WO2024061350A1 - Protéine de fusion et son utilisation - Google Patents
Protéine de fusion et son utilisation Download PDFInfo
- Publication number
- WO2024061350A1 WO2024061350A1 PCT/CN2023/120732 CN2023120732W WO2024061350A1 WO 2024061350 A1 WO2024061350 A1 WO 2024061350A1 CN 2023120732 W CN2023120732 W CN 2023120732W WO 2024061350 A1 WO2024061350 A1 WO 2024061350A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- amino acid
- acid sequence
- seq
- heavy chain
- sequence shown
- Prior art date
Links
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 79
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 76
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims abstract description 62
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 claims abstract description 62
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 263
- 230000027455 binding Effects 0.000 claims description 131
- 210000004027 cell Anatomy 0.000 claims description 106
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 claims description 53
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 claims description 49
- 239000000427 antigen Substances 0.000 claims description 46
- 102000036639 antigens Human genes 0.000 claims description 46
- 108091007433 antigens Proteins 0.000 claims description 46
- 206010028980 Neoplasm Diseases 0.000 claims description 41
- 108090000623 proteins and genes Proteins 0.000 claims description 37
- 239000013598 vector Substances 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 32
- 239000012634 fragment Substances 0.000 claims description 31
- 201000011510 cancer Diseases 0.000 claims description 28
- 150000007523 nucleic acids Chemical class 0.000 claims description 27
- 102000039446 nucleic acids Human genes 0.000 claims description 26
- 108020004707 nucleic acids Proteins 0.000 claims description 26
- 102000004169 proteins and genes Human genes 0.000 claims description 25
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 24
- 101000863873 Homo sapiens Tyrosine-protein phosphatase non-receptor type substrate 1 Proteins 0.000 claims description 24
- 102100029948 Tyrosine-protein phosphatase non-receptor type substrate 1 Human genes 0.000 claims description 24
- 239000003814 drug Substances 0.000 claims description 15
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 238000011282 treatment Methods 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 239000003446 ligand Substances 0.000 claims description 9
- 238000006467 substitution reaction Methods 0.000 claims description 9
- 229940124597 therapeutic agent Drugs 0.000 claims description 9
- 102000025171 antigen binding proteins Human genes 0.000 claims description 8
- 108091000831 antigen binding proteins Proteins 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 206010009944 Colon cancer Diseases 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 210000004408 hybridoma Anatomy 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 206010006187 Breast cancer Diseases 0.000 claims description 5
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- 230000006044 T cell activation Effects 0.000 claims description 5
- 208000029742 colonic neoplasm Diseases 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 230000002538 fungal effect Effects 0.000 claims description 5
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 210000004962 mammalian cell Anatomy 0.000 claims description 5
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 claims description 4
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 4
- 201000005787 hematologic cancer Diseases 0.000 claims description 4
- 102000044459 human CD47 Human genes 0.000 claims description 4
- 201000010893 malignant breast melanoma Diseases 0.000 claims description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 102000006495 integrins Human genes 0.000 claims description 3
- 108010044426 integrins Proteins 0.000 claims description 3
- 210000005253 yeast cell Anatomy 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 101710105077 Agglutinin-1 Proteins 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 101000835928 Homo sapiens Signal-regulatory protein gamma Proteins 0.000 claims description 2
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 claims description 2
- 102100025795 Signal-regulatory protein gamma Human genes 0.000 claims description 2
- 239000000611 antibody drug conjugate Substances 0.000 claims description 2
- 229940049595 antibody-drug conjugate Drugs 0.000 claims description 2
- 239000002246 antineoplastic agent Substances 0.000 claims description 2
- 229940034982 antineoplastic agent Drugs 0.000 claims description 2
- 230000012010 growth Effects 0.000 claims description 2
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 238000001959 radiotherapy Methods 0.000 claims description 2
- 239000000833 heterodimer Substances 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 28
- 230000014509 gene expression Effects 0.000 description 26
- 210000004881 tumor cell Anatomy 0.000 description 25
- 102000004196 processed proteins & peptides Human genes 0.000 description 20
- 239000013612 plasmid Substances 0.000 description 19
- 229920001184 polypeptide Polymers 0.000 description 19
- 238000001514 detection method Methods 0.000 description 17
- 239000006228 supernatant Substances 0.000 description 15
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 14
- 239000000872 buffer Substances 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 108090000695 Cytokines Proteins 0.000 description 11
- 102000004127 Cytokines Human genes 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 11
- 102000053602 DNA Human genes 0.000 description 11
- 108060003951 Immunoglobulin Proteins 0.000 description 11
- 210000001744 T-lymphocyte Anatomy 0.000 description 11
- 102000018358 immunoglobulin Human genes 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 241000700159 Rattus Species 0.000 description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 10
- 238000001890 transfection Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 210000002540 macrophage Anatomy 0.000 description 9
- 238000001994 activation Methods 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 8
- 102000040430 polynucleotide Human genes 0.000 description 8
- 108091033319 polynucleotide Proteins 0.000 description 8
- 239000002157 polynucleotide Substances 0.000 description 8
- 230000004913 activation Effects 0.000 description 7
- 229910002092 carbon dioxide Inorganic materials 0.000 description 7
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 230000001404 mediated effect Effects 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 6
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 6
- 229920002873 Polyethylenimine Polymers 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- 230000002147 killing effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000012636 effector Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- LNEPOXFFQSENCJ-UHFFFAOYSA-N haloperidol Chemical compound C1CC(O)(C=2C=CC(Cl)=CC=2)CCN1CCCC(=O)C1=CC=C(F)C=C1 LNEPOXFFQSENCJ-UHFFFAOYSA-N 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 239000001509 sodium citrate Substances 0.000 description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 101000946860 Homo sapiens T-cell surface glycoprotein CD3 epsilon chain Proteins 0.000 description 4
- 101150036449 SIRPA gene Proteins 0.000 description 4
- 102100035794 T-cell surface glycoprotein CD3 epsilon chain Human genes 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000002998 immunogenetic effect Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 201000007270 liver cancer Diseases 0.000 description 4
- 208000014018 liver neoplasm Diseases 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 229920002477 rna polymer Polymers 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 3
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- 206010057249 Phagocytosis Diseases 0.000 description 3
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 3
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000008782 phagocytosis Effects 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 238000011830 transgenic mouse model Methods 0.000 description 3
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 2
- 238000002965 ELISA Methods 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
- 241000282412 Homo Species 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 241000282567 Macaca fascicularis Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- 238000011284 combination treatment Methods 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 230000009260 cross reactivity Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 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
- 230000003325 follicular Effects 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 210000003714 granulocyte Anatomy 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 208000017805 post-transplant lymphoproliferative disease Diseases 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012216 screening 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
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- VGONTNSXDCQUGY-RRKCRQDMSA-N 2'-deoxyinosine Chemical group C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 VGONTNSXDCQUGY-RRKCRQDMSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FVFVNNKYKYZTJU-UHFFFAOYSA-N 6-chloro-1,3,5-triazine-2,4-diamine Chemical group NC1=NC(N)=NC(Cl)=N1 FVFVNNKYKYZTJU-UHFFFAOYSA-N 0.000 description 1
- 208000002008 AIDS-Related Lymphoma Diseases 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 108010034753 Complement Membrane Attack Complex Proteins 0.000 description 1
- 241000557626 Corvus corax Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 206010018852 Haematoma Diseases 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101100438942 Homo sapiens CD3E gene Proteins 0.000 description 1
- 101001066129 Homo sapiens Glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 101001009603 Homo sapiens Granzyme B Proteins 0.000 description 1
- 101000599940 Homo sapiens Interferon gamma Proteins 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 1
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 1
- 229940123776 Immuno-oncology therapy Drugs 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- 208000025205 Mantle-Cell Lymphoma Diseases 0.000 description 1
- 208000006395 Meigs Syndrome Diseases 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 208000003788 Neoplasm Micrometastasis Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 206010048734 Phakomatosis Diseases 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 229930185560 Pseudouridine Natural products 0.000 description 1
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 208000004337 Salivary Gland Neoplasms Diseases 0.000 description 1
- 206010061934 Salivary gland cancer Diseases 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 206010047741 Vulval cancer Diseases 0.000 description 1
- 208000004354 Vulvar Neoplasms Diseases 0.000 description 1
- 208000033559 Waldenström macroglobulinemia Diseases 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
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000000053 blastoma Diseases 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 1
- 230000009850 completed effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 210000000448 cultured tumor cell Anatomy 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000013583 drug formulation Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 201000008184 embryoma Diseases 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 102000054767 gene variant Human genes 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 201000009277 hairy cell leukemia Diseases 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 102000047486 human GAPDH Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000005249 lung adenocarcinoma Diseases 0.000 description 1
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 208000025113 myeloid leukemia Diseases 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 201000002628 peritoneum cancer Diseases 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000008560 physiological behavior Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 229940043274 prophylactic drug Drugs 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000031337 regulation of inflammatory response Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000037432 silent mutation Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- -1 thromboagglutinin-1 Proteins 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 201000005102 vulva cancer Diseases 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- 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
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
Definitions
- This application relates to the field of biomedicine, specifically to a fusion protein composed of SIRP ⁇ V2 and CD3 antibody.
- T cell engager bisAb is a typical drug of this type. Its mechanism of action is to form an immune synapse (as shown in Figure 1) by simultaneously binding to T cell surface antigens (such as CD3) and tumor cell surface antigens (such as CD19), thus bringing them closer together. The distance between tumor cells and T cells is directly activated, proliferating T cells, and then using T cells to directly kill tumor cells, or releasing cytotoxins to kill tumor cells. Generally speaking, this T cell activation process is simple and direct and does not require the presentation of tumor cell antigens to generate specific T lymphocyte clones. Therefore, it is not restricted by MHC or HLA and has a high clinical transformation rate.
- CD47 is a transmembrane protein widely present on the surface of normal cells and belongs to the immunoglobulin superfamily. It is an important target for a new generation of immuno-oncology therapies.
- CD47 and PD-1 respectively target two major immune cell groups that play a major role in tumor immunotherapy.
- the CD47 signaling pathway mainly regulates macrophages
- the PD-1 signaling pathway mainly regulates T lymphocytes.
- Macrophages are an important part of innate immunity and can act as scavengers in the human body by engulfing aging and dead cells.
- CD47 is highly expressed on a variety of solid tumor cells and malignant hematoma cells, and its expression level is positively correlated with disease progression.
- CD47 pathway drugs can theoretically be used in the treatment of many types of cancers like PD-1 drugs.
- CD47-targeting drugs have been used as single agents and combination treatments in the treatment of common blood tumors and solid tumors such as leukemia, lymphoma, lung cancer, and liver cancer. Under treatment.
- integrins There are three known natural ligands of CD47: integrins, platelet agglutinin-1 and SIRP ⁇ .
- the biological effects it participates in include: cell adhesion, cell migration, regulation of inflammatory responses and inhibition of macrophage phagocytosis.
- SIRP ⁇ V domain in molecular design can help target CD47 and relieve the resistance of cancer cells by preventing CD47 on the surface of cancer cells from binding to SIRP ⁇ on the surface of macrophages or DC cells.
- the inhibitory effect of macrophages induces the phagocytosis of tumor cells by macrophages, promotes the uptake of tumor cells by DC cells, and is beneficial to the presentation of tumor antigens.
- SIRP ⁇ V2 SNP variant Since CD47 is also expressed on the surface of normal cells (especially red blood cells) and the SIRP ⁇ V2 SNP variant shows a selective weak affinity for CD47 on the surface of red blood cells, in order to avoid off-target toxicity, the selection of SIRP ⁇ V2 variants in molecular design can greatly alleviate cell agglutination and lysis, and thrombocytopenia and other issues.
- This application combines SIRP ⁇ V2 and CD3 antibodies to form a fusion protein, thereby activating T cells and bringing tumor cells and T cells closer to induce the killing of tumor cells.
- SIRPa can also activate the inhibitory effect of contact with tumor cells on macrophages and DCs, further weakening the immune evasion of tumor cells.
- this fusion protein we compared the symmetric configuration (D1-TW) and the asymmetric configuration (H3S02). It was found that the asymmetric configuration is significantly more effective than the symmetric configuration, and the release levels of several different cytokines are close to or lower than those of the symmetric configuration molecules.
- this application also provides an asymmetrically configured molecule Mos-D1-TW, which uses a different anti-CD3 sequence, but other parts are the same as D1-TW.
- the application provides an isolated fusion protein including a CD3 binding portion and a CD47 binding portion;
- the CD3 binding portion comprises an amino acid sequence having at least 95% identity with heavy chain variable regions HCDR1, HCDR2 and HCDR3, and the amino acid sequence of HCDR1 is any one of SEQ ID NO: 4, 24, 32 and 40
- the amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO: 5, 25, 33 and 41; the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO: 6, 26, 34 and 42 Item 1, and/or the CD3 binding portion includes an amino acid sequence that is at least 95% identical to the light chain variable regions LCDR1, LCDR2 and LCDR3, and the amino acid sequence of LCDR1 is such as SEQ ID NO: 1, 27, As shown in any one of 35 and 43; the amino acid sequence of the LCDR2 is as shown in any one of SEQ ID NO:2, 28, 36 and 44; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:3, 29, 37 and 45, wherein the CD3 binding moiety is capable of inducing T cell activation.
- the CD47 binding portion comprises an anti-CD47 antibody or an antigen-binding fragment thereof, or a CD47 ligand or a functional variant thereof.
- the CD47 ligand comprises integrin, thromboagglutinin-1, SIRP ⁇ , SIRP ⁇ or functional variants or fragments thereof.
- the CD47 binding moiety includes SIRP ⁇ , or a functional variant thereof, or a fragment thereof.
- the present application provides an isolated fusion protein including a CD3 binding portion and a CD47 binding portion;
- the CD3 binding portion comprises an amino acid sequence having at least 95% identity with heavy chain variable regions HCDR1, HCDR2 and HCDR3, and the amino acid sequence of HCDR1 is any one of SEQ ID NO: 4, 24, 32 and 40
- the amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO: 5, 25, 33 and 41; the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO: 6, 26, 34 and 42 Item 1, and/or the CD3 binding portion includes an amino acid sequence that is at least 95% identical to the light chain variable regions LCDR1, LCDR2 and LCDR3, and the amino acid sequence of LCDR1 is such as SEQ ID NO: 1, 27, As shown in any one of 35 and 43; the amino acid sequence of the LCDR2 is as shown in any one of SEQ ID NO:2, 28, 36 and 44; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:3, 29, As shown in any one of 37 and 45, wherein the CD3 binding portion can induce T cell activation;
- the CD3 binding portion comprises an amino acid sequence having at least 95% identity with the SIRP ⁇ protein
- the amino acid sequence of the SIRP ⁇ protein is shown in SEQ ID NO: 46.
- the present application provides an isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion that specifically binds to human CD47 and human CD3, wherein the CD3 binding portion competes with the reference antigen binding protein for human CD3 Binding, the reference antigen binding protein includes three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein HCDR1 contains as shown in SEQ ID NO:4
- the amino acid sequence of HCDR2 includes the amino acid sequence shown in SEQ ID NO:5;
- HCDR3 includes the amino acid sequence shown in SEQ ID NO:6,
- LCDR1 includes the amino acid sequence shown in SEQ ID NO:1, where LCDR2 includes the amino acid sequence set forth in SEQ ID NO:2, and wherein LCDR3 includes the amino acid sequence set forth in SEQ ID NO:3; and
- the CD47 binding part competes with the SIRP ⁇ protein for binding to human CD47, and the SIRP ⁇ protein includes the amino acid sequence shown in SEQ ID NO: 46.
- the present application provides an isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion, wherein the CD3 binding portion includes three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and three light chain complementation determining regions.
- Region LCDR1, LCDR2 and LCDR3
- HCDR1 includes the amino acid sequence shown in any one of SEQ ID NO:4, 24, 32 and 40
- HCDR2 includes any one of SEQ ID NO:5, 25, 33 and 41
- the amino acid sequence shown in one item wherein HCDR3 includes the amino acid sequence shown in any one of SEQ ID NO:6, 26, 34 and 42, and wherein LCDR1 includes any one of SEQ ID NO:1, 27, 35 and 43.
- LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO:2, 28, 36 and 44, and wherein LCDR3 includes any one of SEQ ID NO:3, 29, 37 and 45. an amino acid sequence represented by an item;
- the CD47 binding part includes the amino acid sequence shown in SEQ ID NO: 46.
- the CD3 binding portion comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3; wherein the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 4 As shown, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:5 and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:6; and/or the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:1, The amino acid sequence of LCDR2 is shown in SEQ ID NO:2 and the amino acid sequence of LCDR3 is shown in SEQ ID NO:3;
- HCDR1 comprises the amino acid sequence shown in SEQ ID NO:24; wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO:25; wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:26, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO:28 (DTS), and wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:29;
- HCDR1 comprises the amino acid sequence shown in SEQ ID NO:32; wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO:33; wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:34, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:35, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:37; or
- HCDR1 comprises the amino acid sequence shown as SEQ ID NO:40; wherein HCDR2 comprises the amino acid sequence shown as SEQ ID NO:41; wherein HCDR3 comprises the amino acid sequence shown as SEQ ID NO:42, wherein LCDR1 comprises the amino acid sequence shown as SEQ ID NO:43, wherein LCDR2 comprises the amino acid sequence shown as SEQ ID NO:44(YTS), and wherein LCDR3 comprises the amino acid sequence shown as SEQ ID NO:45.
- the CD3 binding moiety comprises a heavy chain variable region
- the amino acid sequence of the heavy chain variable region is any one of SEQ ID NOs: 8, 15, 16, 22, 30 and 38 As shown in the item; and/or the CD3 binding portion includes a light chain variable region, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 7, 19, 23, 31 and 39 .
- the CD3 binding portion comprises a heavy chain variable region and a light chain variable region
- the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8
- the light chain The variable region contains the amino acid sequence shown in SEQ ID NO:7;
- the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19;
- the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:16, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:19;
- the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:23;
- the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:31; or
- the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39.
- the CD3-binding portion is an antibody or antigen-binding fragment thereof.
- the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.
- the antigen-binding fragments include Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
- the fusion protein further comprises a heterodimeric Fc portion.
- the Fc is from an Fc of IgGl, IgG2, IgG3 or IgG4.
- the heterodimeric Fc portion is connected by a disulfide bond in the hinge region and a hinge structure of the CH3 domain.
- the CD3 binding portion comprises an antibody heavy chain constant region
- the antibody heavy chain constant region comprises a constant region derived from human IgGl, IgG2, IgG3 or IgG4.
- the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 17.
- the CD3 binding portion comprises an antibody light chain constant region
- the antibody light chain constant region comprises a human Ig ⁇ constant region or a human Ig ⁇ constant region.
- the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 20.
- the CD3 binding portion comprises an antibody heavy chain and a light chain, wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:18, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:21.
- the CD47 binding portion comprises an antibody heavy chain constant region
- the antibody heavy chain constant region comprises a constant region derived from human IgGl, IgG2, IgG3 or IgG4.
- the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 47.
- the CD47 binding portion comprises a heavy chain
- the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 48.
- the present application provides a fusion protein having two heavy chains and one light chain, wherein the first heavy chain has VH-CH1-hinge region-Fc from N-terminus to C-terminus, and the first light chain has VH-CH1-hinge region-Fc from N-terminus to C-terminus. It has VL-CL from end to C end; the second heavy chain has SIRP ⁇ -hinge region-Fc from N end to C end, wherein the VH-CH1 of the first heavy chain and the VL-CL of the first light chain form The antigen binding site binds to CD3, and the SIRP ⁇ of the second heavy chain binds to CD47;
- the first heavy chain includes three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), and the first light chain includes three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), among which HCDR1 includes SEQ ID NO:4 , the amino acid sequence shown in any one of 33, 41 and 49; wherein HCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 5, 34, 42 and 50; wherein HCDR3 includes such as SEQ ID NO: 6, 35, 43 and 51, wherein LCDR1 includes the amino acid sequence shown in any one of SEQ ID NO: 1, 36, 44 and 52, wherein LCDR2 includes SEQ ID NO: 2, 37, 45 and 53, and wherein LCDR3 comprises an amino acid sequence as shown in any one of SEQ ID NO: 3, 38, 46 and 54; and wherein the second heavy chain includes SIRP ⁇ , Wherein the SIRP ⁇ includes the amino acid sequence shown in SEQ ID NO:46.
- the first heavy chain comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3
- the first light chain comprises light chain variable regions LCDR1, LCDR2 and LCDR3
- the amino acid sequence of HCDR1 is as SEQ ID NO:4
- the amino acid sequence of the HCDR2 is shown in SEQ ID NO:5
- the amino acid sequence of the HCDR3 is shown in SEQ ID NO:6
- the amino acid sequence of the LCDR1 is shown in SEQ ID
- the amino acid sequence of the LCDR2 is shown in SEQ ID NO:2 and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:3;
- HCDR1 includes the amino acid sequence shown in SEQ ID NO:24; wherein HCDR2 includes the amino acid sequence shown in SEQ ID NO:25; wherein HCDR3 includes the amino acid sequence shown in SEQ ID NO:26, wherein LCDR1 includes the amino acid sequence shown in SEQ ID NO:25 The amino acid sequence shown in ID NO:27, wherein LCDR2 includes the amino acid sequence shown in SEQ ID NO:28, and wherein LCDR3 includes the amino acid sequence shown in SEQ ID NO:29;
- HCDR1 contains the amino acid sequence shown in SEQ ID NO:32; wherein HCDR2 contains the amino acid sequence shown in SEQ ID NO:33; wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:34, and LCDR1 contains the amino acid sequence shown in SEQ The amino acid sequence shown in ID NO:35, wherein LCDR2 includes the amino acid sequence shown in SEQ ID NO:36, and wherein LCDR3 includes the amino acid sequence shown in SEQ ID NO:37; or
- HCDR1 includes the amino acid sequence shown in SEQ ID NO:40; wherein HCDR2 includes the amino acid sequence shown in SEQ ID NO:41; wherein HCDR3 includes the amino acid sequence shown in SEQ ID NO:42, and LCDR1 includes the amino acid sequence shown in SEQ The amino acid sequence shown in ID NO:43, wherein LCDR2 includes the amino acid sequence shown in SEQ ID NO:44, and wherein LCDR3 includes the amino acid sequence shown in SEQ ID NO:45.
- the first heavy chain comprises a heavy chain variable region
- the amino acid sequence of the heavy chain variable region is any of SEQ ID NOs: 8, 15, 16, 22, 30 and 38. as shown in one item; and/or the first light chain comprises a light chain variable region, and the amino acid sequence of the light chain variable region is as in any one of SEQ ID NO: 7, 19, 23, 31 and 39 shown.
- the first heavy chain includes a heavy chain variable region
- the first light chain includes a light chain variable region
- the heavy chain variable region includes SEQ ID NO: 8
- the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO:7;
- the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:19;
- the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:16, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:19;
- the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:23;
- the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:31; or
- the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 38, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 39.
- first heavy chain and the second heavy chain comprise Fc from IgG.
- the Fc is Fc from IgGl, IgG2, IgG3 or IgG4.
- first heavy chain and the second heavy chain comprise Fc from human IgG.
- the Fc is an Fc from human IgG1, human IgG2, human IgG3 or human IgG4.
- the first heavy chain and the second heavy chain are connected through a disulfide bond in the hinge region and a pestle structure of the CH3 domain.
- first heavy chain and the second heavy chain are human IgG1 isotypes
- one of the first heavy chain or the second heavy chain comprises T366S, L368A and Y407V heavy chain substitutions
- the other of the first heavy chain or the second heavy chain contains the T366W heavy chain substitution, where the residues are numbered according to the EU index.
- the first light chain comprises CL from a human lambda or kappa light chain.
- the first heavy chain comprises the amino acid sequence shown in SEQ ID NO:18
- the first light chain comprises the amino acid sequence shown in SEQ ID NO:21.
- the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 48.
- the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein described in the present application or a fusion protein described in the present application.
- the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) production by clonal recombination, (iii) purification , for example by enzymatic digestion and gel electrophoresis fractionation, or (iv) synthetic, for example by chemical synthesis.
- the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
- nucleic acids encoding the antibodies and antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including but not limited to, using restriction fragment manipulation or using synthetic oligonucleotides.
- restriction fragment manipulation or using synthetic oligonucleotides.
- specific procedures can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.
- the present application provides a vector comprising a nucleic acid according to the present application.
- a nucleic acid may be included in each vector.
- other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
- the vector may contain expression control elements that allow correct expression of the coding region in an appropriate host.
- control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
- the expression control sequences are tunable elements.
- the specific structure of the expression control sequence can vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, GA cap sequence, CAAT sequence, etc.
- the 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid.
- the expression control sequences may also include enhancer sequences or upstream activator sequences.
- suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoters, CMV promoters and artificial hybrid promoters thereof (such as CMV), where the promoter A certain part can be fused with a certain part of the gene promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not include additional introns.
- One or more nucleic acid molecules described herein can be operably linked to the expression control element.
- the vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering.
- the vector is an expression vector.
- each or each host cell contains one or more nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell contains multiple (eg, 2 or more) or multiple (eg, 2 or more) nucleic acid molecules or vectors described herein.
- the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. The vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.
- the present application provides a pharmaceutical composition, which includes the fusion protein described in the present application and a pharmaceutically acceptable carrier.
- the pharmaceutical composition of the present application may contain a safe and effective amount (such as 0.001-99wt%, 0.01-90wt%, or 0.1-80wt%) of the fusion protein described in the present application and a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier may include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
- the drug formulation should match the mode of administration.
- the pharmaceutical composition described in the present application can be prepared in the form of an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other auxiliary agents. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
- the active ingredients are administered in amounts that are therapeutically effective.
- the fusion proteins described herein can also be used with other therapeutic agents.
- the fusion proteins or pharmaceutical compositions described herein may be formulated, administered, and administered in a manner consistent with good medical practice. Considerations in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the etiology of the condition, the site of agent delivery, the method of administration, and other factors known to the medical practitioner.
- the therapeutic agent need not be, but optionally, be formulated and/or administered concurrently with one or more agents currently used to prevent or treat the condition under consideration.
- the effective amount of such other agents depends on the amount of therapeutic agent present in the formulation, the type of condition or treatment, and other factors discussed above. These agents can generally be used at any dose and by any route empirically/clinically determined to be appropriate.
- the dose of antibodies administered in combination treatment can be reduced compared to individual treatments. The progress of this therapy is easily monitored by conventional techniques.
- the present application provides the fusion protein described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the host cell described in the present application, or the pharmaceutical composition described in the present application. Use in the preparation of medicaments for the treatment of cancer.
- the cancer includes solid tumors and hematological tumors.
- cancer is a CD47-expressing cancer.
- the CD47-expressing cancer is selected from the group consisting of at least one of breast cancer, melanoma, and colon cancer.
- the present application provides a method for treating cancer in a subject, the method comprising administering to the subject the fusion protein described in the present application, the isolated nucleic acid molecule described in the present application, the The vector described herein, the host cell described herein, or the pharmaceutical composition described herein, thereby inhibiting the growth of the cancer in the subject.
- the cancer includes solid tumors and hematological tumors.
- cancer is a CD47-expressing cancer.
- the CD47-expressing cancer is selected from the group consisting of at least one of breast cancer, melanoma, and colon cancer.
- it further comprises administering to the subject a second therapeutic agent.
- the second therapeutic agent is an antineoplastic agent, radiation therapy, an antibody drug conjugate, a checkpoint inhibitor, or a combination thereof.
- the present application provides a method for producing the fusion protein described in the present application or the fusion protein described in the present application, wherein the method comprises culturing the host cell described in the present application under conditions capable of expressing the fusion protein.
- the host cell is selected from the group consisting of bacterial cells, fungal cells, plant cells, mammalian cells, or viruses.
- the bacterial cell is E. coli.
- the fungal cell is a yeast cell.
- the mammalian cells are selected from CHO, NSO, BHK or HEK293 cells.
- the cells are hybridoma cells.
- the hybridoma cells are selected from mouse, rat, or rabbit.
- Figure 1 shows the configuration diagrams of the asymmetric anti-CD3 ⁇ SIRPa-Fc Fusion Protein (D1-TW) and symmetric anti-CD3 x SIRPa (H3S02) described in this application.
- Figure 2 shows the vector p2MPT map.
- Figure 3 shows the vector pMPTN map.
- Figure 4 shows a two-step purification diagram of the Mos-D1-TW molecule.
- Figure 5 shows the SDS-PAGE image after purification of Mos-D1-TW molecules.
- Figure 6 shows the two-step purification diagram of the D1-TW molecule.
- Figure 7 shows the SDS-PAGE image after purification of D1-TW molecules.
- Figure 8 shows the purification diagram of H3S02 molecule.
- Figure 9 shows the SDS-PAGE image after purification of H3S02 molecules.
- Figure 10 shows the affinity check results of D1-TW with CD3E/G and CD47.
- Figure 11 shows the ELISA detection results of Mos-D1-TW double antibody.
- Figure 12 shows the FACS detection of the binding of D1-TW to 8226 and HCT-8 tumor cells.
- Figure 13 shows the FACS detection of the binding of mos-D1-TW to target cells 8226 and hCD3E transgenic mouse PBMC.
- Figure 14 shows the killing effect of hPBMC on D1-TW-mediated tumor cells 8226 and HCT-8.
- Figures 15-16 show the release of cytokines during D1-TW-mediated killing of two tumor cell lines, 8226 and HCT-8.
- the term "about" when used in conjunction with a numerical value is intended to encompass a range of numerical values having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.
- SIRP ⁇ is a protein belonging to the SIRP receptor family.
- the full Chinese name of SIRP is Signal regulatory proteins (SIRP). It is mainly expressed on the surface of myeloid cells (monocytes, macrophages, granulocytes, myeloid DC cells, etc.), and is also expressed in neuronal cells of the nervous system.
- SIRP ⁇ contains three extracellular immunoglobulin superfamily domains, including an N-terminal variable region (V region) that binds to CD47 and two C1-type immunoglobulin superfamily domains. It is then connected to the intracellular inhibitory signaling domain through a transmembrane helical domain.
- V region N-terminal variable region
- CD3 generally refers to being part of a T cell receptor complex consisting of three distinct chains, CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
- concentration of CD3 on T cells produced by, for example, its immobilization by anti-CD3 antibodies, results in activation of T cells that is similar to T cell receptor-mediated activation but is independent of the specificity of the TCR clone.
- the vast majority of anti-CD3 antibodies recognize the CD3 epsilon chain.
- the term refers to any native CD3 from any vertebrate (including mammals such as primates (eg, humans)) and rodents (eg, mice and rats), unless otherwise stated.
- CD3 refers to the full length CD3 from human and cynomolgus monkey or a fragment thereof (such as its mature fragment lacking a signal peptide). In a preferred embodiment, CD3 refers to the full length from mouse/rat or a fragment thereof (such as its mature fragment lacking a signal peptide).
- percent (%) amino acid sequence identity or simply “identity” is defined as when the amino acid sequences are aligned (and gaps introduced when necessary) to obtain the maximum percent sequence identity without The percentage of amino acid residues in the candidate amino acid sequence that are identical to the amino acid residues in the reference amino acid sequence after any conservative substitutions are considered part of the sequence identity.
- Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software.
- One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain maximal alignment over the full length of the sequences being compared.
- the term "functional variant” generally refers to a nucleotide comprising an alteration of one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequence of a reference nucleic acid molecule or binding molecule. and/or amino acid sequence of a nucleic acid molecule or protein.
- the modification of the amino acid and/or nucleotide sequence of the reference binding molecule does not significantly affect or change the binding properties of the binding molecule encoded by the nucleotide sequence or containing the amino acid sequence, that is, the binding molecule is still able to recognize and bind to it. target.
- Functional variants of a gene include gene variants with minor changes, such as silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions that do not significantly alter the function of the gene.
- Functional variants can have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can include natural as well as unnatural nucleotides and amino acids.
- immune response generally refers to the effect of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes and the production of soluble macromolecules (including antibodies, cytokines and complements) by these cells or the liver, which The effects result in the selective damage, destruction or removal from the human body of invading pathogens, pathogen-infected cells or tissues, cancer cells or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.
- signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in a signal emanating from a part of the cell. passed to another part of the cell.
- transmission involves specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a cascade of reactions that results in signal transduction. Penultimate processes often involve nuclear events leading to changes in gene expression.
- the terms “activity” or “biological activity”, or the terms “biological property” or “biological characteristic” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, the ability to neutralize or antagonize CD47 activity in vivo or in vitro, IC50, in vivo stability of the antibody, and the immunogenic properties of the antibody.
- Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the targeted peptide, or with other proteins or tissues in general), and the ability to maintain high expression levels of the protein in mammalian cells.
- fusion protein generally refers to an amino acid sequence containing a first polypeptide or protein, or a fragment, analog or derivative thereof, and a heterologous polypeptide or protein (i.e., different from the first polypeptide or protein or of a second polypeptide or protein or a fragment, analog or derivative thereof, or that is generally not part of the first polypeptide or protein or a fragment, analog or derivative thereof) of the amino acid sequence Peptide or protein.
- a fusion protein may comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide.
- the heterologous protein, polypeptide or peptide may or may not be a preventive or therapeutic drug of different types.
- two different proteins, polypeptides or peptides with immunomodulatory activity can be fused together to form a fusion protein.
- the fusion protein retains or has improved activity compared to the activity of the heterologous protein, polypeptide, or original polypeptide or protein prior to fusion.
- the term "antigen-binding protein” generally refers to a protein comprising an antigen-binding portion, and optionally a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to the antigen.
- antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb), immunoconjugates Objects, multispecific antibodies (such as fusion proteins), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they display the required antigen-binding activity.
- An "isolated antigen-binding protein" of the present application may comprise an antigen-binding portion and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding portion by the antigen-binding portion.
- antibody generally refers to any form of antibody possessing the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as fusion proteins), humanized antibodies, fully human antibodies, chimeric Antibodies and camelized single domain antibodies. It is known that the basic antibody structural unit consists of tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy” chain (approximately 50-70 kDa).
- the amino-terminal portion or fragment of each chain may include a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition.
- the carboxyl-terminal portion or fragment of each chain may define the constant region primarily responsible for effector function.
- Human light chains are generally classified as kappa and lambda light chains.
- human heavy chains are typically classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
- the variable and constant regions are connected by a "J" region of about 12 or more amino acids, where the heavy chain also includes a "D” region of about 10 or more amino acids. See generally Chapter 7 of Fundamental Immunology (Ed. Paul, W., 2nd ed. Raven Press, N.Y. (1989)).
- isolated antibody generally refers to the purified state of the bound compound, and in this case means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, sugars or other substances e.g. Cell debris and growth medium.
- isolated does not imply the complete absence of such materials or the absence of water, buffers or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the bound compounds described herein.
- the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies composing the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and target a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
- the modifier "monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
- fusion protein generally refers to an artificially designed antibody, which is composed of components of two different antigen binding sites and can bind to two different antigen binding sites at the same time.
- full-length antibody generally refers to an immunoglobulin molecule that when naturally occurring contains four peptide chains: two heavy (H) chains (approximately 50-70 kDa in full length) and two light (L) chains. ) chains (approximately 25 kDa in full length) are connected to each other by disulfide bonds.
- Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
- the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
- Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
- the light chain constant region consists of one domain, CL.
- the VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conservative regions called framework regions (FRs).
- CDRs complementarity determining regions
- FRs framework regions
- Each VH or VL region consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
- the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
- the constant region of an antibody may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
- the term "antigen-binding fragment" of an antibody includes fragments or derivatives of the antibody, typically including at least one of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody Fragments that retain at least some of the binding specificity of the parent antibody.
- antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
- the binding fragment or derivative When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity.
- the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or greater of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as “conservative variants” or “functionally conserved variants” of the antibody).
- binding compound refers to both an antibody and its binding fragment.
- single-chain Fv or "scFv” antibody generally refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
- the Fv polypeptide generally also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
- CDR complementarity determining region
- CDR CDR
- HCDR1 CDR1
- HCDR2 CDR3
- LCDR1 CDR2
- LCDR3 CDR3
- Fc or “Fc region” or “Fc fragment” generally refers to the CH2 and CH3 domains of IgA, IgD and IgG, or the CH2, CH3 and CH4 domains of IgE and IgM through the hinge. region of the polypeptide.
- the heavy chain Fc fragment of human IgG generally refers to the stretch of polypeptide from A231 to its carboxyl terminus.
- hinge region generally refers to the proline-rich, easily stretchable and bendable polypeptide chain located between CH1 and CH2 in an antibody.
- the recognized hinge region of IgG is a polypeptide chain composed of amino acid residues from 216 to 230.
- domain antibody generally refers to an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region.
- two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
- the two VH regions of a bivalent domain antibody can target the same or different antigens.
- bivalent antibody encompasses 2 antigen-binding sites. In some cases, 2 binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
- the term "diabody” generally refers to a small antibody fragment with two antigen-binding sites contained in the same polypeptide chain (VH-VL or VL-VH) with a light chain variable domain (VH-VL or VL-VH).
- VL linked heavy chain variable domain
- chimeric antibody generally refers to an antibody having a variable domain of a first antibody and a constant domain of a second antibody, where the first and second antibodies are from different species.
- the variable domains are obtained from an antibody such as a rodent (a "parent antibody")
- the constant domain sequences are obtained from a human antibody such that the resulting chimeric antibody induces in human subjects compared to the parent rodent antibody. There is a lower likelihood of adverse immune responses.
- humanized antibody generally refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that is engineered to contain a minimal non-human (e.g., mouse) sequence.
- a humanized antibody is a human immunoglobulin, wherein the residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) with desired specificity, affinity, and ability (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)).
- the Fv framework region (FW) residues of a human immunoglobulin are replaced by corresponding residues from antibodies of non-human species with desired specificity, affinity, and ability.
- the term “fully human antibody” generally refers to an antibody that contains only human immunoglobulin protein sequences. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, fully human antibodies may contain mouse sugar chains. Similarly, “mouse antibodies” refer to antibodies that contain only mouse immunoglobulin sequences. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, fully human antibodies may contain rat sugar chains. Similarly, “rat antibodies” refer to antibodies that contain only rat immunoglobulin sequences.
- isotype of an antibody generally refers to the class of antibody provided by the heavy chain constant region gene (eg, IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where modifications have been made to alter Fc function, for example to enhance or weaken effector function or binding to Fc receptors.
- the heavy chain constant region gene eg, IgM, IgE, IgG such as IgG1, IgG2, or IgG4
- Isotypes also include modified forms of one of these classes, where modifications have been made to alter Fc function, for example to enhance or weaken effector function or binding to Fc receptors.
- epitope generally refers to the region of an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by the tertiary folding of the protein.
- affinity or “binding affinity” generally refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
- the affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon respectively).
- KD equilibrium dissociation constant
- kdis and kon association rate constant
- Affinity can be measured by common methods known in the art.
- One specific method used to measure affinity is the ForteBio kinetic binding assay described herein.
- the term "does not bind" to a protein or cell generally means that it does not bind to the protein or cell, or does not bind to it with high affinity, that is, the KD of the binding protein or cell is 1.0 ⁇ 10 -6 M or higher, More preferably, it is 1.0 ⁇ 10 -5 M or higher, more preferably 1.0 ⁇ 10 -4 M or higher, 1.0 ⁇ 10 -3 M or higher, and more preferably 1.0 ⁇ 10 -2 M or higher.
- high affinity for IgG antibodies generally means that the KD for the antigen is 1.0 ⁇ 10 -6 M or lower, preferably 5.0 ⁇ 10 -8 M or lower, and more preferably 1.0 ⁇ 10 -8 M or less, 5.0 ⁇ 10 -9 M or less, more preferably 1.0 ⁇ 10 -9 M or less.
- "high affinity” binding may vary.
- "high affinity” binding of the IgM subtype means a KD of 10 "6 M or less, preferably 10" 7 M or less, more preferably 10" 8 M or less.
- antibody-dependent cytotoxicity As used herein, the terms “antibody-dependent cytotoxicity”, “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refer to cell-mediated immune defense in which immune system effector cells actively bind cell membrane surface antigens to Target cells to which the antibody binds, such as cancer cells, are lysed.
- complement-dependent cytotoxicity generally refers to the effector functions of IgG and IgM antibodies that, when bound to surface antigens, trigger the typical complement pathway, including formation of the membrane attack complex and target cell lysis. .
- nucleic acid or “polynucleotide” generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single- or double-stranded form.
- the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, belonging to Kariko et al. Human U.S. Patent No. 8,278,036, which discloses an mRNA molecule in which uridine is replaced by pseudouridine, methods of synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo).
- nucleic acid sequence also implicitly includes conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences thereof as well as the sequences explicitly indicated.
- degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and/or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
- construct generally refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single- or double-stranded DNA or RNA polynucleoside acid molecule), derived from any source, capable of integrating with the genome or replicating autonomously, constituting a polynucleotide molecule to which one or more polynucleotide molecules have been linked in a functionally operative manner (i.e., operably linked).
- a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single- or double-stranded DNA or RNA polynucleoside acid molecule derived from any source, capable of integrating with the genome or replicating autonomously, constituting a polynucleotide molecule to which one or more polynucleotide molecules have been linked in
- Recombinant constructs will typically comprise a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in the host cell.
- transcription initiation regulatory sequences that direct transcription of the polynucleotide in the host cell.
- Both heterologous and non-heterologous (ie, endogenous) promoters can be used to direct expression of the nucleic acids of the invention.
- vector generally refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., the introduction of heterologous DNA into a host cell).
- plasmid which is a circular double-stranded DNA loop into which additional DNA segments can be ligated.
- viral vector in which additional DNA segments can be ligated into the viral genome.
- Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
- vectors Upon introduction into a host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate with the host genome. In addition, certain vectors are capable of directing expression of operably linked genes. Such vectors are referred to herein as "expression vectors.”
- expression vector generally refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell.
- the expression vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to provide amplification within the host if desired.
- activation may have the same meaning, for example, a cell or receptor is activated, stimulated, or treated with a ligand, unless the context otherwise or expressly dictates otherwise.
- Ligands include natural and synthetic ligands such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
- Ligand also includes small molecules such as peptide mimetics of cytokines and peptide mimetics of antibodies.
- Activation can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.
- a “response”, such as a response of a cell, tissue, organ or organism, includes a change in biochemical or physiological behavior (such as concentration, density, adhesion or migration, gene expression rate or differentiation status within a biological compartment) in which the change Relating to activation, stimulation or processing, or to internal mechanisms such as genetic programming.
- the term “treating” or “treating” any disease or condition in one embodiment means ameliorating the disease or condition (ie, slowing or arresting or reducing at least one of the progression of the disease or its clinical symptoms).
- “treating” refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discernible by the patient.
- “treating” or “treating” means modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of body parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.
- subject includes any human or non-human animal.
- non-human animals includes all vertebrate animals, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
- cyno or “cyno” refers to the cynomolgus monkey.
- administration in conjunction with one or more other therapeutic agents includes simultaneous (co) administration and sequential administration in any order.
- therapeutically effective amount generally refer to the ability of the antigen-binding protein of the present invention to effectively prevent or an amount that ameliorates the symptoms of one or more diseases or conditions or the progression of that disease or condition.
- a therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an improvement in symptoms, such as an amount that treats, cures, prevents, or ameliorates a related medical condition or increases the rate of treatment, cure, prevention, or amelioration of such a condition.
- the therapeutically effective dose refers only to that ingredient.
- a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether combined, administered sequentially, or administered simultaneously.
- An effective amount of the therapeutic agent will result in an improvement in diagnostic criteria or parameters of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, most preferably at least 50%.
- cancer and “cancerous” generally refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
- cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer, or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer, or uterine cancer, Salivary gland cancer, kidney or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade/follicular non-Hodgkin's disease) King's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL
- the sequences encoding the asymmetric structure of the Linton anti-CD3 antibody heavy chain and the Linton anti-CD3 antibody light chain were inserted into the EcoRI/XbaI expression cassette and NotI/BamHI expression cassette of the vector p2MPT, respectively, to form the recombinant plasmid 1p2MPT-hDL-D1TW-GA;
- the sequence encoding SIRPa-Fc Fusion protein was connected to the NotI/BamHI expression cassette of the vector p2MPT to form recombinant plasmid 2 p2MPT-hS1-GA.
- the QIAGEN Plasmid Midi Kit was used to prepare the correctly constructed plasmid 1 and plasmid 2.
- the sequences encoding the competitor anti-CD3 antibody heavy chain and the competitor anti-CD3 antibody light chain are each connected to the NotI/BamHI expression cassette of the vector pMPTN to form recombinant plasmid 3pMPTN-MD1TW-HC and plasmid 4pMPTN-MD1TW-LC respectively.
- the Endo-Free PlasmidMaxi Kit (Omega) kit was used to prepare the correctly constructed plasmid 3 and plasmid 4.
- sequences encoding the heavy chain and light chain of the anti-CD3 x SIRPa antibody with a symmetrical structure were connected to the NotI/BamHI expression cassette and EcoRI/XbaI expression cassette of the vector p2MPT respectively, forming recombinant plasmid 5 p2MPT-H3S02-HC-C3002LC.
- the QIAGEN PlasmidMidi Kit was used to prepare the correctly constructed plasmid 5.
- CHO-DG44-express stored in liquid nitrogen is recovered and cultured (Ex- cell Advanced CHO Fed-Batch Medium, Sigma), culture conditions were 37°C, 180 rpm, 75% RH, 5% CO2. The transfection and expression operation was performed after the cell viability stabilized to above 98%.
- the cell density was 3.0 ⁇ 10 6 cells/ml
- the transfection medium was proCHO5 medium (Lonza)
- the transfection reagent was 25K polyethylenimine (PEI, sigma)
- the DNA dosage was 3ug/mL (cell Culture volume, the dosage of helper plasmid pBase is 10% of the total DNA amount), and the dosage ratio to PEI is 1:3.
- the dosage of Linton anti-CD3 plasmid and SIRPa-Fc Fusion plasmid is 1:1. 24 hours after transfection, the medium was replaced with Ex-cell Advanced CHO Fed-Batch Medium (Sigma), and puromycin (Enzo) was used for screening.
- the seeding cell density was 5*10 ⁇ 5 cells/mL, and the final concentration of puromycin was is 10ug/mL.
- antibody expression is performed. After expression for 7 days at 31°C, 180 rpm, 75% RH, 5% CO2, the supernatant was collected by centrifugation.
- Expi293F (gibco) cells stored in liquid nitrogen were recovered and cultured (Wayne 293 Transfection Medium, Kang Tianshenghe).
- the culture conditions were 37°C, 110 rpm, 75% RH, and 6% CO2.
- the transfection and expression operation was performed after the cell viability stabilized to above 98%.
- the cell density is 1.5 ⁇ 10 6 cells/ml
- the transfection reagent is 25K polyethylenimine (PEI, sigma)
- the DNA dosage is 1ug/mL (cell culture volume)
- the dosage ratio to PEI is 1: 4.
- the dosage ratio of Competitor anti-CD3 heavy and light chain plasmid and SIRPa-Fc Fusion plasmid is 1:1:1.
- the supernatant was collected by centrifugation.
- Mos-D1TW molecules were purified using cation chromatography. Equilibrate the SPHP column with 20mM sodium citrate buffer (pH 5.4), and then flow the enriched antibody solution through the SPHP column at 1 ml/min. After the injection is completed, continue to use 20mM sodium citrate buffer (pH 5.4) to equilibrate the column. Finally, linear elution from A: 20mM sodium citrate buffer (pH5.4) to 35% B: 20mM sodium citrate buffer, 1M NaCl (pH5.4), 20CV, samples were collected respectively, and peak 1 (approximately 110kda), peak 2 (impurity).
- the D1-TW molecule was purified using a hydrophobic column.
- the UniHR Phenyl-30L column was equilibrated with 50mM sodium phosphate, 1M ammonium sulfate (pH6.8) buffer, and then the enriched antibody solution was added with 2M ammonium sulfate (pH6.8) at a ratio of 1:1, mixed and flowed through the UniHR Phenyl-30L column at 1ml/min. After the injection, the column was equilibrated with 50mM sodium phosphate buffer, 1M ammonium sulfate (pH6.8).
- Elisabuffer was diluted to 2ug/ml, 100ul per well, and placed in a 37°C incubator for 1 hour; take out the test plate, wash it 3 times with PBST, and add anti-his-HRP (Genscript, A00612, 1:1K), 100ul per well.
- D1-TW has the affinity to bind to both CD3E/G and CD47.
- Coat hCD3E-his (nearshore, C578) 1ug/ml with 0.05M CB solution and keep overnight at 4°C; wash 3 times with PBST the next day, add 1xPBS+1% BSA Elisa buffer to block, 200ul per well, put at 37°C Incubate for 1 hour; prepare the sample to be tested, Mos-D1-TW, and dilute D1-TW with Elisa buffer.
- the concentration starts from 10ug/ml and is diluted 10 times, including a total of 5 concentration points at 0; put the sealed Elisa test plate Take it out from the incubator, pat it dry, add the diluted antibody, 100ul per well, and place it in a 37°C incubator for 1 hour; take out the detection plate, wash it 3 times with PBST, add anti-SIRPa-mFc (abcam, ab267409), and use Elisa buffer Dilute to 1ug/ml, 100ul per well, and place in a 37°C incubator for 1 hour; take out the detection plate, wash it 3 times with PBST, and add Goat anti-mouse Fc-HRP (Jacksonimmuno, 115-035-071, 1:5K).
- the expressed and purified mos-D-TW is a complete and effective double antibody.
- Dilute D1-TW with 3% BSA FACS buffer the starting concentration is 50ug/ml, 3X gradient dilution, including 0ug/ml, a total of 9 concentration points; digest tumor cells HCT-8 with trypsin, and collect 8226 cells at the same time, 200g Centrifuge for 5 minutes, resuspend with 3% BSA, count, adjust the cell density to 2E6 cells/ml, add 100ul per well to a 96-well U-shaped plate, centrifuge at 200g for 5 minutes, remove the supernatant; add the diluted antibody, 100ul per well.
- Dilute D1-TW with 3% BSA FACS buffer the starting concentration is 50ug/ml, 5X gradient dilution, including 0ug/ml, a total of 6 concentration points; collect 8226 cells, centrifuge at 200g for 5 minutes, resuspend with 3% BSA, and count , adjust the cell density to 2E6 cells/ml, add 100ul per well to a 96-well U-shaped plate, centrifuge at 200g for 5 minutes, and remove the supernatant; at the same time, take the whole blood of hCD3E transgenic mice, add red blood cell lysis solution to lyse red blood twice, each time 10 minutes, wash twice with 1xPBS, resuspend the cells, add 100ul to each well, centrifuge at 200g for 5 minutes, remove the supernatant; add diluted antibodies, 100ul per well, resuspend, and set up negative control wells without antibodies, 4 degrees React for 30 minutes, centrifuge at 200g for 5 minutes,
- Mos-D1-TW has a certain affinity with target cells 8226 and CD3E on the cell surface.
- the frozen PBMCs were revived, counted, and the cell density was adjusted to 2 ⁇ 10 ⁇ 6 cells/ml. 50ul/well was added to a 96-well U-shaped plate, i.e. 1 ⁇ 10 ⁇ 5 cells/well;
- the starting concentration is 40000ng/ml, 5-fold gradient dilution. Take 50 ⁇ l of each concentration of antibody solution into a 96-well plate; then the final concentration is 10000ng/ml, 5-fold gradient dilution, including 0ng/ml, 11 concentration points in total; single wells of PBMC wells and tumor cell wells are set at the same time;
- CCK8 After culturing for 24h, 48h, 72h, and 96h respectively in a carbon dioxide incubator (37°C, 5% CO2), the culture supernatant was aspirated for cytokine kit detection; the cultured cells were washed with PBS. Wash away the PBMC several times, then add 100 ⁇ l/well of CCK8 working solution, incubate at 37°C for 2 to 4 hours and then measure the OD450; for the 8226 culture system, add 1ul of PI staining reagent to each well, protect from light for 10 minutes, and put on the flow cytometer.
- D1-TW can mediate efficient killing of two tumor cell lines, 8226 and HCT-8, and is more effective than the symmetrically structured H3S02.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
Abstract
L'invention concerne une protéine de fusion constituée de SIRPαV2 et d'un anticorps CD3.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211163564 | 2022-09-23 | ||
CN202211163564.8 | 2022-09-23 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2024061350A1 true WO2024061350A1 (fr) | 2024-03-28 |
Family
ID=90453887
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2023/120732 WO2024061350A1 (fr) | 2022-09-23 | 2023-09-22 | Protéine de fusion et son utilisation |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2024061350A1 (fr) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104098698A (zh) * | 2013-04-08 | 2014-10-15 | 中国人民解放军第二军医大学 | 一种抗cd3抗体及其制法和应用 |
CN104704004A (zh) * | 2012-10-08 | 2015-06-10 | 罗切格利卡特公司 | 包含两个Fab片段的无Fc的抗体及使用方法 |
CN111423515A (zh) * | 2020-03-23 | 2020-07-17 | 倍而达药业(苏州)有限公司 | 一种cd20/cd47双特异性抗体及应用 |
WO2021190441A1 (fr) * | 2020-03-23 | 2021-09-30 | 倍而达药业(苏州)有限公司 | Anticorps dirigé contre cd47/cd47 humanisé ou fragment de liaison à l'antigène ou fragment immunologiquement actif de celui-ci et utilisation associée |
WO2022199555A1 (fr) * | 2021-03-23 | 2022-09-29 | Guangzhou Lintonpharm Co., Ltd. | Protéine de liaison à un antigène multispécifique se liant à cd3 et son utilisation |
WO2023072217A1 (fr) * | 2021-10-28 | 2023-05-04 | Guangzhou Lintonpharm Co., Ltd. | Protéines de fusion ciblant cd3 et cd47 |
-
2023
- 2023-09-22 WO PCT/CN2023/120732 patent/WO2024061350A1/fr unknown
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104704004A (zh) * | 2012-10-08 | 2015-06-10 | 罗切格利卡特公司 | 包含两个Fab片段的无Fc的抗体及使用方法 |
CN104098698A (zh) * | 2013-04-08 | 2014-10-15 | 中国人民解放军第二军医大学 | 一种抗cd3抗体及其制法和应用 |
CN111423515A (zh) * | 2020-03-23 | 2020-07-17 | 倍而达药业(苏州)有限公司 | 一种cd20/cd47双特异性抗体及应用 |
WO2021190441A1 (fr) * | 2020-03-23 | 2021-09-30 | 倍而达药业(苏州)有限公司 | Anticorps dirigé contre cd47/cd47 humanisé ou fragment de liaison à l'antigène ou fragment immunologiquement actif de celui-ci et utilisation associée |
WO2022199555A1 (fr) * | 2021-03-23 | 2022-09-29 | Guangzhou Lintonpharm Co., Ltd. | Protéine de liaison à un antigène multispécifique se liant à cd3 et son utilisation |
WO2023072217A1 (fr) * | 2021-10-28 | 2023-05-04 | Guangzhou Lintonpharm Co., Ltd. | Protéines de fusion ciblant cd3 et cd47 |
Non-Patent Citations (1)
Title |
---|
XU LIJUN; WANG SHANLONG; LI JIE; LI BINGYU: "CD47/SIRPα blocking enhances CD19/CD3-bispecific T cell engager antibody-mediated lysis of B cell malignancies", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ELSEVIER, AMSTERDAM NL, vol. 509, no. 3, 1 January 1900 (1900-01-01), Amsterdam NL , pages 739 - 745, XP085579685, ISSN: 0006-291X, DOI: 10.1016/j.bbrc.2018.12.175 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
ES2801873T3 (es) | Anticuerpo de PDL-1, composición farmacéutica del mismo y sus usos | |
WO2021063330A1 (fr) | Anticorps de ciblage de cd3, anticorps bispécifique et son utilisation | |
CN102574921B (zh) | 双特异性死亡受体激动型抗体 | |
WO2018095428A1 (fr) | Anticorps cd47, fragment de liaison à l'antigène de l'anticorps cd47 et leur utilisation médicale | |
JP2021508676A (ja) | 抗tigit抗体並びに治療剤及び診断剤としてのその使用 | |
CN108290958A (zh) | 多特异性抗体 | |
JP7533897B2 (ja) | 融合タンパク質およびその使用 | |
KR20200012920A (ko) | 항-cd40 항체, 이의 항원 결합 단편 및 이의 의학적 용도 | |
CN114269782B (zh) | 抗tigit抗体及其应用 | |
WO2022174813A1 (fr) | Anticorps trispécifique anti-gprc5d × bcma × cd3 et son utilisation | |
CA2958672A1 (fr) | Traitement de tumeurs a l'aide d'un anticorps anti-l1 specifique | |
JP2024512957A (ja) | Cd3に結合する多重特異性抗原結合タンパク質とその使用方法 | |
KR20210099027A (ko) | 항-cd40 항체, 이의 항원-결합 단편 및 약학적 용도 | |
CN113056486A (zh) | 改善的抗flt3抗原结合蛋白 | |
JP2024506626A (ja) | 抗cd112r抗体及びその用途 | |
KR20220167331A (ko) | 항-flt3 항체 및 조성물 | |
CA3159155A1 (fr) | Anticorps anti-clec-1 a et fragment de liaison a l'antigene de ceux-ci | |
WO2023227033A1 (fr) | Anticorps anti-bdca2 et son utilisation | |
JP7510209B2 (ja) | ヘルパーT細胞TGF-βシグナルを特異的に中和する二重特異性抗体、その薬物組成物およびその使用 | |
WO2024061350A1 (fr) | Protéine de fusion et son utilisation | |
KR20220143869A (ko) | 항-il-2 항체, 이의 항원-결합 단편 및 이의 의학적 용도 | |
WO2024061352A1 (fr) | Protéine de liaison à l'antigène isolée et son utilisation | |
WO2023051727A1 (fr) | Anticorps se liant à cd3, et utilisation associée | |
WO2024094151A1 (fr) | Anticorps multi-spécifique et son utilisation médicale | |
WO2022037582A1 (fr) | Anticorps bispécifique anti-cd3 et anti-cldn-18.2 et son utilisation |
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: 23867635 Country of ref document: EP Kind code of ref document: A1 |