CN111138522B - Tumor antigen short peptides derived from AFP - Google Patents
Tumor antigen short peptides derived from AFP Download PDFInfo
- Publication number
- CN111138522B CN111138522B CN201911071527.2A CN201911071527A CN111138522B CN 111138522 B CN111138522 B CN 111138522B CN 201911071527 A CN201911071527 A CN 201911071527A CN 111138522 B CN111138522 B CN 111138522B
- Authority
- CN
- China
- Prior art keywords
- peptide
- pmhc
- cells
- cell
- complex
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 143
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 29
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 63
- 108091007433 antigens Proteins 0.000 title abstract description 28
- 102000036639 antigens Human genes 0.000 title abstract description 28
- 239000000427 antigen Substances 0.000 title abstract description 27
- 201000011510 cancer Diseases 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 9
- 229960005486 vaccine Drugs 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 3
- 201000007270 liver cancer Diseases 0.000 claims description 3
- 208000014018 liver neoplasm Diseases 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 208000030062 persistent idiopathic facial pain Diseases 0.000 claims 2
- 108010013476 HLA-A24 Antigen Proteins 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 61
- 108091008874 T cell receptors Proteins 0.000 description 50
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 50
- 230000027455 binding Effects 0.000 description 34
- 238000000034 method Methods 0.000 description 31
- 210000001744 T-lymphocyte Anatomy 0.000 description 28
- 229920001184 polypeptide Polymers 0.000 description 24
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 19
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 19
- 150000007523 nucleic acids Chemical class 0.000 description 19
- 239000012634 fragment Substances 0.000 description 17
- 150000001413 amino acids Chemical class 0.000 description 15
- 239000000203 mixture Substances 0.000 description 14
- 239000013598 vector Substances 0.000 description 14
- 210000004881 tumor cell Anatomy 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 108020004707 nucleic acids Proteins 0.000 description 12
- 102000039446 nucleic acids Human genes 0.000 description 12
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 239000012071 phase Substances 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 8
- 210000003000 inclusion body Anatomy 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 238000004153 renaturation Methods 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 108010090804 Streptavidin Proteins 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 230000006287 biotinylation Effects 0.000 description 5
- 238000007413 biotinylation Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000008194 pharmaceutical composition Substances 0.000 description 5
- 229940124597 therapeutic agent Drugs 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- -1 D-amino acids) Chemical class 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000017854 proteolysis Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000011510 Elispot assay Methods 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 210000000612 antigen-presenting cell Anatomy 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 210000004443 dendritic cell Anatomy 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 239000000032 diagnostic agent Substances 0.000 description 3
- 229940039227 diagnostic agent Drugs 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000004481 post-translational protein modification Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 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 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 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 2
- 238000002965 ELISA Methods 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 2
- 108010075704 HLA-A Antigens Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 2
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 2
- 230000002998 immunogenetic effect Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 235000000638 D-biotin Nutrition 0.000 description 1
- 239000011665 D-biotin Substances 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- SYAYROHMAIHWFB-KBIXCLLPSA-N Glu-Ser-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SYAYROHMAIHWFB-KBIXCLLPSA-N 0.000 description 1
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 1
- 108010027412 Histocompatibility Antigens Class II Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- XLXPYSDGMXTTNQ-UHFFFAOYSA-N Ile-Phe-Leu Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 XLXPYSDGMXTTNQ-UHFFFAOYSA-N 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- ZFNYWKHYUMEZDZ-WDSOQIARSA-N Lys-Trp-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCCCN)N ZFNYWKHYUMEZDZ-WDSOQIARSA-N 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 241000713862 Moloney murine sarcoma virus Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 1
- BZQFBWGGLXLEPQ-UHFFFAOYSA-N O-phosphoryl-L-serine Natural products OC(=O)C(N)COP(O)(O)=O BZQFBWGGLXLEPQ-UHFFFAOYSA-N 0.000 description 1
- KZRQONDKKJCAOL-DKIMLUQUSA-N Phe-Leu-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KZRQONDKKJCAOL-DKIMLUQUSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 241000934136 Verruca Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000000260 Warts Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000007998 bicine buffer Substances 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000010370 cell cloning Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000004671 cell-free system Anatomy 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000006240 deamidation Effects 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000011223 gene expression profiling Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000006525 intracellular process Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000009149 molecular binding Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000002062 molecular scaffold Substances 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 238000002170 nanoflow liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- USRGIUJOYOXOQJ-GBXIJSLDSA-N phosphothreonine Chemical compound OP(=O)(O)O[C@H](C)[C@H](N)C(O)=O USRGIUJOYOXOQJ-GBXIJSLDSA-N 0.000 description 1
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical compound OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 201000010153 skin papilloma Diseases 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 210000001325 yolk sac Anatomy 0.000 description 1
Images
Classifications
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
- Microbiology (AREA)
- Reproductive Health (AREA)
- General Engineering & Computer Science (AREA)
- Public Health (AREA)
- Gynecology & Obstetrics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Hematology (AREA)
- Veterinary Medicine (AREA)
- Pregnancy & Childbirth (AREA)
Abstract
The present invention relates to a short peptide of a tumor antigen derived from AFP, a complex formed between the short peptide and an MHC molecule and the use of the short peptide and the complex. The invention also relates to molecules which bind to the above-mentioned short peptides or complexes, and to the use of these molecules.
Description
Technical Field
The present invention relates to a short peptide of tumor antigen derived from AFP, in particular, it relates to a newly-discovered short peptide derived from tumor antigen AFP, the complex formed by said short peptide and MHC molecule and the application of said short peptide and complex. The invention also relates to molecules which bind to the above-mentioned short peptides or complexes, and to the use of these molecules.
Background
It is well known that in many pathological conditions, such as infections, cancers, autoimmune diseases, etc., there is inappropriate expression of some specific molecules. Thus, these molecules become "markers" for pathological or abnormal conditions. These molecules can be used not only as markers for disease diagnosis, but also for the production of diagnostic and/or therapeutic agents. For example, markers for cancer are used to generate specific antibodies. In addition, these molecules can also efficiently elicit specific immune responses from Cytotoxic T Lymphocytes (CTLs) to exert antitumor efficacy, and at the same time, can also be used to obtain T Cell Receptors (TCRs) capable of binding to the "markers" as therapeutic agents via activated CTLs. Therefore, these molecules play a very important role in the diagnosis and treatment of related diseases.
For tumors, many different endogenous tumor antigen molecules have been published in the literature. However, this is not a true target for the relevant disease, since it is not the complete tumor antigen molecule that elicits the CTL immune response, but rather a specific CTL Epitope (Epitope) from the antigen. In general, tumor antigens are processed intracellularly by proteolysis into polypeptide fragments of 8-16 amino acids in length, i.e., CTL epitopes, which are then bound to major histocompatibility complex (MHC, human MHC is often referred to as HLA gene or HLA complex) molecules in the lumen of the endoplasmic reticulum to form polypeptide-MHC complexes (pMHC), which are finally presented to the cell surface for CD8 + TCR recognition on the T cell surface. Although the relevant endogenous tumor antigen molecules have been published for a long time, we are not aware of the specific polypeptide fragments that are presented. Therefore, whether as a vaccine or a diagnostic or therapeutic agent for the production of the relevant disease, it is crucial to identify these 8-16 amino acid long polypeptide fragments presented on the cell surface, i.e. CTL epitopes. One skilled in the art is directed to the discovery and identification of these polypeptide fragments that are presented to the surface of a target cell.
The discovery and identification of such presented polypeptide fragments is a complex process, since the presentation of polypeptides by HLA is a combined result of proteolysis of the antigen protein and the interaction of the polypeptide fragment with HLA. This indicates that the complete tumor antigen molecule does not provide any information for the discovery and identification of polypeptide fragments. A number of documents have published methods using computer simulations, such as the public databases SYFPEITHI (Rammensee, et al, immunogenetics.1999 (50): 213-219) and BIMAS (Parker, et al, J.Immunol.1994.152: 163), which provide predictive algorithms to identify which polypeptide fragments are likely to be presented. However, this is only a prediction and has great uncertainty, since it is not a true intracellular process and post-translational modification process. After the tumor tissue is treated, the polypeptide fragments presented on the surface of the tumor cells can be directly identified by using a mass spectrometer, and although the process is a complex process, the obtained result is very reliable. At the same time, the sensitivity of the mass spectrometer is sufficient to be able to identify low concentrations of polypeptide fragments and post-translational modifications, and thus it is an ideal tool for the discovery and determination of polypeptide fragments on tumor surfaces.
AFP (alpha Fetoprotein), also called alpha Fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver, and is subsequently inhibited. In hepatocellular carcinoma, AFP expression is activated (Butterfield et al.J. Immunol.,2001, apr 15 (8): 5300-8). Thus, peptides derived from AFP, as targets for the above hepatocellular carcinoma, can be used not only as markers for the diagnosis of the above diseases, but also for the production of preventive and/or therapeutic agents for the above diseases, such as antibodies or T-cell receptors. The invention utilizes mass spectrometer analysis to identify and firstly discovers a polypeptide fragment derived from a tumor antigen AFP presented on the surface of a tumor cell.
Disclosure of Invention
The invention aims at providing a newly discovered short peptide derived from a tumor antigen AFP, a complex formed by the short peptide and an MHC molecule and application of the short peptide and the complex.
In a first aspect of the invention, there is provided a peptide comprising the amino acid sequence: KWVESIFLI (SEQ ID NO: 1);
in another preferred embodiment, the peptide is capable of forming a complex with an MHC molecule.
In another preferred embodiment, the peptide consists of 9 or 10 amino acids.
In another preferred embodiment, the peptide consists of 9 amino acids.
In another preferred embodiment, the amino acid sequence of the peptide is SEQ ID NO 1.
In a second aspect of the invention, there is provided a pMHC complex comprising a peptide according to the first aspect of the invention.
In another preferred embodiment, the amino acid sequence of the peptide in the pMHC complex is SEQ ID NO 1.
In another preferred embodiment, the MHC molecule is of the type HLA-base:Sub>A 24.
In another preferred embodiment, the MHC molecule is of the type HLA-base:Sub>A 2402.
In another preferred embodiment, the pMHC complex is a multimer.
In another preferred embodiment, the pMHC complex is soluble.
In another preferred embodiment, the pMHC complex is biotinylated.
In a third aspect of the invention, there is provided an isolated cell which presents on its surface a pMHC complex according to the second aspect of the invention.
In a fourth aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide according to the first aspect of the invention or a complement thereof.
In a fifth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the fourth aspect of the invention.
In a sixth aspect of the invention, there is provided a host cell comprising a vector according to the fifth aspect of the invention.
In a seventh aspect of the present invention, there is provided a molecule capable of binding a peptide according to the first aspect of the present invention and/or a pMHC complex according to the second aspect of the present invention.
In another preferred embodiment, the molecule is capable of specifically binding to a peptide according to the first aspect of the invention and/or a pMHC complex according to the second aspect of the invention.
In another preferred embodiment, the molecule is a T cell receptor.
In another preferred embodiment, the T cell receptor is soluble.
In another preferred embodiment, the molecule is an antibody or binding fragment thereof.
In another preferred embodiment, the antibody is a monoclonal antibody.
In an eighth aspect of the present invention there is provided an isolated monoclonal T cell isolated using a peptide according to the first aspect of the invention and/or a pMHC complex according to the second aspect of the invention.
In another preferred embodiment, the monoclonal T cells specifically bind to the pMHC complex of the second aspect of the invention.
In a ninth aspect, the present invention provides the use of a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention or a cell according to the third aspect of the invention, for activating and/or isolating T-cells.
In a tenth aspect of the invention there is provided the use of a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, for screening a library of T cell receptors or antibodies.
In an eleventh aspect, the present invention provides the use of a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, a cell according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a molecule according to the seventh aspect of the invention or a T cell according to the eighth aspect of the invention, in the manufacture of a medicament for the prophylaxis or treatment of cancer.
In a twelfth aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, a cell according to the third aspect of the invention, a molecule according to the seventh aspect of the invention or a T cell according to the eighth aspect of the invention.
In another preferred embodiment, the pharmaceutical composition is a vaccine.
In a thirteenth aspect of the invention, there is provided a method of preventing or treating a disease, comprising administering to a subject in need thereof an amount of a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, a cell according to the third aspect of the invention, a molecule according to the seventh aspect of the invention or a T cell according to the eighth aspect of the invention.
In a fourteenth aspect of the present invention, there is provided a method of obtaining a molecule which binds to a pMHC complex according to the second aspect of the present invention, comprising:
contacting a candidate molecule with a pMHC complex according to the second aspect of the invention;
(ii) selecting the molecules which bind to the pMHC complex of (i).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a typical mass spectrum for identifying short peptides of the present invention.
FIG. 2 is a Native gel map of a soluble pMHC complex of the invention. The left band is BSA control and the right band is pMHC complex.
FIG. 3 shows the double positive staining results of monoclonal cells for CD8+ and tetramer-PE.
FIG. 4 is a diagram showing the results of an Elispot functional experiment of monoclonal cells obtained using the short peptide of the present invention on tumor cell lines and T2 cells.
FIG. 5 is a kinetic profile of soluble TCR molecule binding to a pMHC complex of the invention.
Detailed Description
The present invention, through extensive and intensive studies, has resulted in the obtainment of peptides derived from the antigen AFP, which are presented by MHC molecules to the surface of tumor cells as tumor markers. Thus, the present invention provides peptides derived from the antigen AFP, complexes formed between the peptides and MHC molecules and uses of the peptides and complexes. Also, the present invention relates to a molecule bound to the above peptide or complex. It is understood that in the present invention, the peptide of the invention, used interchangeably with the polypeptide of the invention or the short peptide of the invention, refers to the peptide provided by the invention derived from the antigen AFP.
Specifically, the first aspect of the present invention provides a peptide having an amino acid sequence of: KWVESIFLI (SEQ ID NO: 1).
It is known to those skilled in the art that the peptides of the invention may be post-translationally modified at one or more positions between the amino acid sequences. Examples of post-translational modifications can be found in Engelhard et al Curr Opin Immunol.2006, month 2; 18 (1): 92-7 and include phosphorylation, acetylation, and deamidation.
Preferably, the peptides of the invention bind to MHC a peptide binding site of an MHC molecule. Generally, the modified amino acids described above do not disrupt the ability of the peptide to bind to MHC. In a preferred embodiment, the amino acid modification increases the ability of the peptide to bind to MHC. For example, mutations may occur at the binding site of a peptide to MHC. These binding sites and preferred residues on the binding sites are known in the art, especially for those peptides that bind HLA-A.sub.24 (see, e.g., parkhurst et al, J.Immunol.157:2539-2548 (1996)).
The peptides of the invention may consist of or consist essentially of KWVESIFLI (SEQ ID NO: 1), which corresponds to the position of the full length 2-10 residues of the AFP protein.
The invention also provides SEQ ID NO:1, or an analog of a protein or peptide as set forth in claim 1. These analogs may differ from the native peptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These peptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the peptides of the present invention are not limited to the representative peptides exemplified above.
Modified (generally without altering primary structure) forms include: chemically derivatized forms of the peptide such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those peptides that result from glycosylation modifications during synthesis and processing of the peptide or during further processing steps. Such modification may be accomplished by exposing the peptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are peptides that have been modified to improve their resistance to proteolysis or to optimize solubility.
The peptides of the invention can be simply synthesized by the Merrifield synthesis method (also known as polypeptide solid phase synthesis method). GMP-grade peptides can be synthesized using solid phase synthesis techniques of the polypeptide system (Multiple Peptide Systems, san Diego, calif.). Alternatively, the peptides may be recombined and, if desired, synthesized using methods known in the art. Typical such methods involve the use of a vector comprising a nucleic acid sequence encoding a polypeptide, which is expressed in vivo; for example, in bacterial, yeast, insect or mammalian cells. Alternatively, expression can also be performed using an in vitro cell-free system. Such systems are known in the art and are commercially available. The peptides may be isolated and/or provided in substantially pure form. For example, they may be provided in a form that is substantially free of other peptides or proteins.
Tumor antigens are processed intracellularly by proteolysis into polypeptide fragments of 8-16 amino acids in length, i.e., CTL epitopes, which are then bound to MHC molecules in the lumen of the endoplasmic reticulum to form polypeptide-MHC complexes (pMHC) which are presented together on the cell surface. Accordingly, a second aspect of the present invention provides a pMHC complex comprising a peptide according to the first aspect of the invention. Preferably, the polypeptide binds to the peptide binding groove of an MHC molecule. The MHC molecule may be an MHC class I molecule or an MHC class ii molecule, preferably the MHC molecule is an MHC class I molecule. inbase:Sub>A preferred embodiment, the MHC molecule is HLA-base:Sub>A 24, more preferably the MHC molecule is HLA-base:Sub>A 2402.
pMHC complexes described herein may exist in multimeric form, for example, dimeric, or tetrameric, or pentameric, or hexameric, or octameric, or larger. Suitable methods for generating pMHC multimers can be found in the relevant literature, e.g. (Green et al, clin. Diagnostic Lab. Immunol.2002: 216-220).
Typically, pMHC multimers can be generated by complexing a pMHC complex with biotin residues to streptavidin via fluorescent labeling. Alternatively, the pMHC multimer may also be formed by an immunoglobulin as a molecular scaffold. In this system, the extracellular domain of the MHC molecule is joined to the constant region of the immunoglobulin heavy chain by a short linker (linker). Alternatively, the formation of pMHC multimers may utilize carrier molecules such as dextran (WO 02072631). pMHC multimers help to improve detection of binding moieties, such as T cell receptors, thereto. Alternatively, the effect of the pMHC complex in related applications, such as the activation of T cells, is enhanced.
The pMHC complexes described herein may be provided in soluble form. To obtain the soluble form of the pMHC complex, preferably the MHC molecules in the pMHC complex are devoid of a transmembrane region. In particular, in pMHC complexes, MHC class i molecules may consist of the extracellular domain of their light chain and all or part of their heavy chain. Alternatively, an MHC molecule is a fragment comprising only its functional domain.
Methods of producing soluble pMHC complexes of the invention are known to those skilled in the art and include, but are not limited to, the methods described in example 2 of the invention. The MHC molecules in the soluble pMHC complexes of the invention may also be produced synthetically and then refolded with the peptides of the invention. By determining whether a peptide is capable of refolding with an MHC molecule, it can be determined with which class of MHC molecule the peptide of the invention is capable of forming a complex.
The soluble pMHC complexes of the invention can be used to screen for or detect molecules, such as TCRs or antibodies, that bind to them. The method comprises contacting the pMHC complex with a binding moiety to be tested and determining whether the binding moiety to be tested binds to the complex. Assays for binding of pMHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensing technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, the binding may be detected by functional assays for biological responses resulting from the binding, such as cytokine release or apoptosis.
Likewise, the soluble pMHC complexes of the invention may also be used to screen TCR or antibody libraries. The construction of Antibody libraries using phage display technology is well known in the art, as described in the references Aitken, antibody phase display: methods and Protocols (2009, humana, new York). In a preferred embodiment, pMHC complexes of the invention are used to screen diverse TCR libraries displayed on the surface of phage particles. The TCRs displayed by the library may contain unnatural mutations.
Thus, the soluble pMHC complexes of the invention may be immobilised to a suitable solid support by means of a linker. Examples of solid supports include, but are not limited to, beads, membranes, sepharoses, magnetic beads, substrates, tubes, columns. The pMHC complex can be immobilized on ELISA reaction plate, magnetic beads, or surface plasmon resonance biosensor chip. Methods of immobilising pMHC complexes to solid phase carriers are known to those skilled in the art and include, for example, the use of affinity binding pairs such as biotin and streptavidin, or antibodies and antigens. In a preferred embodiment, the pMHC complex is labeled with biotin and is immobilized on a streptavidin-coated surface.
The peptides of the invention may be presented to the cell surface together with MHC complexes. Accordingly, the present invention also provides a cell capable of presenting a pMHC complex of the invention to its surface. Such cells may be mammalian cells, preferably cells of the immune system, and preferably specialized antigen presenting cells, such as dendritic cells or B cells. Other preferred cells include T2 cells (Hosken, et al., science.1990.248: 367-70). The cells presenting the peptides or pMHC complexes of the invention may be isolated, preferably in the form of a population of cells, or provided in substantially pure form. The cell may not naturally present the complex of the invention, or the cell may present the complex at a higher level than in its natural state. Such cells can be obtained by pulsing with the peptides of the invention. The pulse treatment involves incubating the cells with the peptide for several hours, preferably at a concentration of 10 -5 -10 -12 And M. In addition, the cells may be transduced with HLA-A x 24 molecules to further induce peptide presentation. Presenting bookThe cells of the pMHC complexes of the invention can be used to isolate T cells and T cell receptors, which are activated by the cells and further sorted out, and can also obtain the T cell receptors expressed on the surface of the T cells.
In a preferred embodiment, the method of obtaining the above-described T cells comprises stimulating fresh blood obtained from healthy volunteers with the above-described cells presenting the pMHC complex of the invention. Several rounds of stimulation may be performed, such as 3-4 rounds. Identification of activated T cells cytokine release can be measured by the presence of peptide-pulsed T2 cells of the invention (e.g., IFN- γ ELISpot assay). Using labeled antibodies, activated cells can be sorted by flow cytometry (FACS), and the sorted cells can be expanded and further validated, for example, by ELISpot detection and/or cytotoxicity against target cells and/or pMHC multimer staining. TCR chains from validated T cell clones can be amplified by cDNA end Rapid Amplification (RACE) and sequenced.
The invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide of the invention. The nucleic acid may be cDNA. The nucleic acid molecule may consist essentially of a nucleic acid sequence encoding a peptide according to the invention, or may encode only a peptide according to the invention. Such nucleic acid molecules can be synthesized using methods known in the art. Due to the degeneracy of the genetic code, it will be understood by those skilled in the art that nucleic acid molecules of different nucleic acid sequences may encode the same amino acid sequence.
The invention also provides a vector, wherein the vector comprises the nucleic acid sequence. Suitable vectors are known in the art of vector construction and include promoter selection and other regulatory elements, such as enhancer elements. The vectors of the invention include sequences suitable for introduction into a cell. For example, the vector may be an expression vector in which the coding sequence for the polypeptide is under the control of its own cis-acting regulatory elements, designed to facilitate gene integration or gene replacement in a host cell, and the like.
It will be appreciated by those of ordinary skill in the art that in the present invention, the term "vector" includes DNA molecules, such as plasmids, phages, viruses or other vectors, which contain one or more heterologous or recombinant nucleic acid sequences. Suitable phage and viral vectors include, but are not limited to: lambda-phage, EMBL phage, simian virus, verruca bovis, epstein-Barr virus, adenovirus, herpes virus, murine sarcoma virus, murine mammary carcinoma virus, lentivirus, etc.
The invention also provides a binding molecule which may be used as an immunotherapeutic or diagnostic agent. The binding molecule may be bound to the peptide alone or to a complex formed between the peptide and an MHC molecule. In the latter case, the binding molecule may be partially bound to an MHC molecule, while it is also bound to a peptide of the invention. Binding moieties of the invention may be isolated and/or soluble, and/or non-naturally occurring, i.e., without an equivalent in nature, and/or pure, and/or synthetically produced.
In a preferred embodiment of the invention, the binding molecule is a T Cell Receptor (TCR). TCRs can be described using the international immunogenetic information system (IMGT). Native α β heterodimeric TCRs have an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linker region and a constant region, and the beta chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered part of the linker region.
The TCRs of the invention may be in any form known in the art. For example, the TCR may be heterodimeric, or present as a single chain. The TCR may be in soluble form (i.e. without a transmembrane or cytoplasmic region), in particular the TCR may comprise all or part of the TCR extracellular domain. The TCR may also be a full length chain comprising its transmembrane region. The TCR may be provided to the cell surface, such as a T cell.
Soluble TCRs can be obtained in combination with techniques known in the art, for example, by introducing an artificial disulfide bond between the constant domains of the α and β chains of the α β TCR, or between the α chain variable region and the β chain constant region of the α β TCR.
The TCRs of the invention are useful for delivering cytotoxic or immunostimulatory agents to target cells, or are transformed into T cells, enabling T cells expressing the TCR to destroy tumor cells for administration to patients in a course of treatment known as adoptive immunotherapy. In addition, the TCR of the invention may also comprise a mutation, preferably the mutated TCR has an improved affinity for the pMHC complex of the invention. The TCRs of the invention may be used alone or may be covalently or otherwise associated, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the pMHC complex of the invention), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination or conjugate of any of the above. The inventive TCR may also be bound, preferably covalently, to an anti-CD 3 antibody to redirect T cells, thereby killing the target cell.
In another preferred embodiment, the binding molecule of the invention is an antibody. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain specific binding sites, which can be wholly natural, or partially or wholly artificial. The term "antibody" includes antibody fragments, derivatives, functional equivalents, as well as homologous antibodies, humanized antibodies, and fragments comprising an immunoglobulin binding region that is or is homologous to an antibody binding region. It may be all natural, partially synthetic, or fully synthetic. A humanized antibody may be a modified antibody comprising the variable regions of a non-human antibody (e.g., mouse) and the constant regions of a human antibody.
Examples of antibodies may be isotypic immunoglobulins (e.g., igG, igE, igM, igD, and IgA) and their isotypic subclasses; fragments include antigen binding regions, such as Fab, scFv, fv, dAb, fd; and diabodies. The antibody may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
Methods for making such TCRs and antibodies are known to those skilled in the art and include, but are not limited to, expression from e.coli cells or insect cells and purification.
In a further aspect, the invention further provides the use of the peptides, pMHC complexes, nucleic acid molecules, vectors, cells and binding molecules of the invention in the manufacture of a medicament. The peptide, pMHC complex, nucleic acid, vector, cell or binding molecule may be used to treat or prevent cancer, preferably melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, squamous cell carcinoma and the like.
The invention also provides a pharmaceutical composition comprising a peptide of the invention, a pMHC complex, a nucleic acid molecule of the invention, a cell of the invention or a binding molecule of the invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the method of administration required by the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits typically (but not necessarily) contain instructions for use. It may comprise a plurality of said unit dosage forms.
The pharmaceutical composition is suitable for any suitable route of administration, such as injection (including subcutaneous, intramuscular, intraperitoneal or intravenous), inhalation or oral, or nasal, or anal. The compositions may be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with the carrier or excipient under sterile conditions.
The dosage of the formulation of the present invention to be administered may vary within wide limits depending on the disease or disorder to be treated (e.g., cancer, viral infection or autoimmune disease), the individual age and condition of the patient, and the like. The appropriate dosage to be administered will be ultimately determined by the physician.
According to the state of the art, peptides presented to the cell surface together with MHC molecules, pMHC complexes or cells presenting pMHC complexes can activate T cells or B cells to function.
Thus, the peptides, pMHC complexes or cells presenting pMHC complexes of the invention may be provided in the form of vaccine compositions. The vaccine composition may be used for the treatment or prevention of cancer. All such compositions are included in the present invention. It will be appreciated that the vaccine may be in a variety of forms (Schlom J.J Natl Cancer Inst.2012 (8): 599-613). For example, the peptides of the invention can be used directly in immunized patients (Salgaleler ML. Cancer Res.1996.56 (20): 4749-57and Marchand M. Int J cancer.1999.80 (2): 219-230). The vaccine composition may comprise additional peptides such that the peptide of the invention is one of a mixture of peptides. The vaccine composition may be adjuvanted to enhance the immune response. Alternatively, the vaccine composition may be in the form of an antigen presenting cell presenting a complex of a peptide of the invention and an MHC. Preferably, the antigen presenting cell is an immune cell, more preferably a dendritic cell. The peptides may also be pulsed onto the surface of cells (Thurner bi.et al, j.exp.med.1999.190: 1669), or nucleic acids encoding the peptides of the invention may be introduced into dendritic cells, for example, using electroporation (Van endeloo, vf.et al, blood 2001.98.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.
The materials used in the examples of the present invention are all commercially available products unless otherwise specified.
Example 1 identification of polypeptides derived from AFP antigen by Mass Spectrometry
Before mass spectrometric identification, the invention utilizes a digital single-molecule multiplex gene expression profiling system to carry out detection (nanostring), and further verifies the mass expression of the AFP antigen in liver cancer cells.
Commercial antibody A11.1M was used to purify HLA-short peptide complexes. Specifically, tumor cells were lysed with a buffer containing the nonionic surfactant Triton X-100 (1% v/v), 1ml of the lysate was added to 2X 10^7 cells, and incubated at 4 ℃ for 1 hour with rolling. Cell debris was removed by centrifugation, and the supernatant was incubated with the antibody, followed by addition of rProtein A-Sepharose to capture the "antibody-HLA-short peptide complex". And (4) passing through a column to collect the rProtein A-Sepharose-antibody-HLA-short peptide complex. Washing the column with low-salt and high-salt buffer solutions, eluting HLA-short peptide complex suspended on the immunoaffinity column with 10% acetic acid, heating at 95 deg.C, and filtering with 10KDa (amicon R Centrifugal Filters, MILLIPORE) ultrafiltration membrane to remove macromolecules to obtain polypeptide mixture.
The polypeptide mixture was fractionated by Agilent 1260 hplc: ZORBAX 300SB-C18;1.0 x 150mm,3.5um; mobile phase a was 98% water, 2% acetonitrile, 0.1% trifluoroacetic acid, mobile phase B was 98% acetonitrile, 2% water, 0.1% trifluoroacetic acid, and mobile phase gradient was mobile phase B increased from 5% to 70% within 10 minutes. One fraction was collected every minute. The total run time was 30 minutes.
Concentrating the HPLC fraction of the polypeptide, and analyzing by a sample introduction nanoLC-MSMS system:
eksigent nanolC-AB Sciex Triple TOF 5600 System: the mass spectrum was analyzed by IDA analysis. The liquid chromatography adopts the following steps: pre-column: (eksingent) NanoLC Trap column.5 μm C18.100 μm 2.5cm,910-00050, analytical column: (Eksigent) C18-CL-120,3 μm,
0.075×150mm，805-00120。
dionex Ultimate3000-Thermo QE Plus System: mass spectrometry adopts ddms2 analytical method liquid chromatography adopts: pre-column: (Thermo) Acclaim
100,100um X2cm, nanoViper, C18,5um,100A,164564, analytical column: (Thermo) Acclaim
100，75um×15cm,nanoViper，C18,3um,100A，164568。
The mobile phase a of the nanofluidic chromatography of the two systems described above was 98% water, 2% acetonitrile, 0.1% formic acid, the mobile phase B was 98% acetonitrile, 2% water, 0.1% formic acid, and the mobile phase gradient was that the mobile phase B rose from 5% to 50% in 74 minutes. The total run time was 90 minutes.
And (3) searching a Uniprot database of human proteins by virtue of library searching software Proteinpilot and Peaks according to the mass spectrum analysis result. According to the results of the software, the antigen short peptide sequence of the invention is finally obtained by comprehensive analysis, as shown in figure 1.
Example 2 preparation of soluble pMHC complexes
HLA class i-base:Sub>A 2402 molecules heavy and light chains (β 2 m) were expressed in e.coli (e.coli) as inclusion bodies, respectively. It should be noted that the heavy chain of the HLA-base:Sub>A 2402 molecule used in this example does not comprise its transmembrane and cytoplasmic domains in order to obtain soluble pMHC complexes. In addition, for the convenience of subsequent biotinylation of soluble pMHC complexes, a biotinylation tag may be added at the C-terminus of the heavy chain. The specific procedure for preparing the soluble pMHC complexes of the invention is as follows:
a. purification of
Collecting 100ml E.coli liquid for inducing expression of heavy chain or light chain, centrifuging at 4 ℃ for 10min at 8000g, washing the thalli once with 10ml PBS, then resuspending the thalli with 5ml BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, rotatably incubating at room temperature for 20min, centrifuging at 4 ℃ for 15min at 6000g, discarding supernatant, and collecting inclusion body.
Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5min; adding 30ml of BugBuster diluted by 10 times, mixing uniformly, and centrifuging at 6000g at 4 ℃ for 15min; discarding supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing, centrifuging at 4 ℃ for 15min, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mixing, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.
b. Renaturation
The peptide KWVESIFLI (synthesized by Beijing Saibobai Gene technology Co., ltd.) of the present invention was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized using 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. KWVESIFLI peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in this order (final concentration, heavy chain was added in three portions, 8 h/time), and renaturation was carried out at 4 ℃ for at least 3 days to completion.
c. Purification after renaturation
The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5 ml bed volume). The protein was eluted using a linear gradient of 0-400mM NaCl in 20mM Tris pH 8.0 using an Akta purifier (GE general electric), pMHC was eluted at approximately 250mM NaCl, and the peak fractions were collected. The Native gel pattern of the soluble pMHC complexes of the present invention obtained is shown in FIG. 2, and the bands are very uniform.
d. Biotinylation of the compound
The purified pMHC molecules were concentrated using Mill ipore ultrafilter tubes while the buffer was replaced with 20mM Tris pH 8.0, and then biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu.M D-Biotin, 100. Mu.g/ml BirA enzyme (GST-BirA) was added, the mixture was incubated overnight at room temperature, and SDS-PAGE was performed to determine whether biotinylation was complete.
e. Purification of biotinylated complexes
The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, the HiPrepTM 16/60S200 HR column (GE general electric) was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric), loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min.
Example 3T cell clone obtained Using the short peptide of the present invention
This example provides an illustration of the use of the pMHC complexes of the invention to obtain monoclonal T cells.
A variety of methods for obtaining TCRs are well known to those skilled in the art, including, but not limited to, isolating the sequences of TCR α and β chains from T cell clones stimulated by cells presenting the pMHC complex of the invention. The TCR sequences obtained can be cloned into a suitable vector and then expressed in e.coli, e.g. e.
The short peptide of the invention is used for stimulating peripheral blood lymphocytes of healthy volunteers, sorting and then carrying out monoclonal culture by a limiting dilution method to obtain T cell clones, and the double positive staining results of CD8+ and tetramer-PE are shown in figure 3.
Example 4 function of T cell cloning
The function and specificity of the T cell clone were further tested by ELISPOT assay. Meanwhile, the specific reaction of the T cell clone obtained by using the short peptide of the invention on a tumor cell line can also show that the short peptide of the invention is really presented on the surface of the tumor cell by MHC.
Methods for detecting cell function using the ELISPOT assay are well known to those skilled in the art. The effector cells used in the IFN-gamma ELISPOT experiment in the present example are T cell clones obtained in the present invention, the target cells are T2 (A2402) cells loaded with the short peptide of the present invention (transferring HLA A2402 to T2), T2 (A2402) cells loaded with other short peptides, positive tumor cell lines SNU398 (transfected with AFP antigen) and HEPG2, and negative tumor cell lines K562 (A24) (transfected with HLA A2402) and NCI-H266.
Firstly, preparing an ELISPOT plate, wherein the ELISPOT experiment steps are as follows: the components of the assay were added to the ELISPOT plates in the following order: the amount of short peptide added to the T2 experimental group was 20. Mu.l, and 20. Mu.l of the culture medium (test medium) was added to the tumor cell line group, and 2 wells were set, with 20,000 target cells/well and 2000 effector cells/well. Then incubated overnight (37 ℃,5% 2 ). The plates were then washed and subjected to secondary detection and color development, the plates were dried for 1 hour, and spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID Co.).
The results of the experiment are shown in FIG. 4, and the obtained T cell clone specific to the specific antigen has a specific response to T2 cells loaded with the short peptide of the present invention, but has no substantial response to T2 cells loaded with other irrelevant peptides. In addition, T cell clones obtained by using the short peptide of the invention have specific reaction on positive tumor cell lines and have no reaction on negative tumor cell lines.
Example 5 TCR binding the pMHC Complex of the invention
Using Quick-RNA TM MiniPrep (ZYMO research) extracts the total RNA of the above T cell clone and obtains the TCR sequence. To further verify that the obtained TCRs were able to bind to the pMHC complex of the invention, soluble TCR proteins were expressed in e. It should be noted that soluble TCRs can be obtained according to the prior art, including, but not limited to, that described in patent document PCT/CN 2015/093806.
The binding activity of soluble TCR proteins to pMHC complexes was tested using a BIAcore T200 real-time assay system. Anti-streptavidin antibody (GenScript) was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine in hydrochloric acid to complete the coupling process at a coupling level of about 15,000RU. A low concentration of streptavidin was flowed over the surface of the antibody-coated chip, and then pMHC prepared as described in example 2 was flowed over the detection channel, the other channel served as a reference channel, and 0.05mM biotin was flowed over the chip at a flow rate of 10. Mu.L/min for 2min to block the remaining binding sites of streptavidin.
Kinetic parameters were calculated using BIAcore Evaluation software, and the kinetic profile of the binding of soluble TCR molecules of the invention to pMHC complexes of the invention is shown in FIG. 5, which shows the binding of the two. Meanwhile, the method is used for detecting the binding activity of the soluble TCR molecule and the short peptides of other unrelated antigens and the HLA complex, and the result shows that the TCR molecule is not bound with other unrelated antigens.
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.
Sequence listing
<110> Guangdong Xiangxue accurate medical technology Limited
<120> AFP-derived tumor antigen short peptide
<130> P2019-1918
<150> CN201811314554.3
<151> 2018-11-06
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 1
Lys Trp Val Glu Ser Ile Phe Leu Ile
1 5
Claims (10)
1. Use of a peptide or a pMHC complex for the preparation of a medicament for the prevention or treatment of AFP-associated cancer;
the amino acid sequence of the peptide is SEQ ID NO 1;
said pMHC complex comprises said peptide;
the AFP-related cancer is liver cancer.
2. Use according to claim 1, wherein the MHC molecule is of the type HLA-base:Sub>A 24.
3. Use according to claim 2, wherein the MHC molecule is of the type HLA-base:Sub>A 2402.
4. The use of any one of claims 1 to 3, wherein the pMHC complex is a multimer.
5. The use according to any one of claims 1 to 3, wherein the pMHC complexes are soluble.
6. The use according to any one of claims 1 to 3, which comprises a peptide or pMHC complex, and a pharmaceutically acceptable carrier;
the medicament is a vaccine;
the amino acid sequence of the peptide is SEQ ID NO 1;
the pMHC complex comprises the peptide.
7. Use according to claim 6, wherein the MHC molecule class is HLA-A24.
8. Use according to claim 7, wherein the MHC molecule is of the type HLA-A2402.
9. The use of claim 6, wherein the pMHC complex is a multimer.
10. The use of claim 6, wherein the pMHC complexes are soluble.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2018113145543 | 2018-11-06 | ||
| CN201811314554 | 2018-11-06 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111138522A CN111138522A (en) | 2020-05-12 |
| CN111138522B true CN111138522B (en) | 2023-01-24 |
Family
ID=70517002
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911071527.2A Active CN111138522B (en) | 2018-11-06 | 2019-11-05 | Tumor antigen short peptides derived from AFP |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111138522B (en) |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2089423B1 (en) * | 2006-09-21 | 2016-10-26 | Vaxil Biotherapeutics Ltd. | Antigen specific multi epitope vaccines |
| CN104745629A (en) * | 2015-03-10 | 2015-07-01 | 中国人民解放军第四军医大学 | Preparation method of AFP/HBsAg double-targeting Dexosome anti-hepatoma anti-tumor vaccine |
| GB201520575D0 (en) * | 2015-11-23 | 2016-01-06 | Immunocore Ltd & Adaptimmune Ltd | Peptides |
| CN105524884A (en) * | 2016-02-29 | 2016-04-27 | 时宏珍 | Preparation method of HLA-A0201 restriction AFP antigen specific CTL |
| US20210147550A1 (en) * | 2017-08-18 | 2021-05-20 | Joshua Michael Francis | Antigen-binding proteins targeting shared antigens |
-
2019
- 2019-11-05 CN CN201911071527.2A patent/CN111138522B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN111138522A (en) | 2020-05-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111138521B (en) | Short peptides derived from AFP antigen | |
| WO2017089763A1 (en) | Peptides | |
| CN107936109B (en) | Tumor antigen short peptide derived from SAGE1 | |
| WO2017089771A1 (en) | Peptides from piwil1 | |
| WO2017089782A1 (en) | Peptides | |
| CN108218977B (en) | Short peptide derived from tumor antigen SAGE1 | |
| CN106478797B (en) | Tumour antigen small peptide from SAGE1 | |
| WO2022105922A1 (en) | Ssx2 antigen derived short peptides | |
| EP3380488A1 (en) | Peptides | |
| WO2017089766A1 (en) | Peptides from npsr1 | |
| CN108250289B (en) | Tumor antigen short peptides derived from MAGE B6 | |
| CN111138522B (en) | Tumor antigen short peptides derived from AFP | |
| CN112300261B (en) | Tumor antigen short peptide derived from AFP | |
| CN110862449B (en) | SSX-derived tumor antigen short peptides | |
| CN108250286B (en) | Tumor antigen short peptide derived from PASD1 | |
| CN107266552B (en) | Tumor antigen short peptide derived from PRAME | |
| CN108203460B (en) | Short peptides derived from the tumor antigen PRAME | |
| CN108218976B (en) | Tumor antigen short peptide derived from LMP1 | |
| WO2017089777A1 (en) | Peptides derived from lengsin (lgsn), complexes comprising such peptides bound to mhc molecules | |
| CN108997481B (en) | Antigen short peptide derived from LMP1 | |
| WO2017089758A1 (en) | Peptides of page5 | |
| WO2017089762A1 (en) | Peptides | |
| CN112898399A (en) | Short peptides derived from AFP antigens | |
| CN116948001A (en) | Antigen oligopeptide | |
| CN116948002A (en) | Peptide and complex thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210409 Address after: 519031 2715 office building, no.3000 Huandao East Road, Hengqin New District, Zhuhai City, Guangdong Province Applicant after: Xiangxue Life Science Technology (Guangdong) Co.,Ltd. Address before: 510663 No.2, jinfengyuan Road, Science City, Guangzhou hi tech Industrial Development Zone, Guangdong Province Applicant before: GUANGDONG XIANGXUE LIFE SCIENCES, Ltd. |
|
| TA01 | Transfer of patent application right | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |