14所示;其中,轻链氨基酸(SEQ ID NO:14)序列中的1-214位为曲妥珠单抗的轻链氨基酸序列;第222-396位为G-CSF氨基酸序列。
在另一优选例中,本发明的曲妥珠单抗HC-G-CSF融合蛋白的轻链核苷酸序列如SEQ ID NO:7所示,轻链氨基酸序列如SEQ ID NO:12所示。
在另一优选例中,本发明的曲妥珠单抗LC-G-CSF或LC-linker-G-CSF融合蛋白的重链核苷酸序列如SEQ ID NO:8所示,重链氨基酸序列如SEQ ID NO:13所示。
如本文所用,“分离的”是指物质从其原始环境中分离出来(如果是天然的物质,原始环境即是天然环境)。如活体细胞内的天然状态下的多核苷酸和多肽是没有分离纯化的,但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开,则为分离纯化的。
如本文所用,“分离的重组融合蛋白”是指重组融合蛋白基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化重组融合蛋白。基本上纯的蛋白在非还原聚丙烯酰胺凝胶上能产生单一的主带。
如本文所用,术语“融合蛋白”还包括具有上述活性的融合蛋白(如SEQ ID NO.:1或2所示的序列)的变异形式。这些变异形式包括(但并不限于):1-3个(通常为1-2个,更佳地1个)氨基酸的缺失、插入和/或取代,以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内,较佳地为2个以内,更佳地为1个以内)氨基酸。例如,在本领域中,用性能相近或相似的氨基酸进行取代时,通常不会改变蛋白质的功能。又比如,在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外,所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述融合蛋白的活性片段、衍生物和类似物。如本文所用,术语 “片段”、“衍生物”和“类似物”是指基本上保持本发明融合蛋白的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽,或(ii)在一个或多个氨基酸残基中具有取代基团的多肽,或(iii)抗原肽与另一个化合物(比如延长多肽半衰期的化合物,例如聚乙二醇)融合所形成的多肽,或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6×His等标签序列融合而形成的融合蛋白)。根据本文的教导,这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I或式II的氨基酸序列相比,有至多3个,较佳地至多2个,更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 | 代表性的取代 | 优选的取代 |
Ala(A) | Val;Leu;Ile | Val |
Arg(R) | Lys;Gln;Asn | Lys |
Asn(N) | Gln;His;Lys;Arg | Gln |
Asp(D) | Glu | Glu |
Cys(C) | Ser | Ser |
Gln(Q) | Asn | Asn |
Glu(E) | Asp | Asp |
Gly(G) | Pro;Ala | Ala |
His(H) | Asn;Gln;Lys;Arg | Arg |
Ile(I) | Leu;Val;Met;Ala;Phe | Leu |
Leu(L) | Ile;Val;Met;Ala;Phe | Ile |
Lys(K) | Arg;Gln;Asn | Arg |
Met(M) | Leu;Phe;Ile | Leu |
Phe(F) | Leu;Val;Ile;Ala;Tyr | Leu |
Pro(P) | Ala | Ala |
Ser(S) | Thr | Thr |
Thr(T) | Ser | Ser |
Trp(W) | Tyr;Phe | Tyr |
Tyr(Y) | Trp;Phe;Thr;Ser | Phe |
Val(V) | Ile;Leu;Met;Phe;Ala | Leu |
本发明还提供本发明融合蛋白的类似物。这些类似物与SEQ ID NO.:11、12、13或14所示的多肽的差别可以是氨基酸序列上的差异,也可以是不影响序列的修饰形式上的差异,或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物,以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解,本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括:体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化,如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸,磷酸丝氨酸,磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
多核苷酸
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
本发明还涉及上述多核苷酸的变异体,其编码与本发明有相同的氨基酸序列的蛋白质片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的,等位变异体是一个多核苷酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,但不会从实质上改变其编码多肽的功能。
如本文所用,术语“引物”指的是在与模板配对,在DNA聚合酶的作用下能以其为起点进行合成与模板互补的DNA链的寡居核苷酸的总称。引物可以是天然的RNA、DNA,也可以是任何形式的天然核苷酸。引物甚至可以是非天然的核苷酸如LNA或ZNA等。引物“大致上”(或“基本上”)与模板上一条链上的一个特殊的序列互补。引物必须与模板上的一条链充分互补才能开始延伸,但引物的序列不必与模板的序列完全互补。比如,在一个3'端与模板互补的引物的5'端加上一段与模板不互补的序列,这样的引物仍大致上与模板互补。只要有足够长的引物能与模板充分的结合,非完全互补的引物也可以与模板形成引物-模板复合物,从而进行扩增。
根据本发明提供的氨基酸序列,本技术领域人员可方便地用各种已知方法制得本发明的融合蛋白。这些方法例如但不限于:重组DNA法,人工合成等。
本发明融合蛋白的元件(如抗Her-2抗体活性片段或G-CSF)的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法,可根据已公开的有关核苷酸序列,尤其是开放阅读框序列来设计引物,并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板,扩增而得有关序列。当序列较长时,常常需要进行两次或多次PCR扩 增,然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择,并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
载体和宿主细胞
本发明也涉及包含本发明的多核苷酸的载体,以及用本发明的载体或融合蛋白编码序列经基因工程产生的宿主细胞,以及经重组技术产生本发明所述蛋白质的方法。
通过常规的重组DNA技术,可利用本发明的多核苷酸序列可用来表达或生产重组蛋白。一般来说有以下步骤:
(1).用本发明的编码本发明蛋白的多核苷酸(或变异体),或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞;
(2).在合适的培养基中培养的宿主细胞;
(3).从培养基或细胞中分离、纯化蛋白质。
本领域的技术人员熟知的方法能用于构建含本发明蛋白的编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上,以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外,表达载体优选地包含一个或多个选择性标记基因,以提供用于选择转化的宿主细胞的表型性状,如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP),或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体,可以用于转化适当的宿主细胞,以使其能够表达蛋白质。
宿主细胞可以是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞;或是高等真核细胞,如哺乳动物细胞。代表性例子有:大肠杆菌,链霉菌属的细菌细胞;真菌细胞如酵母;植物细胞;果蝇S2或Sf9的昆虫细胞;CHO、COS、或293细胞的动物细胞等。
一种特别优选的细胞为人和非人哺乳动物的细胞,尤其是免疫细胞,包括T细胞、NK细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。另一种方法是使用MgCl
2。如果需要,转化也可用电穿孔的方法进行。当宿主是真核生物,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养,表达本发明的基因所编码的多肽。根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。
在上面的方法中的蛋白质可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
肽接头
本发明的双功能融合蛋白可任选地含有或不含有肽接头。肽接头大小和复杂性可能会影响蛋白的活性。通常,肽接头应当具有足够的长度和柔韧性,以保证连接的两个蛋白在空间上有足够的自由度以发挥其功能。同时避免肽接头中形成α螺旋或β折叠等对融合蛋白的稳定性的影响。
肽接头的长度一般为0-20个氨基酸,较佳地1-15个氨基酸。
优选的肽接头的例子包括(但并不限于):GSGGGGS,(GGGGS)
n,其中n为1-8的整数,优选n为1、2或3。
在本发明的一个具体实施例中,肽接头的氨基酸序列为:曲妥珠单抗LC-linker-G-CSF氨基酸序列(SEQ ID NO:14)中的第215-221位。
药物组合物及施用方法
本发明还提供了一种组合物,它含有(a)有效量的本发明融合蛋白和/或有效量的本发明的免疫细胞,以及药学上可接受的载体。
通常,可将本发明的融合蛋白配制于无毒的、惰性的和药学上可接受的水 性载体介质中,其中pH通常约为5-8,较佳地,pH约为6-8。
如本文所用,术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量,如0.001-99wt%;较佳的0.01-95wt%;更佳的,0.1-90wt%。
当本发明的药物组合物含有免疫细胞时,“有效量”或“有效剂量”是指1×10
3-1×10
7个所述的免疫细胞/ml。
如本文所用,“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的,即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体,包括各种赋形剂和稀释剂。
本发明的药物组合物含有安全有效量的本发明的融合蛋白以及药学上可接受的载体。这类载体包括(但并不限于):盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配,本发明的药物组合物可以被制成针剂形式,例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。
本发明融合蛋白的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于:本发明融合蛋白的药代动力学参数例如生物利用率、代谢、半衰期等;患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常,当本发明的融合蛋白每天以约5mg-20mg/kg动物体重(较佳的5mg-10mg/kg动物体重)的剂量给予,能得到令人满意的效果。例如,由治疗状况的迫切要求,可每天给予若干次分开的剂量,或将剂量按比例地减少。
本发明融合蛋白特别适合用于治疗肿瘤等疾病。代表性的肿瘤包括(但并不限于):乳腺癌肿瘤、胃癌肿瘤、膀胱癌肿瘤、胰腺癌肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、黑色素肿瘤。
本发明的主要优点包括
(1)本发明构建了一种抗Her-2抗体与G-CSF的全新组合,该融合蛋白靶向表达Her-2的肿瘤组织,通过阻断Her-2的功能,从而抑制肿瘤的生长;或通过ADCC或CDC功能杀伤肿瘤细胞。具有精确识别、免疫治疗、毒性可控、ADCC增强的优势。
(2)本发明的融合蛋白能把具有生物活性的细胞因子特异性递送到肿瘤 部位。这些细胞因子具有调节免疫细胞活性的功能,因此,能增加免疫细胞肿瘤组织浸润以及增强免疫细胞活性,使肿瘤,例如乳腺癌、胃癌等,生长得到抑制。由于细胞因子主要局限在肿瘤组织部位,给患者造成的毒性相对较小。
下面结合具体实施例,进一步陈述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
本发明融合蛋白的序列
实施例1.抗体-G-CSF融合蛋白表达质粒的构建
以曲妥珠单抗作为Her-2抗体的示例。编码曲妥珠单抗重链和轻链的完整cDNA由GenScrip(USA)公司合成,分别克隆在pUC57载体上。人G-CSF的cDNA购自OpenBiosystems(美国)。
1.曲妥珠单抗重链-G-CSF表达质粒的构建
有大量报道显示,在单克隆抗体表达和制备过程中,绝大部分抗体的重链C-末端赖氨酸被降解掉,所以在构建抗体重链-G-CSF融合蛋白时,去掉了这个赖氨酸,使的抗体-细胞因子融合蛋白能保持完整性。
编码曲妥珠单抗重链的基因与编码G-CSF的基因用两步聚合酶链式反应技术(PCR)方法连接起来。第一步,用PCR方法(高保真聚合酶Pfx,Invitrogen)以人工合成的抗体重链DNA为底物扩增重链基因:
5’端引物M13-F(SEQ ID NO:1):5'-TGTAAAACGACGGCCAGT-3',位于pUC57载体上。
3’端引物KDP004(SEQ ID NO:2):5'-TCCTGGGGACAGTGACAGTG-3',是抗体重链基因专一引物。
同样,用PCR方法扩增成熟G-CSF蛋白部分(Ala30-Pro207)的基因:
5’端引物KDP047(SEQ ID NO:3):
5'-CACTGTCACTGTCCCCAGGAGCCACCCCCCTGGGCCC-3';
3’端引物BGH-R(SEQ ID NO:4):
5'-AACTAGAAGGCACAGTCGAGGC-3',位于底物质粒载体上。
其中引物KDP047的头20个核苷酸序列与引物KDP004的核苷酸序列互补,这样在第二步的重叠延伸PCR过程中,可以把这2个PCR片段连接起来。
上面2个PCR片段经DNA胶纯化后(天根生化科技有限公司,北京),进行第二步重叠PCR。5’端引物M13-F(SEQ ID NO:1),3’端引物KDP048(SEQ ID NO:5),5'-TGGTGGTGTCTAGAGACTCAGGGCTGGGCAAGGTGG-3',含有用于克隆的Xba I酶切序列。
曲妥珠单抗重链基因转录起始位点前有Not I的酶切位点,这样,在胶纯化重叠PCR得到的片段后,进行Not I/Xba I双酶切(Takara)。然后把酶切的PCR片段克隆到同样酶切的哺乳动物细胞表达载体上。此哺乳动物细胞表达载体是改进的pcDNA3.1(Invitrogen),pcDNA3.1里的抗neomycin(新霉素)基因被大鼠谷氨 酰胺合成酶基因取代,改进后的载体适用于筛选稳定转染蛋白高表达的哺乳动物细胞。将重组质粒转染进DH5a感受态细菌,用菌落PCR方法鉴定含有正确重组质粒的阳性菌落,提纯重组质粒。经酶切和测序鉴定,曲妥珠单抗重链-G-CSF重组基因具有正确的序列。
2.曲妥珠单抗轻链-G-CSF表达质粒的构建
编码曲妥珠单抗轻链-linker-G-CSF的基因由苏州金唯智生物科技有限公司(中国)合成,克隆到上述改进的pcDNA3.1载体中。
3.曲妥珠单抗重链和轻链表达质粒的构建
分别把在pUC57载体里的曲妥珠单抗重链和轻链表达基因用亚克隆的方法克隆到上述改进的pcDNA3.1载体中。克隆酶是Not I和Xba I。
实施例2.构建抗体-G-CSF融合蛋白稳定表达细胞株
用于稳定表达这些抗体-细胞因子融合蛋白的宿主细胞为中国仓鼠卵巢细胞CHO-KS。CHO-KS是生长在含胎牛血清(FBS)培养基里的CHO-K1细胞经过逐渐降低培养基中FBS含量的培养直至无FBS培养基培养,最终驯化成在不含FBS的OptiCHO培养基(Invitrogen)中悬浮生长的细胞。含有抗体-细胞因子融合蛋白基因的pcDNA3.1载体中的抗新霉素基因用大鼠谷氨酰胺合成酶基因取代,采用电转染(Bio-Rad,Gene Pulser Xcell)的方法把重链和轻链表达质粒共转染进CHO-KS细胞,转染的细胞在培养24-48个小时后,用有限稀释法在96孔培养板上对转染的细胞进行筛选培养。筛选培养基是OptiCHO,5μg/ml重组人胰岛素和10M氨基亚砜蛋氨酸(MSX)。在37℃,8%CO
2的培养箱里培养细胞。3个星期后,用ELISA方法(碱性磷酸酶偶联的羊抗人IgG Fc抗体,Jackson ImmunoResearch Lab)对每个长有细胞群的孔的细胞培养液进行分析,把抗体-细胞因子融合蛋白表达阳性的细胞群进一步扩增,再ELISA检测,再扩增,最后得到抗体-细胞因子融合蛋白表达稳定细胞株。
实施例3.曲妥珠单抗-G-CSF融合蛋白的制备和鉴定
将实施例2所得重链-G-CSF曲妥珠单抗和轻链-G-CSF曲妥珠单抗高表达的细胞株培养扩增。离心细胞培养液,收集上清,用Protein-A亲和层析柱从上清中纯化融合蛋白。
结果与分析
图2A的非还原SDS-PAGE电泳胶显示重链-G-CSF曲妥珠单抗融合蛋白的分子量大于曲妥珠单抗,很接近其理论值183kDa。图2B的还原SDS-PAGE电泳胶显示重链-G-CSF曲妥珠单抗融合蛋白的重链分子量大于曲妥珠单抗重链,与其理 论分子量一致(68kDa)。重链-G-CSF曲妥珠单抗融合蛋白的轻链与曲妥珠单抗的轻链相同。
实施例4.曲妥珠单抗-G-CSF融合蛋白与Her-2体外结合研究
用ELISA方法研究重链-G-CSF曲妥珠单抗与重组人Her-2胞外段(ECD)的体外结合活性。
用PBS(pH7.4)溶解重组人Her-2 ECD(北京义翘神州科技股份有限公司)至终浓度0.8μg/mL,然后加入50μL/孔于96-孔ELISA板,4℃冰箱里过夜。第二天,用PBST(PBS含0.05%Tween-20)洗ELISA板3次后,加入100μL/孔PBST含3%BSA的封闭溶液,于37℃恒温箱里放置1小时。分别将重链-G-CSF曲妥珠单抗和曲妥珠单抗稀释在PBST含1%BSA的结合溶液里,制备3倍系列稀释的溶液。倒掉封闭液,分别加入稀释的抗体,50μL/孔,在37℃恒温箱里反应1小时。倒掉溶液,ELISA板用PBST清洗3次,加入50μL/孔的二抗(碱性磷酸酶偶联的羊抗人IgG Fab抗体,Jackson ImmunoResearch Lab),在37℃恒温箱里反应1小时。倒掉显色抗体,向ELISA板上加200μL/孔PBST清洗溶液,ELISA板于水平摇床上放置5分钟,转速100转/分钟,倒掉清洗溶液,再重复清洗4次。加50μL/孔抗体显色液(PNPP)后ELISA板置于37℃恒温箱里进行显色。用酶标仪在波长405nm/490nm下读板。
用流式细胞仪方法(flow cytometry)研究重链-G-CSF曲妥珠单抗与细胞膜上Her-2蛋白的结合。人乳腺癌细胞BT-474(购自中国科学院细胞库)是Her-2高表达细胞株。取适量的BT-474细胞,用预冷的FACS工作液(PBS含0.1%FBS)调整其细胞密度为2×10
6个/ml,分装100μL/管,冰上封闭1小时。然后用FACS工作液稀释曲妥珠单抗-G-CSF融合蛋白和曲妥珠单抗到100μg/mL,然后系列稀释,取系列稀释的10μL加到100μL的细胞悬液中,使抗体终浓度分别为10、3、1、0.3、0.1、0.03和0μg/mL。冰上孵育30分钟后,向每管细胞悬液加1mL FACS工作液,涡旋混匀细胞,离心5分钟,1200rpm/min,弃去上清,重复洗涤一遍。用FACS工作液稀释FITC标记的羊抗人IgG Fc抗体(Jackson ImmunoResearch Lab),每管细胞悬液加10μL抗体,使其终浓度为1μg/mL,避光,冰上孵育30分钟。孵育完成后,向每管细胞悬液加1mL FACS工作液,涡旋混匀细胞,离心5分钟,1200rpm/min,弃去上清,重复洗涤一遍。用流式细胞仪C6(BD Biosciences)检测细胞。
结果与分析
ELISA试验结果显示(图3A):重链-G-CSF曲妥珠单抗融合蛋白能特异性结合Her-2 ECD,EC
50是72ng/mL。
流式细胞术试验显示:重链-G-CSF曲妥珠单抗融合蛋白能够结合细胞膜上的Her-2,结合能力和曲妥珠单抗相当,见图3B。
本发明的融合蛋白完好保留了曲妥珠单抗与Her-2结合的特性。
实施例5.曲妥珠单抗-G-CSF融合蛋白对Her-2阳性人乳腺癌BT-474细胞生长的影响
在体外曲妥珠单抗能抑制Her-2阳性人肿瘤细胞的生长,比如人乳腺癌细胞BT-474。研究了曲妥珠单抗-G-CSF融合蛋白在体外对BT-474细胞生长的影响。BT-474细胞培养在含10%FBS的RPMI1660培养基中。BT-474细胞在96-孔培养板里培养1天后,加入系列稀释的曲妥珠单抗-G-CSF融合蛋白,再继续培养5天。然后加入细胞活性检测试剂CCK-8,用酶标仪在双波长450nm/655nm下读板。
结果与分析
结果显示,重链-G-CSF曲妥珠单抗抑制Her-2阳性人乳腺癌细胞BT-474的体外生长,抑制活性IC
50为0.58μg/mL。见图4。
实施例6.曲妥珠单抗-G-CSF融合蛋白的G-CSF细胞生物学活性研究
小鼠髓性白血病淋巴细胞(NFS-60)的生长依赖G-CSF,研究了曲妥珠单抗-G-CSF融合蛋白对NFS-60细胞体外生长的影响。NFS-60细胞来自中国科学院典型培养物保藏委员会细胞库,细胞培养在RPMI1640/10%FBS(Gibco)培养基里。NFS-60细胞在96-孔培养板里培养1天后,加入系列稀释的曲妥珠单抗-G-CSF融合蛋白或重组人G-CSF(rhG-CSF),再继续培养3天。然后加入细胞活性检测试剂CCK-8,用酶标仪在双波长450nm/655nm下读板。
结果与分析
结果显示,重链-G-CSF曲妥珠单抗能刺激NFS-60细胞体外生长,刺激细胞生长活性EC
50为6.0pmole/L,与重组人G-CSF的活性相当(4.6pmole/L)。见图5。
实施例7.曲妥珠单抗-G-CSF融合蛋白小鼠体内刺激中性粒细胞增殖研究
8只10-12周龄小鼠C57BL/6分为2组,分别静脉给予PBS和2.5mg/kg剂量的重链-G-CSF曲妥珠单抗融合蛋白,72小时后取血,放入含抗凝血剂的试管中。然后加入FITC荧光标记的抗鼠CD45抗体和PE荧光标记的抗鼠Gr-1抗体,用流式细胞仪分析荧光抗体结合的血液细胞。
结果与分析
PBS组小鼠血液里中性粒细胞(Gr-1阳性)的含量约28%,而重链-G-CSF曲 妥珠单抗融合蛋白组小鼠血液里中性粒细胞的含量约50%,表明重链-G-CSF曲妥珠单抗融合蛋白能促进小鼠血液里中性粒细胞增殖(结果见图6)。
实施例8.人Her-2稳定表达小鼠肿瘤细胞的构建
小鼠黑色素瘤细胞B16来自中国科学院典型培养物保藏委员会细胞库,细胞培养在RPMI1640/10%FBS(Gibco)培养基里。
人Her-2表达基因克隆在表达载体pcDNA3.1中(Invitrogen),重组质粒用Lipofectmaine 3000(Invitrogen)转染进小鼠黑色素瘤细胞B16里,转染的细胞在含G418(Sigma)的RPMI/10%FBS培养基里培养,得到稳转细胞池。从稳转细胞池中用流式细胞分选仪(Influx,BD Biosciences)筛选出表达人Her-2的单克隆稳转细胞株B16/Her-2。
实施例9.曲妥珠单抗-G-CSF融合蛋白在小鼠体内抑制表达人Her-2的小鼠黑色素瘤B16生长
C57BL/6小鼠来自上海斯莱克有限公司,饲养在SPF级别环境里。
将32只6-7周龄的C57BL/6小鼠,分成4组,每组8只,雌雄各半。用皮下接种的方法,在小鼠腋下注射B16/Her-2细胞1 x 10
6细胞/只。接种细胞8天后(D8),尾静脉给予小鼠PBS(对照组)或曲妥珠单抗-G-CSF融合蛋白10mg/kg或曲妥珠单抗10mg/kg或联用曲妥珠单抗8mg/kg和rhG-CSF 2mg/kg(曲妥珠单抗-G-CSF融合蛋白分子里曲妥珠单抗与G-CSF的质量比是4:1),每周给药2次,共给药4次。每次给药时测量瘤体积,称小鼠重量。在接种细胞后第23天结束实验,引颈脱臼处死小鼠,摘眼球取血,收集小鼠血液,并解剖小鼠,记录肿瘤重量、脾脏重量大小,同时,记录各组肿瘤照片。
结果与分析
图7显示,与对照组小鼠肿瘤平均体积相比,在D20时,重链-G-CSF曲妥珠单抗融合蛋白组的小鼠肿瘤相对增殖率为30%,显著抑制B16/Her-2肿瘤在小鼠体内的生长。曲妥珠单抗在剂量10mg/kg时对肿瘤生长几乎没有影响。实验证明了重链-G-CSF曲妥珠单抗融合蛋白体内抑制B16/Her-2肿瘤生长的效果远要好于曲妥珠单抗。
同时,用小鼠体重的变化评估曲妥珠单抗-G-CSF融合蛋白对小鼠的毒性。结果显示,3个组小鼠平均体重的变化没有显著区别,表明重链-G-CSF曲妥珠单抗融合蛋白对小鼠没有明显的毒性。
实施例10.曲妥珠单抗-G-CSF融合蛋白在小鼠体内对Her-2阳性人胃癌细胞 NCI-N87肿瘤生长的影响
裸鼠(nude)来自上海斯莱克有限公司,饲养在SPF级别环境里。
将32只6-7周龄的裸鼠,分成3组,每组8只,雌雄各半。用皮下接种的方法,在小鼠腋下注射NCI-N87细胞1 x 10
6细胞/只。接种细胞8天后(D8),尾静脉给予小鼠PBS(对照组)或曲妥珠单抗-G-CSF融合蛋白10mg/kg或曲妥珠单抗10mg/kg或联用曲妥珠单抗8mg/kg和rhG-CSF 2mg/kg(曲妥珠单抗-G-CSF融合蛋白分子里曲妥珠单抗与G-CSF的质量比是4:1),每周给药2次,共给药4次。每次给药时测量瘤体积,称小鼠重量。在接种细胞后第23天结束实验,引颈脱臼处死小鼠,摘眼球取血,收集小鼠血液,并解剖小鼠,记录肿瘤重量、脾脏重量大小,同时,记录各组肿瘤照片。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (15)
- 一种融合蛋白单链,其特征在于,所述融合蛋白单链包括融合在一起的以下元件:(a)第一蛋白元件;(b)第二蛋白元件;以及(c)任选的位于第一蛋白元件和第二蛋白元件之间的接头元件;其中,所述第一蛋白元件为抗原识别模块;第二蛋白元件为粒细胞集落刺激因子。
- 如权利要求2所述的融合蛋白,其特征在于,所述融合蛋白为同源或异源二聚体。
- 如权利要求2所述的融合蛋白,其特征在于,所述的肽接头的长度为0-20个氨基酸,较佳地1-15个氨基酸。
- 如权利要求2所述的融合蛋白,其特征在于,所述的H-Chain含有或具有SEQ ID NO:11中的第1-449位,所述的V-Chain含有或具有SEQ ID NO:14中的第1-214位,所述的G-CSF含有或具有SEQ ID NO:11中的第450-624位,或SEQ ID NO:14中的第222-396位。
- 如权利要求2所述的融合蛋白,其特征在于,所述融合蛋白的序列选自下组:(1)如SEQ ID NO:11所示的H-chain-M的序列;和如SEQ ID NO:12所示的V-chain的序列;(2)如SEQ ID NO:13所示的H-chain的序列;和如SEQ ID NO:14所示的V-chain-M的序列;和(3)与上述序列≥80%同源性(优选地,≥90%的同源性;等优选地≥95%的同源性;最优选地,≥97%的同源性,如98%以上,99%以上)的衍生序列。
- 一种分离的多核苷酸,其特征在于,所述的多核苷酸编码如权利要求2所述的融合蛋白。
- 一种载体,其特征在于,它含有如权利要求7所述的多核苷酸。
- 一种宿主细胞,其特征在于,它含有如权利要求5所述的载体或基因组中整合有如权利要求7所述的多核苷酸。
- 一种产生如权利要求2所述融合蛋白的方法,它包括步骤:(1)在适合表达的条件下,培养如权利要求9所述的宿主细胞,从而表达如权利要求2所述的融合蛋白;和(2)任选地分离所述融合蛋白。
- 一种药物组合物,其特征在于,所述组合物包含:如权利要求2所述的融合蛋白,以及药学上可接受的载体。
- 一种免疫细胞,其特征在于,所述的免疫细胞携带如权利要求2所述的融合蛋白。
- 一种药物组合物,其特征在于,所述的组合物包含:如权利要求12所述的免疫细胞,以及药学上可接受的载体。
- 如权利要求2所述的融合蛋白或如权利要求12所述的免疫细胞的用途,用于制备治疗肿瘤的药物。
- 如权利要求14所述的用途,其特征在于,所述的肿瘤包括:乳腺癌肿瘤、胃癌肿瘤、膀胱癌肿瘤、胰腺癌肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、黑色素肿瘤。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110902623.8A CN115703840A (zh) | 2021-08-06 | 2021-08-06 | 抗Her-2抗体-粒细胞调节因子融合蛋白及其制法和应用 |
CN202110902623.8 | 2021-08-06 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023011662A1 true WO2023011662A1 (zh) | 2023-02-09 |
Family
ID=85154870
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2022/110926 WO2023011662A1 (zh) | 2021-08-06 | 2022-08-08 | 抗Her-2抗体-粒细胞调节因子融合蛋白及其制法和应用 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN115703840A (zh) |
WO (1) | WO2023011662A1 (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1311332A (zh) * | 2001-02-27 | 2001-09-05 | 李欣越 | 用基因工程方法生产红细胞刺激因子/粒细胞集落刺激因子融合蛋白 |
CN1410450A (zh) * | 2001-10-01 | 2003-04-16 | 旭华(上海)生物研发中心有限公司 | 生物活性提高的人粒细胞集落刺激因子的Fc融合蛋白 |
US20030187225A1 (en) * | 2002-03-21 | 2003-10-02 | The Regents Of The University Of California | Antibody fusion proteins: effective adjuvants of protein vaccination |
CN103755812A (zh) * | 2012-12-14 | 2014-04-30 | 上海柯莱逊生物技术有限公司 | 一种gm-csf-her2重组蛋白及其方法和应用 |
-
2021
- 2021-08-06 CN CN202110902623.8A patent/CN115703840A/zh active Pending
-
2022
- 2022-08-08 WO PCT/CN2022/110926 patent/WO2023011662A1/zh unknown
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1311332A (zh) * | 2001-02-27 | 2001-09-05 | 李欣越 | 用基因工程方法生产红细胞刺激因子/粒细胞集落刺激因子融合蛋白 |
CN1410450A (zh) * | 2001-10-01 | 2003-04-16 | 旭华(上海)生物研发中心有限公司 | 生物活性提高的人粒细胞集落刺激因子的Fc融合蛋白 |
US20030187225A1 (en) * | 2002-03-21 | 2003-10-02 | The Regents Of The University Of California | Antibody fusion proteins: effective adjuvants of protein vaccination |
CN103755812A (zh) * | 2012-12-14 | 2014-04-30 | 上海柯莱逊生物技术有限公司 | 一种gm-csf-her2重组蛋白及其方法和应用 |
Non-Patent Citations (3)
Title |
---|
CRUZ, J. S. D. ET AL.: "Anti-HER2/neu IgG3–(IL-2) and anti-HER2/neu IgG3–(GM-CSF) promote HER2/neu processing and presentation by dendritic cells: Implications in immunotherapy and vaccination strategies", MOLECULAR IMMUNOLOGY, vol. 43, 23 May 2005 (2005-05-23), pages 667 - 676, XP025037173, DOI: 10.1016/j.molimm.2005.04.007 * |
FANG YONGXIANG, FENG HAIYAN, MOSCOW, JING ZHIZHONG: "Cytokine Fusion Protein Technology and Its Application Prospects", CHINESE JOURNAL OF CELLULAR AND MOLECULAR IMMUNOLOGY, FOURTH MILITARY MEDICAL UNIVERSITY, XI'AN, CN, vol. 25, no. 9, 31 December 2009 (2009-12-31), CN , pages 856 - 859, XP093031509, ISSN: 1007-8738, DOI: 10.13423/j.cnki.cjcmi.005177856 * |
GUO XIN: " Protective Immune Response against HER2/neu Expressing Tumors Induced by Inoculation of Novel Anti-HER2/neu Cytokine Fusion Proteins with Soluble Protein Antigen Conjugates", INTERNATIONAL JOURNAL OF BIOLOGICALS, vol. 29, no. 6, 31 December 2006 (2006-12-31), pages 279 - 279, XP093031514 * |
Also Published As
Publication number | Publication date |
---|---|
CN115703840A (zh) | 2023-02-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR102652247B1 (ko) | 항체 중쇄불변부위 이종이중체 (heterodimeric Fc)에 융합된 사이토카인 (heterodimeric Fc-fused cytokine) 및 이를 포함하는 약제학적 조성물 | |
RU2711979C2 (ru) | Белковый комплекс интерлейкина 15 и его применение | |
JP7282383B2 (ja) | タンパク質性ヘテロ二量体及びその用途 | |
CN105121474B (zh) | 融合免疫调节蛋白及其制备方法 | |
US7169904B2 (en) | Immunocytokine sequences and uses thereof | |
JP2021522786A (ja) | インターロイキン15融合タンパク質、およびその組成物ならびに治療方法 | |
US10167328B2 (en) | Methods for cancer therapy using mutant light molecules with increased affinity to receptors | |
KR20180057715A (ko) | 치료 화합물 및 방법 | |
CN108250303B (zh) | 单域抗体融合蛋白及其应用 | |
JP6242484B2 (ja) | 特定の改善されたヒト二重特異性EGFRvIII抗体結合分子 | |
HU229039B1 (en) | Cd19xcd3 specific polypeptides and uses thereof | |
CN112142859A (zh) | 一种人白细胞介素10-Fc融合蛋白及其编码基因与应用 | |
CN110835374A (zh) | 抗ccr8×ctla-4双特异性抗体及其应用 | |
JP2006517970A (ja) | 癌免疫療法のための組成物及び方法 | |
KR20230070256A (ko) | 이중특이성 재조합 단백질 및 이의 용도 | |
CN115916831A (zh) | 包含抗lag-3抗体和il-2的融合蛋白及其用途 | |
AU2021212558A1 (en) | Fusion protein comprising anti-TAA antibody, anti-PD-L1 antibody, and IL-2, and uses thereof | |
CN113248625A (zh) | 融合蛋白、编码融合蛋白的基因、重组载体及它们的应用 | |
WO2022100585A1 (zh) | 抗Her-2抗体-趋化因子融合蛋白及其制法和应用 | |
WO2023011662A1 (zh) | 抗Her-2抗体-粒细胞调节因子融合蛋白及其制法和应用 | |
KR20230044130A (ko) | Pd-1 및 il-21을 포함하는 융합단백질 이량체 및 이의 용도 | |
KR20240032847A (ko) | Cldn18.2 및 cd3에 결합하는 이중특이 결합제 | |
CN112166122A (zh) | 抗体-肿瘤坏死因子α融合蛋白及其制法和应用 | |
WO2021031736A1 (zh) | 多功能抗体、其制备及其用途 | |
US20230322882A1 (en) | Fusion Polypeptide and Polypeptide Dimer, and Use Thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22852372 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |