CN114106136A - 一种肿瘤新生抗原表位肽Pep2及其多聚体和应用 - Google Patents
一种肿瘤新生抗原表位肽Pep2及其多聚体和应用 Download PDFInfo
- Publication number
- CN114106136A CN114106136A CN202111202851.0A CN202111202851A CN114106136A CN 114106136 A CN114106136 A CN 114106136A CN 202111202851 A CN202111202851 A CN 202111202851A CN 114106136 A CN114106136 A CN 114106136A
- Authority
- CN
- China
- Prior art keywords
- peptide
- tumor
- antigen
- pmhc
- epitope
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 177
- 239000000427 antigen Substances 0.000 title claims abstract description 97
- 108091007433 antigens Proteins 0.000 title claims abstract description 96
- 102000036639 antigens Human genes 0.000 title claims abstract description 96
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 72
- 101100189913 Caenorhabditis elegans pept-1 gene Proteins 0.000 title claims abstract description 21
- CQTGBCFGAAYOCY-ZCRNMIQFSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-3-phenylpropanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-amino-5-oxopentanoyl]amino]-4-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-amino-3-methyl-1-oxobutan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-4-oxobutanoic acid Chemical compound CC(C)C[C@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@@H](NC(C)=O)C(C)C)C(C)C)[C@@H](C)O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](C(C)C)C(N)=O CQTGBCFGAAYOCY-ZCRNMIQFSA-N 0.000 title claims abstract description 18
- 230000009707 neogenesis Effects 0.000 title claims abstract description 12
- 229920000642 polymer Polymers 0.000 title abstract description 18
- 238000007792 addition Methods 0.000 claims abstract description 6
- 238000012217 deletion Methods 0.000 claims abstract description 6
- 230000037430 deletion Effects 0.000 claims abstract description 6
- 238000006467 substitution reaction Methods 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 10
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 45
- 238000002360 preparation method Methods 0.000 claims description 29
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 25
- 239000000178 monomer Substances 0.000 claims description 25
- 238000002965 ELISA Methods 0.000 claims description 23
- 238000012216 screening Methods 0.000 claims description 21
- 108010090804 Streptavidin Proteins 0.000 claims description 19
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 claims description 14
- 230000035772 mutation Effects 0.000 claims description 14
- 239000011616 biotin Substances 0.000 claims description 13
- 229960002685 biotin Drugs 0.000 claims description 13
- 230000000890 antigenic effect Effects 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 12
- 230000005847 immunogenicity Effects 0.000 claims description 12
- 235000020958 biotin Nutrition 0.000 claims description 10
- 229920001184 polypeptide Polymers 0.000 claims description 10
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 7
- 230000004936 stimulating effect Effects 0.000 claims description 6
- 108090001008 Avidin Proteins 0.000 claims description 5
- 238000013329 compounding Methods 0.000 claims description 3
- 239000012642 immune effector Substances 0.000 claims description 3
- 229940121354 immunomodulator Drugs 0.000 claims description 3
- 230000000379 polymerizing effect Effects 0.000 claims description 2
- 239000013598 vector Substances 0.000 claims description 2
- 238000009169 immunotherapy Methods 0.000 abstract description 12
- 201000011510 cancer Diseases 0.000 abstract description 10
- 238000001514 detection method Methods 0.000 abstract description 10
- 239000003814 drug Substances 0.000 abstract description 10
- 229940079593 drug Drugs 0.000 abstract description 4
- 238000012827 research and development Methods 0.000 abstract description 4
- 229940126585 therapeutic drug Drugs 0.000 abstract description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 54
- 210000004027 cell Anatomy 0.000 description 23
- 230000000694 effects Effects 0.000 description 21
- 239000007788 liquid Substances 0.000 description 17
- 239000000523 sample Substances 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 14
- 239000003153 chemical reaction reagent Substances 0.000 description 13
- 238000011534 incubation Methods 0.000 description 10
- 230000002147 killing effect Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 102000004127 Cytokines Human genes 0.000 description 8
- 108090000695 Cytokines Proteins 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 238000011156 evaluation Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 101150105104 Kras gene Proteins 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 210000004881 tumor cell Anatomy 0.000 description 7
- ICUTTWWCDIIIEE-BQBZGAKWSA-N Gly-Met-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)CN ICUTTWWCDIIIEE-BQBZGAKWSA-N 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- 239000012131 assay buffer Substances 0.000 description 6
- 238000007789 sealing Methods 0.000 description 6
- 239000011534 wash buffer Substances 0.000 description 6
- -1 consumable Substances 0.000 description 5
- 230000003211 malignant effect Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 4
- 102100030708 GTPase KRas Human genes 0.000 description 4
- 206010064571 Gene mutation Diseases 0.000 description 4
- 101000584612 Homo sapiens GTPase KRas Proteins 0.000 description 4
- PHRWFSFCNJPWRO-PPCPHDFISA-N Ile-Leu-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N PHRWFSFCNJPWRO-PPCPHDFISA-N 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 210000002865 immune cell Anatomy 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- ICRHGPYYXMWHIE-LPEHRKFASA-N Arg-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ICRHGPYYXMWHIE-LPEHRKFASA-N 0.000 description 3
- 235000000638 D-biotin Nutrition 0.000 description 3
- 239000011665 D-biotin Substances 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- RYAOJUMWLWUGNW-QMMMGPOBSA-N Gly-Val-Gly Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O RYAOJUMWLWUGNW-QMMMGPOBSA-N 0.000 description 3
- HKXSZKJMDBHOTG-CIUDSAMLSA-N Lys-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CCCCN HKXSZKJMDBHOTG-CIUDSAMLSA-N 0.000 description 3
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 3
- UQFYNFTYDHUIMI-WHFBIAKZSA-N Ser-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CO UQFYNFTYDHUIMI-WHFBIAKZSA-N 0.000 description 3
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 3
- VIWQOOBRKCGSDK-RYQLBKOJSA-N Trp-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N)C(=O)O VIWQOOBRKCGSDK-RYQLBKOJSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 210000000612 antigen-presenting cell Anatomy 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 108010016616 cysteinylglycine Proteins 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 108010015792 glycyllysine Proteins 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 208000014018 liver neoplasm Diseases 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000012089 stop solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 2
- PZVMBNFTBWQWQL-DCAQKATOSA-N Arg-His-Cys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N PZVMBNFTBWQWQL-DCAQKATOSA-N 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- BCSYBBMFGLHCOA-ACZMJKKPSA-N Cys-Glu-Cys Chemical compound SC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(O)=O BCSYBBMFGLHCOA-ACZMJKKPSA-N 0.000 description 2
- KVGPYKUIHZJWGA-BQBZGAKWSA-N Cys-Met-Gly Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)NCC(O)=O KVGPYKUIHZJWGA-BQBZGAKWSA-N 0.000 description 2
- 238000012286 ELISA Assay Methods 0.000 description 2
- SOYWRINXUSUWEQ-DLOVCJGASA-N Glu-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O SOYWRINXUSUWEQ-DLOVCJGASA-N 0.000 description 2
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 2
- IXHQLZIWBCQBLQ-STQMWFEESA-N Gly-Pro-Phe Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IXHQLZIWBCQBLQ-STQMWFEESA-N 0.000 description 2
- WSEITRHJRVDTRX-QTKMDUPCSA-N His-Met-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC1=CN=CN1)N)O WSEITRHJRVDTRX-QTKMDUPCSA-N 0.000 description 2
- 206010069755 K-ras gene mutation Diseases 0.000 description 2
- LJBVRCDPWOJOEK-PPCPHDFISA-N Leu-Thr-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LJBVRCDPWOJOEK-PPCPHDFISA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- FXBKQTOGURNXSL-HJGDQZAQSA-N Met-Thr-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(O)=O FXBKQTOGURNXSL-HJGDQZAQSA-N 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- LNICFEXCAHIJOR-DCAQKATOSA-N Pro-Ser-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LNICFEXCAHIJOR-DCAQKATOSA-N 0.000 description 2
- DGHFNYXVIXNNMC-GUBZILKMSA-N Ser-Gln-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CO)N DGHFNYXVIXNNMC-GUBZILKMSA-N 0.000 description 2
- 108010057517 Strep-avidin conjugated horseradish peroxidase Proteins 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 101150080074 TP53 gene Proteins 0.000 description 2
- CAJFZCICSVBOJK-SHGPDSBTSA-N Thr-Ala-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAJFZCICSVBOJK-SHGPDSBTSA-N 0.000 description 2
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 2
- RTJPAGFXOWEBAI-SRVKXCTJSA-N Val-Val-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RTJPAGFXOWEBAI-SRVKXCTJSA-N 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 description 2
- 108010030694 avidin-horseradish peroxidase complex Proteins 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 2
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- KRHRBKYBJXMYBB-WHFBIAKZSA-N Ala-Cys-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O KRHRBKYBJXMYBB-WHFBIAKZSA-N 0.000 description 1
- HIIJOGIBQXHFKE-HHKYUTTNSA-N Ala-Thr-Ala-Pro Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O HIIJOGIBQXHFKE-HHKYUTTNSA-N 0.000 description 1
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 1
- IIABBYGHLYWVOS-FXQIFTODSA-N Arg-Asn-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O IIABBYGHLYWVOS-FXQIFTODSA-N 0.000 description 1
- UULLJGQFCDXVTQ-CYDGBPFRSA-N Arg-Pro-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UULLJGQFCDXVTQ-CYDGBPFRSA-N 0.000 description 1
- VLDRQOHCMKCXLY-SRVKXCTJSA-N Asn-Ser-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O VLDRQOHCMKCXLY-SRVKXCTJSA-N 0.000 description 1
- UWOPETAWXDZUJR-ACZMJKKPSA-N Asp-Cys-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(O)=O UWOPETAWXDZUJR-ACZMJKKPSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 101150037241 CTNNB1 gene Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 241000724252 Cucumber mosaic virus Species 0.000 description 1
- PJWIPBIMSKJTIE-DCAQKATOSA-N Cys-His-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CS)N PJWIPBIMSKJTIE-DCAQKATOSA-N 0.000 description 1
- 101100347633 Drosophila melanogaster Mhc gene Proteins 0.000 description 1
- 238000011510 Elispot assay Methods 0.000 description 1
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 1
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 1
- CUXJIASLBRJOFV-LAEOZQHASA-N Glu-Gly-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O CUXJIASLBRJOFV-LAEOZQHASA-N 0.000 description 1
- ZALGPUWUVHOGAE-GVXVVHGQSA-N Glu-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZALGPUWUVHOGAE-GVXVVHGQSA-N 0.000 description 1
- PUUYVMYCMIWHFE-BQBZGAKWSA-N Gly-Ala-Arg Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PUUYVMYCMIWHFE-BQBZGAKWSA-N 0.000 description 1
- RLFSBAPJTYKSLG-WHFBIAKZSA-N Gly-Ala-Asp Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O RLFSBAPJTYKSLG-WHFBIAKZSA-N 0.000 description 1
- GQGAFTPXAPKSCF-WHFBIAKZSA-N Gly-Ala-Cys Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(=O)O GQGAFTPXAPKSCF-WHFBIAKZSA-N 0.000 description 1
- XUDLUKYPXQDCRX-BQBZGAKWSA-N Gly-Arg-Asn Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O XUDLUKYPXQDCRX-BQBZGAKWSA-N 0.000 description 1
- UTYGDAHJBBDPBA-BYULHYEWSA-N Gly-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN UTYGDAHJBBDPBA-BYULHYEWSA-N 0.000 description 1
- DENRBIYENOKSEX-PEXQALLHSA-N Gly-Ile-His Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 DENRBIYENOKSEX-PEXQALLHSA-N 0.000 description 1
- 101150050733 Gnas gene Proteins 0.000 description 1
- FFYYUUWROYYKFY-IHRRRGAJSA-N His-Val-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O FFYYUUWROYYKFY-IHRRRGAJSA-N 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 101000891649 Homo sapiens Transcription elongation factor A protein-like 1 Proteins 0.000 description 1
- YSGBJIQXTIVBHZ-AJNGGQMLSA-N Ile-Lys-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O YSGBJIQXTIVBHZ-AJNGGQMLSA-N 0.000 description 1
- SAVXZJYTTQQQDD-QEWYBTABSA-N Ile-Phe-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N SAVXZJYTTQQQDD-QEWYBTABSA-N 0.000 description 1
- KBDIBHQICWDGDL-PPCPHDFISA-N Ile-Thr-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N KBDIBHQICWDGDL-PPCPHDFISA-N 0.000 description 1
- 230000004163 JAK-STAT signaling pathway Effects 0.000 description 1
- 229940126685 KRAS G12R Drugs 0.000 description 1
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 1
- BABSVXFGKFLIGW-UWVGGRQHSA-N Leu-Gly-Arg Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N BABSVXFGKFLIGW-UWVGGRQHSA-N 0.000 description 1
- DSFYPIUSAMSERP-IHRRRGAJSA-N Leu-Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DSFYPIUSAMSERP-IHRRRGAJSA-N 0.000 description 1
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 1
- ILDSIMPXNFWKLH-KATARQTJSA-N Leu-Thr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ILDSIMPXNFWKLH-KATARQTJSA-N 0.000 description 1
- LJADEBULDNKJNK-IHRRRGAJSA-N Lys-Leu-Val Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LJADEBULDNKJNK-IHRRRGAJSA-N 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- QWTGQXGNNMIUCW-BPUTZDHNSA-N Met-Asn-Trp Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QWTGQXGNNMIUCW-BPUTZDHNSA-N 0.000 description 1
- 241000713862 Moloney murine sarcoma virus Species 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- LWPMGKSZPKFKJD-DZKIICNBSA-N Phe-Glu-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O LWPMGKSZPKFKJD-DZKIICNBSA-N 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- HRNQLKCLPVKZNE-CIUDSAMLSA-N Ser-Ala-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O HRNQLKCLPVKZNE-CIUDSAMLSA-N 0.000 description 1
- WSTIOCFMWXNOCX-YUMQZZPRSA-N Ser-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CO)N WSTIOCFMWXNOCX-YUMQZZPRSA-N 0.000 description 1
- BUYHXYIUQUBEQP-AVGNSLFASA-N Ser-Phe-Glu Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CO)N BUYHXYIUQUBEQP-AVGNSLFASA-N 0.000 description 1
- GZGFSPWOMUKKCV-NAKRPEOUSA-N Ser-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO GZGFSPWOMUKKCV-NAKRPEOUSA-N 0.000 description 1
- SGAOHNPSEPVAFP-ZDLURKLDSA-N Thr-Ser-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SGAOHNPSEPVAFP-ZDLURKLDSA-N 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- SYOMXKPPFZRELL-ONGXEEELSA-N Val-Gly-Lys Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N SYOMXKPPFZRELL-ONGXEEELSA-N 0.000 description 1
- XBRMBDFYOFARST-AVGNSLFASA-N Val-His-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C(C)C)C(=O)O)N XBRMBDFYOFARST-AVGNSLFASA-N 0.000 description 1
- AEFJNECXZCODJM-UWVGGRQHSA-N Val-Val-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)NCC([O-])=O AEFJNECXZCODJM-UWVGGRQHSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 210000002203 alpha-beta t lymphocyte Anatomy 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011337 individualized treatment Methods 0.000 description 1
- 230000005732 intercellular adhesion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 108700042226 ras Genes Proteins 0.000 description 1
- 102000016914 ras Proteins Human genes 0.000 description 1
- 108010014186 ras Proteins Proteins 0.000 description 1
- 230000031539 regulation of cell division Effects 0.000 description 1
- 230000021014 regulation of cell growth Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 102200006531 rs121913529 Human genes 0.000 description 1
- 102200006539 rs121913529 Human genes 0.000 description 1
- 102200006538 rs121913530 Human genes 0.000 description 1
- 102200006540 rs121913530 Human genes 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000006228 supernatant 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
- 230000002194 synthesizing effect Effects 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000003260 vortexing Methods 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
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Oncology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于分子生物学和免疫学领域,具体地,涉及一种肿瘤新生抗原表位肽Pep2及其多聚体和应用。本发明的肿瘤新生抗原表位肽Pep2的氨基酸序列如SEQ ID NO.10所示;或者所述肿瘤新生抗原表位肽的氨基酸序列通过氨基酸替换、缺失或者增加而与SEQ ID NO.10所示序列具有90%以上相似性的序列;或者所述肿瘤新生抗原表位肽的氨基酸序列包含如SEQ ID NO.10所示序列或者包含通过氨基酸替换、缺失或者增加而与SEQ ID NO.10所示序列具有90%以上相似性的序列。本发明的多聚体的抗原肽为肿瘤新生抗原表位肽Pep2。本发明一方面为临床过继性免疫治疗癌症提供了思路,同时也为药物研发提供了检测手段,可以更好的服务于药物研发,使有效的治疗药物尽快的服务于癌症患者。
Description
技术领域
本发明属于分子生物学和免疫学领域,具体地,涉及一种肿瘤新生抗原表位肽Pep2及其多聚体和应用。
背景技术
本领域现有对免疫细胞的鉴定与分析主要包括以下几大类技术:
酶联免疫吸附斑点(Elispot)分析
如图1所示,Elispot的孔板已被细胞因子特异的一抗进行包被,外源加入的抗原在Elispot的孔板中被递呈细胞递呈给目的检测的T细胞。经过孵育,特异性的T细胞通过识别对应的特异性抗原进而产生效应性细胞因子,细胞因子通过被包被的抗体捕获最终可以以斑点的形式被检测出来。所以Elispot检测能提供关于特异性抗原的免疫反应的频率、细胞因子轮廓和抗原的特异性的信息。
Elispot只能通过细胞因子的分泌量来反映杀伤性免疫细胞的量,不能确定分泌细胞因子的是哪一细胞亚群,即不能区分样品中T细胞与其他细胞如NK。因为Elispot检测的是分泌细胞因子的细胞,很难同时进行表型分析,所以无法鉴定分析出特异性的T细胞,且每个分泌细胞因子的T细胞的数量很难量化。Elispot只能鉴定抗原刺激产生的特异性免疫细胞,无法将特异的免疫杀伤性T细胞进行分选,从而无法获得纯度较高的特异性T细胞。
四聚体分析技术
如图2所示,抗原在APC细胞内经过泛素化降解、pMHC组装及分泌等多个步骤,最终以pMHC复合物的形式表达于APC的细胞膜上,T细胞表达的TCR可识别并结合APC表面的pMHC随后向T细胞内传递活化信号,活化的CTL通过识别靶细胞表面结合有抗原肽的MHC I类分子而杀伤靶细胞。在体外水平,抗原肽与MHC-I结合形成单体复合物后可被T细胞表面的TCR识别,但单体复合物被T细胞识别的活性有限,将pMHC单体复合物制备成多聚体可有效增强被T细胞识别的能力。利用1摩尔链霉亲和素可结合4摩尔生物素这一模式,通过将生物素标记在MHC-I重链的α3末端,再使用偶联有荧光素的链霉亲和素招募捕获pMHC-biotin单体复合物,可制备得到pMHC的四聚体Tetramer。
pMHC四聚体技术在单细胞水平上可标记特异性T细胞作为目标检测细胞,通过流式细胞技术对细胞进行分析,可快速的将抗原特异性T细胞检测出来,且能同时对抗原特异性T细胞进行定性及定量分析,并能通过流式筛选将抗原特异性T细胞分选出来,从而获得纯度较高的特异性T细胞。
T细胞的抗原识别方式及特点:
方式:初始T细胞不能直接识别抗原,需要抗原提呈细胞处理。由于T细胞双识别的特点,决定了对其特异性的研究需要考虑抗原肽的性质和提呈抗原肽的MHC分子型别。即初始αβT细胞需要通过同时识别并结合pMHC复合物中的多肽和MHC来完成对递呈抗原的识别,随后激活T细胞的免疫杀伤活性。
特点:αβT细识别抗原具有MHC限制性。
因为TCR亲和力较低,跟pMHC复合体解离速度较快,单体的pMHC复合物很难用于检测抗原特异性T细胞。所以开发了四聚体技术用于检测抗原特异性T细胞。四聚体增强了TCR的亲和力,使得使用流式细胞术就可以在体外准确的检测抗原特异性T细胞。利用1摩尔链霉亲和素可结合4摩尔生物素这一模式,通过将生物素标记在MHC-I重链的α3末端,再使用偶联有荧光素的链霉亲和素招募捕获pMHC-biotin单体复合物,可制备得到pMHC的四聚体Tetramer。四聚物中的4个pMHC单体都可以与特异性的T细胞受体结合,有效增强了亲和活性。
抗原特异性T细胞鉴定的应用价值,一是肿瘤免疫治疗中的潜在应用(DC-CTL和TCR-T的筛选和制备)
肿瘤能逃脱免疫杀伤往往是由于T细胞不能有效识别肿瘤相关抗原或肿瘤特异性抗原导致,因此通过刺激或改造T细胞使其能识别肿瘤抗原可达到T细胞重新杀伤肿瘤细胞的效果。
DC-CTL疗法是一种过继性免疫治疗方法,通过具有最强抗原递呈能力的树突细胞(DC)来递呈抗原肽给T细胞并激活杀伤性T细胞(CTL),随后CTL杀伤靶细胞。而DC递呈的抗原肽的特异性决定了CTL对肿瘤的特异性杀伤效果。本文中的抗原肽是由肿瘤细胞突变基因编码的新生抗原,主要由基因点突变等产生的与正常细胞表达的蛋白不一样的新的异常蛋白。这些蛋白经过酶解之后形成的多肽片段作为抗原肽可以递呈给T细胞,可促使T细胞变为特异性地识别肿瘤新抗原的成熟活化T细胞。
TCR-T疗法是另一种过继性免疫细胞治疗方法,TCR-T疗法的特点是对T细胞的TCR进行改造使能特异性地识别加工递呈出来的抗原,在靶点的选择上TCR-T更广谱一些。通过改造TCR可提高T淋巴细胞特异性识别肿瘤相关抗原的能力,进而使T淋巴细胞能够高效的识别特异性靶细胞。本文中的基于新抗原制备的Tetramer可有效的鉴定工程改造的TCR-T,从而初步评估TCR-T的有效性。
二是肿瘤免疫治疗伴随诊断中的应用(结合其他表型和功能的标志物)。
过继性免疫治疗方法是通过体外改造T细胞后再回输给患者来增强免疫杀伤活性,持续的杀伤活性依赖于回输后T细胞的高存续能力。过继性免疫治疗后患者体内是否还有特异性杀伤T细胞,及这部分T细胞是否有浸润到肿瘤组织很有必要得到进一步的确定。基于肿瘤新抗原制备的Tetramer可用来示踪特异性识别这些抗原的CTL或TCR-T,从而来反映患者体内是否还存在持续的免疫杀伤能力及确定这部分T细胞的分布。因此,Tetramer技术可以很好地服务于肿瘤过继性免疫治疗后的伴随诊断,更具象地反映体内特异性杀伤的活性。
KRASpG12D突变(共计6种突变)在各种肿瘤中发生频率(选几个有代表性的癌种)以及不同癌肿频率覆盖。
在不同的癌症当中存在不同的KRAS突变的比例:胰腺癌(90%)、结肠癌(50%)、肺癌(30%)、卵巢癌(15%)、甲状腺癌(50%)、膀胱癌(6%)肝癌等癌症以及某些的白血病(Leukemia)都有较高的突变水平。KRAS基因突变发生在肿瘤恶变的早期,且原发灶和转移灶的KRAS基因高度保持一致。
KRAS第十二位G的突变在胰腺癌中都占有比例,如KRAS G12D占35.5%,G12V占27.9%,G12C占1.6%,G12R占15.7%等。而KRAS基因这个点的突变在其它癌种中也有不同频率的覆盖。因为KRAS突变在不同癌种中都有发生,所以根据KRAS的基因突变所得到的序列来作为新抗原表位肽可以覆盖多种癌种。
KRASpG12D特异性T细胞鉴定的难点:具有免疫原性的表位肽难以确定,识别抗原的T细胞多样性强(一人多T、多人多T)。
T细胞抗原表位是T细胞抗原上具有特殊功能的肽段,能够特异地被T细胞识别,从而激起T细胞的免疫应答。所以表位肽包含了一个抗原上所有表位的肽段,也就是说表位是抗原肽的一部分。基于上述原理,T细胞与抗原结合时并不是识别整个抗原,而是主要识别抗原分子上的线型表位。但一个抗原上会有许多表位,所以确定T细胞针对抗原上哪个表位可以起到最好的抗原识别及产生最强的杀伤效果是关键点。
发明内容
为了解决现有技术中存在的问题,本发明提供一种肿瘤新生抗原表位肽及其多聚体和应用。本发明筛选出肿瘤特异性的抗原肽;基于这些抗原肽可以开发过继性免疫疗法DC-CTL或TCR-T;基于这些抗原肽制备多聚体(包括但不限于Tetramer)来检测DC-CTL或TCR-T的抗原特异性,同时来为临床治疗提供伴随诊断。
为了实现上述目的,本发明采用如下技术方案:
本发明的肿瘤新生抗原表位肽Pep2,其氨基酸序列如SEQ ID NO.10所示;或者所述肿瘤新生抗原表位肽的氨基酸序列通过氨基酸替换、缺失或者增加而与SEQ ID NO.10所示序列具有90%以上相似性的序列;或者所述抗原肽的氨基酸序列包含如SEQ ID NO.10所示序列或者包含通过氨基酸替换、缺失或者增加而与SEQ ID NO.10所示序列具有90%以上相似性的序列。
本发明的上述肿瘤新生抗原表位肽的筛选方法,包括以下步骤:
1)采用抗原表位预测算法,对在肿瘤中高频发生的突变位点,进行新生抗原表位预测,得到候选的抗原表位多肽序列,通过对预测的表位肽与HLA-A0201型MHC复合物的亲和力评分进行筛选,得到第一批候选的高亲和力表位肽;
2)将步骤1)得到的第一批候选的高亲和力表位肽,采用构象酶联免疫的实验方法评价候选表位肽与HLA-A0201型MHC复合,进一步筛选到具有较强亲和力的第二批候选的高亲和力表位肽;
3)使用HLA-A0201型别的肿瘤患者PBMC样本,采用酶联免疫斑点法,检测步骤2)筛选到的第二批候选的高亲和力表位肽是否具有免疫原性,刺激PBMC分泌免疫效应因子IFNγ,筛选出的具有免疫原性的新生抗原表位肽,获得肿瘤新生抗原表位肽。
本发明还提供了一种多聚体,所述多聚体的抗原肽为上述的肿瘤新生抗原表位肽Pep2。其中优选的,所述多聚体的聚合单位为pMHC,所述多聚体可以但不限于为四聚体、五聚体或六聚体或通过其他载体聚合pMHC制备的多聚体等。
根据本发明所述的多聚体,作为一种优选,所述多聚体为四聚体,所述四聚体还包括链酶亲和素,链酶亲和素与四组连接在MHCα3重链上的生物素结合,每个MHC上特异性连接肿瘤新生抗原表位肽Pep2。
本发明还优选提供了上述四聚体的制备方法,包括以下步骤:
1)pMHC制备及肽置换:
采用pMHC单体制备法制备携带生物素的敏感肽psenseMHC,以制备得到的敏感肽psenseMHC为基础,通过紫外照射后与作为靶肽的肿瘤新生抗原表位肽Pep2孵育,制备得到靶肽pMHC单体复合物;
2)将步骤1)制得的pMHC单体复合物与链酶亲和素共孵育,使链霉亲和素与连在MHCα3重链上的生物素结合,制得四聚体。
根据本发明所述的制备方法,其中优选的,所述敏感肽序列为KILGFVFJV。
根据本发明所述的制备方法,其中优选的,步骤1)添加亲和素之前,先将靶肽pMHC通过与包被有β2M抗体的ELISA板孵育,使靶肽pMHC被捕获到ELISA板上,通过ELISA方法检测制备得到的靶肽pMHC,确认靶肽pMHC制备成功后再进一步制备四聚体。
根据本发明所述的制备方法,其中优选的,步骤2)所述链酶亲和素为带有荧光的链酶亲和素。
本发明还提供了所述肿瘤新生抗原表位肽Pep2在检测DC-CTL或TCR-T的抗原特异性方面的应用。例如,在制备用于过继性免疫疗法DC-CTL或TCR-T中的药物或检测试剂中的应用。
本发明还进一步提供了所述多聚体在检测DC-CTL或TCR-T的抗原特异性方面的应用。例如,在制备用于过继性免疫疗法DC-CTL或TCR-T中的药物或检测试剂中的应用。
本文中的肿瘤新抗原是根据KRAS等多个基因位点的不同突变产生的,基于这种肿瘤新抗原制备多聚体(例如但不限于,Tetramer)。在DC-CTL中,通过DC递呈抗原肽共同刺激T细胞使产生特异性的CTL,多聚体可以检测抗原肽刺激产生特异性CTL的效果;及在TCR-T治疗中,基于新抗原筛选特异性TCR基因序列并制备TCR-T,再通过多聚体来检验TCR-T对特异性抗原的活性。
本发明首先通过自主研发的抗原表位预测算法并搭配分子水平和细胞水平的评价,筛选到一批癌症特异性抗原肽,基于这些抗原肽可以为癌症尤其是肝癌提供更具特异性的过继性免疫治疗,包括DC-CTL、TCR-T等,为癌症治愈提供了可能。在此基础上搭建了基于抗原肽的多聚体制备技术,制备得到的多聚体可以有效的鉴定这些抗原肽刺激制备的DC-CTL等T细胞的抗原特异性,即为非临床药效评价和药理阐释提供了检测手段。同时,药物上临床后可以提供伴随诊断,即使用基于抗原肽制备的多聚体(例如,Tetramer)来评价给药患者体内药物的存续能力及分布,可以更直观的评价药物活性。因此,该发明一方面为临床过继性免疫治疗癌症,尤其是肝癌,提供了思路,同时也为药物研发提供了检测手段,可以更好的服务于药物研发,使有效的治疗药物尽快的服务于癌症患者。
附图说明
图1为Elispot实验流程图;
图2为四聚体技术路线图;
图3为本发明的新生抗原高亲和力、高免疫原性表位肽筛选流程;
图4为本发明的实验验证表位肽-MHC亲和力的技术原理;
图5为本发明的高亲和力新生抗原表位肽与HLA-A0201的结合力评价结果(On-Rate);
图6为本发明高亲和力新生抗原表位肽与HLA-A0201的结合力评价结果(Off-Rate);
图7为本发明的高免疫原性新生抗原表位肽激发HLA-A0201型PBMC分泌IFNγ;
图8为本发明的多肽置换活性检测的ELISA检测结果(Pos,自制CMV源抗原肽组;Neg,阴性肽组;UV only,紫外照射未添加新肽组;Pos_monomer,未经紫外照射的pMHC;Blank,空白对照组);
图9为本发明的Tetramer活性评价的ELISA检测结果(Pos,自制CMV源抗原肽组;Neg,阴性肽组;UV only,紫外照射未添加新肽组;Pep2,肿瘤新生抗原表位肽Pep2组;Pos_monomer,未经紫外照射的pMHC;Blank,空白对照组);
图10为本发明的自制CMV Tetramer与现有商业CMV Tetramer的流式细胞仪分析染色结果;
图11为本发明的肿瘤新生抗原表位肽Pep2的Tetramer的流式细胞仪分析染色结果。
具体实施方式
本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。具体可参照《分子克隆实验指南》(第四版,J.萨姆布鲁克等著)一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行;下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
缩略语和关键术语定义(本申请中所涉及到的)
TCR,T cell receptor,T细胞受体。
APC,antigen-presenting cell,抗原递呈细胞。
CTL,cytotoxic T lymphocyte,杀伤性T淋巴细胞。
MHC,major histocompatibility complex,组织相容性复合物。
Monomer:是一种单体复合物,由MHC和肽形成的MHC-肽(pMHC)结构单元。
多聚体:借助链霉亲和素-生物素(1:4)系统,将pMHC结构单元聚合为复合物。
KRAS基因:Kras是一种鼠类肉瘤病毒癌基因,定位在12号染色体上,K-ras因编码21kD的ras蛋白又名p21基因。Kras基因也是人体肿瘤中常见的致癌基因,该基因的体细胞突变常见于多种恶性肿瘤,在肺癌患者中的突变率为15%-30%,在结直肠癌患者中为20-50%。作为EGFR信号通路下游最重要得效应因子,KRAS在肿瘤信号转导中发挥重要作用。突变是史上最恶性、最难对付的致癌突变,所以对KRAS基因突变得检测,可以为肿瘤患者的个体化治疗提供更确切的依据。
TP53基因:又称为P53,是被确认的第一种抑癌基因,由该基因编码的蛋白质是一种转录因子,调控细胞的生长、增殖、损伤修复。肿瘤细胞中该基因往往会发生突变,导致肿瘤细胞的增殖及损伤修复等不受控制,导致肿瘤的恶性演进。
抗原特异性T细胞:在防御病毒感染、肿瘤发生以及自身免疫性疾病方面都具有重要作用的细胞。检测细胞的活动以鉴别淋巴细胞对特异刺激的反应,在研究这些疾病的发病机制及治疗方面都有重要意义。适应性免疫反应和免疫发病机制是基于T细胞对特异性抗原的反应能力。
CTNNB1基因:又名β-catenin,该编码蛋白调控基因转录和细胞间的粘附,对细胞的形态维持有调控作用。该基因发生突变可通过影响肿瘤细胞的上皮间充质转变来促进肿瘤的恶性演进。
GNAS基因:介导G蛋白偶联受体的信号转导,可促进cAMP水平的升高,参与调控细胞生长和细胞分裂。该基因表达上调及突变与肿瘤恶性研究显著相关。
JAK基因:一种胞内的酪氨酸激酶,介导JAK-STAT信号通路,调控细胞的增殖、分化、凋亡及免疫调节等功能。在肿瘤细胞中,JAK-STAT信号通路上调。
本发明的具体实施方式按流程分为抗原肽预测筛选验证、Tetramer制备、Tetramer活性评价共计三个部分。其中抗原肽的预测筛选验证是通过预测算法来预测,随后通过分子水平和细胞水平来验证预测的抗原肽的免疫原性及抗原特异性T细胞对对应抗原表达肿瘤细胞的杀伤活性。Tetramer制备采用原核表达系统和体外组装方式制备得到紫外敏感肽psense-MHC(pMHC)单体复合物,该敏感肽单体在366nm紫外照射下,单体复合物上的敏感肽会断裂脱落从而暴露肽结合位点,为靶肽或其他肽的结合提供可能。Tetramer活性评价采用CMV病毒产生的多肽制备Tetramer,并与商业化的同类CMV抗原活性产品在同一种样品上染色比较来测试自制Tetramer活性,在得到验证后制备抗原肽的Tetramer并在抗原肽刺激产生的DC-CTL上检测靶向活性。
实施例1抗原肽预测、筛选、验证
抗原表位肽的发现,是制备多肽-MHC四聚体的关键。与MHC具有较高亲和力以及具有激发免疫响应的较高免疫的表位肽,用于制备识别该抗原特异性T细胞的多肽-MHC四聚体的优选多肽。本发明中筛选高亲和力和高免疫原性表位肽,用于制备pMHC的流程如图3所示:
采用自主研发的抗原表位预测算法(专利公布号CN112002374A),对在肿瘤中高频发生的突变位点(KRASpG12D、KRASpG12V、KRASpG12R、KRASpG12C、Tp53pR249S、TP53pR175H等),进行新生抗原表位预测,得到候选的抗原表位多肽序列,通过对预测的表位肽与HLA-A0201型MHC复合物的亲和力评分进行筛选,共计得到40条候选的高亲和力表位肽,见表1。
表1.抗原表位预测分析得到的40条表位肽信息及筛选数据
合成40条候选表位肽,采用构象酶联免疫的实验方法评价候选表位肽与HLA-A0201型MHC复合。具体实验原理是:当候选表位肽与MHC复合物结合时,可激发荧光信号,结合力越强、荧光信号越强(技术原理见图4)。
实验筛选得到10条(SEQ ID NO.1、2、4、10、13、14、16、19、25、28)候选表位,具有较强的亲和力,见表1、图5和图6。
进一步获得HLA-A0201型别的肿瘤患者PBMC样本,采用酶联免疫斑点法,检测上一步筛选到的高亲和力表位肽是否具有免疫原性,刺激PBMC分泌疫效应因子IFNγ,进而筛选得到1条新生抗原表位肽具有较强的免疫原性,结果见图7。
通过上述筛选步骤得到1条高亲和力、高免疫原性的新生抗原表位肽,序列如下(表2):
表2.高亲和力、高免疫原性新生抗原表位肽信息
实施例2Tetramer制备
1.pMHC制备及肽置换
本部分采用已有的pMHC单体制备法(专利公布号CN109293779A、CN106397549A)制备携带生物素的敏感肽pMHC,其中敏感肽的序列为:KILGFVFJV(HLA-A0201型,其中J指3-氨基-3-(2硝基)苯丙酸)。以制备得到的敏感肽psense-MHC为基础,通过366nm紫外照射后与靶肽(CMV源抗原肽)再孵育制备得到靶肽pMHC,通过与包被有β2M抗体的ELISA板孵育,pMHC被捕获到ELISA板上,随后添加亲和素-HRP使亲和素与连在MHCα3重链上的生物素结合,最后添加HRP底物通过吸光值来反映pMHC的含量,进而可反映紫外照射后敏感肽脱离程度和再孵育后新生成的靶肽pMHC效果。具体试验操作如下:
试剂、耗材、物料和仪器设备
试剂:PBS缓冲液(pH7.4)、Peptide样品、psense-MHC、偶联有链霉亲和素的辣根过氧化物酶(Avidin HRP)、DMSO、Assay Buffer A、Wash Buffer、Substrate Solution F、StopSolution。
耗材:包被有β2M抗体的ELISA条、96孔板(聚苯乙烯,V型孔)、封口膜、1.5mL EP管。
仪器设备:紫外灯(含366nm波长)、水浴锅、低温离心机(适用于96孔板及1.5mL EP管)、移液枪(单枪和排枪)、摇床、酶标仪(含450和570nm两个模块)。
实验步骤
A.多肽抗原置换
将实验所需试剂拿出并冰浴放置,使温度保持在0℃;
用PBS将目标肽稀释到400μM,冰浴放置待用;
取一个V型孔的96孔板,用移液枪在孔内添加1.5μL稀释好的目标肽和1.5μL的psense-MHC(200μg/mL),移液枪吹打使混匀(阴性对照和UV only组加1.5μL对应肽(HLA-A0201的阴性肽序列为IVTDFSVIK)和PBS与1.5μL的psense-MHC混合);
封盖,4℃环境下2500g离心2分钟使液体沉到孔底;
去盖,96孔板冰浴状态下正置于紫外灯下紫外(366nm)照射30min;
随后,封盖,并将孔板在37℃、避光环境下孵育30min使目标肽充分结合;
离心,收集孔内液体待用。(液体中含制备好的pMHC monomer复合物,每孔中含300ng pMHC monomer复合物,浓度为100μg/mL)。
B.多肽置换活性检测
用ddH2O将20×Wash Buffer稀释至1×Wash Buffer;用Assay Buffer A稀释上一步中制备好的pMHC复合物样品使单体复合物的浓度到50ng/mL,此为单体复合物样品母液;
将所需要的试剂拿出并放至室温温度,计算好需要的ELISA条数并拿出放入ELISA板中;
用96μL Assay Buffer A溶4μL的pMHC monomer复合物母液制备适宜浓度样品(2ng/mL),Assay Buffer A设为对照液;
在ELISA板的孔内先加50μL的Assay Buffer A,随后在相应孔内继续分别添加50μL的样品液或对照液;(此步操作后样品浓度为1ng/mL,每个样品设3个复孔)(添加一组Pos对照,该组为未处理过的psense-MHC;及一组空白对照,孔内添加等量的Assay Buffer A);
用封口膜将ELISA板封口,随后室温环境下在摇床上摇动孵育30min(可选择220rpm)或平面上静置孵育2h;
随后弃掉孔内液体,并用200μL 1×Wash Buffer/孔洗板4次,其中每次弃液时尽量控干液体,可以将板向平铺在桌子上的吸水纸上倒扣使液体被吸干;
最后一步控干液体后,向每个孔内添加100μL亲和素-HRP试剂,封口膜封口后室温环境下在摇床上摇动孵育30min(可选择220rpm)或平面上静置孵育30min;
随后弃掉孔内液体,并按前述洗板步骤洗5次,其中最后一次添加Wash Buffer后使液体在孔内浸泡30s–1min以使背景干扰降到最低;
最后一步控干液体后,向每个孔内添加100μL的底物溶液F并在室温环境下避光静置孵育10min;(底物添加后实验孔液体颜色应变蓝,并随着样品浓度增加蓝色逐渐加深;此步骤可选择性封口孵育);
孵育结束后,向每个孔内添加100μL的终止液;(终止液添加后液体颜色由蓝转黄);
终止显色后,在30min内使用酶标仪检测OD450和OD570两个波段的吸光值。(计算时用OD450的吸光值-OD570的吸光值)。
实验结果及分析
如图8所示,从结果看:1.紫外照射后相对于未经紫外照射组,完整的psense-MHC含量显著下降,且几乎全部消失;2.CMV源抗原肽添加组相对于UV only组,新生成的pMHC量显著增加,且几乎与Pos_monomer组持平;3.Neg组几乎没有新生成的完整pMHC。可以得出结论:1.psense-MHC在紫外照射下,敏感肽可降解并导致psense-MHC复合体解散;2.敏感肽脱离后,靶肽可有效结合到MHC上暴露的肽结合位点;3.制备的MHC对抗原肽有选择性,即只结合型别对应的有亲和力的肽。因此,psense-MHC制备成功。
2.Tetramer制备
试剂、耗材、物料和仪器设备
试剂:PBS缓冲液(pH7.4)、Peptide样品、psense-MHC、DMSO、50mM D-Biotin、10%(w/v)NaN3、Fluorophore-conjugated Streptavidin。
耗材:1.5mL EP管。
仪器设备:紫外灯(含366nm波长)、水浴锅、低温离心机(适用于96孔板及1.5mL EP管)、移液枪(单枪和排枪)、摇床。
实验步骤
按“1.pMHC制备及肽置换”中的实验条件制备36μL pMHC复合物并移到1.5mL的EP管中,随后添加3.96μL偶联有荧光的链霉亲和素APC-streptavidin,移液枪上下吹打使混匀,避光冰浴30min;
在上述步骤的孵育阶段,制备封闭液:将1.6μL浓度为50mM的D-Biotin和6μL质量体积比为10%的NaN3添加到192.4μL的PBS中,涡旋震荡使混匀,此为制备好的封闭液,留存待用。单体复合物同链霉亲和素孵育结束后,向反应液中加2.88μL封闭液,用移液枪上下吹打使充分混匀;
在2-8℃环境下继续避光孵育过夜或避光冰浴30min,此步反应后可获得Tetramers。
实施例3Tetramer活性评价
本部分使用本发明自制的CMV源抗原肽制备的Tetramer与商业化的同类CMV源抗原活性产品在同一批DC-CTL上染色,两组数据比较来评价自制Tetramer的靶向活性。在得到验证后使用前面筛选到的1条肽(本发明的氨基酸序列如SEQ ID NO.10所示的肿瘤新生抗原表位肽Pep2)制备各自对应的Tetramer,然后分别在这些抗原肽刺激制备的DC-CTL上染色评价这条肽制备的Tetramer的活性及DC-CTL制备后的特异性。具体操作步骤如下:
试剂、耗材、物料和仪器设备
试剂:PBS缓冲液(pH7.4)、Peptide样品、psense-MHC、DMSO、50mM D-Biotin、10%(w/v)NaN3、Fluorophore-conjugated Streptavidin、Cell Staining Buffer、偶联有链霉亲和素的辣根过氧化物酶(Avidin HRP)、Assay Buffer A、Wash Buffer、SubstrateSolution F、Stop Solution、荧光标记的膜蛋白抗体(CD3-perCP-cy5.5、CD4-APC-CY7、CD8-BV510、CD56-BV605)。
耗材:包被有β2M抗体的ELISA条、96孔板(聚苯乙烯,V型孔)、封口膜、1.5mL EP管。
仪器设备:紫外灯(含366nm波长)、水浴锅、低温离心机(适用于96孔板及1.5mL EP管)、移液枪(单枪和排枪)、摇床、37℃培养箱、酶标仪(含450和570nm两个模块)、流式细胞仪、生物安全柜、CO2培养箱、K2细胞计数仪。
实验步骤
按实施例2中“1.pMHC制备及肽置换”中的实验条件制备37μL pMHC复合物,取出1μL用于ELISA评价pMHC制备的成功性,得到验证后将剩余的36μL pMHC复合物移到1.5mL的EP管中,并按照实施例2中“2.Tetramer制备”步骤制备得到各抗原肽对应的Tetramer。
准备好需要的DC-CTL细胞并计数;
准备1×106个细胞/样,放到12×75mm的流式管内,用细胞染色缓冲液补齐体积到200μL。组别中需要有空白组、对照组、阴性对照组、实验组,其中Blank不加抗体及Tetramer、对照组只加CD3&CD4$CD8&CD56荧光抗体、阴性对照组(Negtive组)在4个荧光抗体基础上加阴性肽制备的Tetramer、实验组是在4个荧光抗体基础上加实验肽制备的Tetramer。按膜蛋白荧光抗体CD3-perCP-cy5.5、CD4-APC-CY7、CD8-BV510、CD56-BV605各2μL、Tetramers试剂量10μL体积分别加到对应的细胞样中,用移液枪上下吹打混匀后避光在相应温度环境下孵育。其中,对照组将抗体混好加到样品中避光冰浴30min即可上样;阴性对照及实验组先加四种抗体避光冰浴20min,buffer洗2次后随后添加对应Tetramer避光室温孵育10min,buffer洗2次后上机,在2h内流式细胞仪检测,未上机样品避光冰浴放置。其中洗样步骤为:350g、4℃离心5min,弃上清后用1mL染色缓冲液重悬。
实验结果及分析
如图9-11所示,从结果看:1.现有商业CMV源抗原肽和本发明的抗原肽均有效生成了pMHC;2.本发明制备的Tetramer_CMV染色阳性率略高于商业同品,说明自制Tetramer成功;3.本发明制备的抗原肽对应Tetramer均能有效靶向标记这些肽刺激制备的DC-CTL,说明本发明的Tetramer可以用于DC-CTL药物质量的评价。
序列表
<110> 北京臻知医学科技有限责任公司
<120> 一种肿瘤新生抗原表位肽Pep2及其多聚体和应用
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> Artificial
<400> 1
Gly Met Asn Trp Arg Pro Ile Leu Thr Ile
1 5 10
<210> 2
<211> 10
<212> PRT
<213> Artificial
<400> 2
Cys Met Gly Gly Met Asn Trp Arg Pro Ile
1 5 10
<210> 3
<211> 9
<212> PRT
<213> Artificial
<400> 3
Met Asn Trp Arg Pro Ile Leu Thr Ile
1 5
<210> 4
<211> 10
<212> PRT
<213> Artificial
<400> 4
Lys Leu Val Val Val Gly Ala Cys Gly Val
1 5 10
<210> 5
<211> 9
<212> PRT
<213> Artificial
<400> 5
Leu Val Val Val Gly Ala Cys Gly Val
1 5
<210> 6
<211> 10
<212> PRT
<213> Artificial
<400> 6
Gly Ala Cys Gly Val Gly Lys Ser Ala Leu
1 5 10
<210> 7
<211> 9
<212> PRT
<213> Artificial
<400> 7
Gly Arg Asn Ser Phe Glu Val His Val
1 5
<210> 8
<211> 10
<212> PRT
<213> Artificial
<400> 8
Leu Gly Arg Asn Ser Phe Glu Val His Val
1 5 10
<210> 9
<211> 10
<212> PRT
<213> Artificial
<400> 9
Leu Leu Gly Arg Asn Ser Phe Glu Val His
1 5 10
<210> 10
<211> 10
<212> PRT
<213> Artificial
<400> 10
Lys Leu Val Val Val Gly Ala Asp Gly Val
1 5 10
<210> 11
<211> 9
<212> PRT
<213> Artificial
<400> 11
Leu Val Val Val Gly Ala Asp Gly Val
1 5
<210> 12
<211> 10
<212> PRT
<213> Artificial
<400> 12
Gly Ala Asp Gly Val Gly Lys Ser Ala Leu
1 5 10
<210> 13
<211> 10
<212> PRT
<213> Artificial
<400> 13
Lys Leu Val Val Val Gly Ala Val Gly Val
1 5 10
<210> 14
<211> 9
<212> PRT
<213> Artificial
<400> 14
Leu Val Val Val Gly Ala Val Gly Val
1 5
<210> 15
<211> 9
<212> PRT
<213> Artificial
<400> 15
Tyr Lys Leu Val Val Val Gly Ala Val
1 5
<210> 16
<211> 9
<212> PRT
<213> Artificial
<400> 16
His Met Thr Glu Val Val Arg His Cys
1 5
<210> 17
<211> 10
<212> PRT
<213> Artificial
<400> 17
His Met Thr Glu Val Val Arg His Cys Pro
1 5 10
<210> 18
<211> 10
<212> PRT
<213> Artificial
<400> 18
Ser Gln His Met Thr Glu Val Val Arg His
1 5 10
<210> 19
<211> 10
<212> PRT
<213> Artificial
<400> 19
Ser Gln His Met Thr Glu Val Val Arg His
1 5 10
<210> 20
<211> 9
<212> PRT
<213> Artificial
<400> 20
Gly Ala Thr Ala Thr Ala Pro Ser Leu
1 5
<210> 21
<211> 9
<212> PRT
<213> Artificial
<400> 21
Gly Ile His Ser Gly Ala Thr Ala Thr
1 5
<210> 22
<211> 10
<212> PRT
<213> Artificial
<400> 22
Leu Leu Arg Cys His Val Leu Thr Ser Gly
1 5 10
<210> 23
<211> 9
<212> PRT
<213> Artificial
<400> 23
Asp Leu Leu Arg Cys His Val Leu Thr
1 5
<210> 24
<211> 10
<212> PRT
<213> Artificial
<400> 24
His Val Leu Thr Ser Gly Ile Phe Glu Thr
1 5 10
<210> 25
<211> 10
<212> PRT
<213> Artificial
<400> 25
Lys Leu Val Val Val Gly Ala Arg Gly Val
1 5 10
<210> 26
<211> 9
<212> PRT
<213> Artificial
<400> 26
Leu Val Val Val Gly Ala Arg Gly Val
1 5
<210> 27
<211> 10
<212> PRT
<213> Artificial
<400> 27
Gly Ala Arg Gly Val Gly Lys Ser Ala Leu
1 5 10
<210> 28
<211> 10
<212> PRT
<213> Artificial
<400> 28
Gly Met Asn Arg Ser Pro Ile Leu Thr Ile
1 5 10
<210> 29
<211> 9
<212> PRT
<213> Artificial
<400> 29
Ser Pro Ile Leu Thr Ile Ile Thr Leu
1 5
<210> 30
<211> 10
<212> PRT
<213> Artificial
<400> 30
Cys Met Gly Gly Met Asn Arg Ser Pro Ile
1 5 10
<210> 31
<211> 10
<212> PRT
<213> Artificial
<400> 31
Ser Gly Ala Thr Thr Thr Ala Pro Ser Leu
1 5 10
<210> 32
<211> 9
<212> PRT
<213> Artificial
<400> 32
Gly Ala Thr Thr Thr Ala Pro Ser Leu
1 5
<210> 33
<211> 9
<212> PRT
<213> Artificial
<400> 33
Thr Thr Ala Pro Ser Leu Ser Gly Lys
1 5
<210> 34
<211> 10
<212> PRT
<213> Artificial
<400> 34
Gly Ile Asp Cys Glu Cys Gly Pro Phe Ile
1 5 10
<210> 35
<211> 10
<212> PRT
<213> Artificial
<400> 35
Leu Leu Ala Arg Glu Gly Ile Asp Cys Glu
1 5 10
<210> 36
<211> 9
<212> PRT
<213> Artificial
<400> 36
Cys Glu Cys Gly Pro Phe Ile Lys Leu
1 5
<210> 37
<211> 9
<212> PRT
<213> Artificial
<400> 37
Gly Met Asn Trp Arg Pro Ile Leu Thr
1 5
<210> 38
<211> 9
<212> PRT
<213> Artificial
<400> 38
Ala Val Gly Val Gly Lys Ser Ala Leu
1 5
<210> 39
<211> 10
<212> PRT
<213> Artificial
<400> 39
Ser Gly Ala Thr Ala Thr Ala Pro Ser Leu
1 5 10
<210> 40
<211> 9
<212> PRT
<213> Artificial
<400> 40
Gly Met Asn Arg Ser Pro Ile Leu Thr
1 5
Claims (10)
1.一种肿瘤新生抗原表位肽Pep2,其特征在于,所述肿瘤新生抗原表位肽Pep2的氨基酸序列如SEQ ID NO.10所示;或者所述肿瘤新生抗原表位肽的氨基酸序列通过氨基酸替换、缺失或者增加而与SEQ ID NO.10所示序列具有90%以上相似性的序列;或者所述抗原肽的氨基酸序列包含如SEQ ID NO.10所示序列或者包含通过氨基酸替换、缺失或者增加而与SEQ ID NO.10所示序列具有90%以上相似性的序列。
2.一种权利要求1所述肿瘤新生抗原表位肽的筛选方法,包括以下步骤:
1)采用抗原表位预测算法,对在肿瘤中高频发生的突变位点,进行新生抗原表位预测,得到候选的抗原表位多肽序列,通过对预测的表位肽与HLA-A0201型MHC复合物的亲和力评分进行筛选,得到第一批候选的高亲和力表位肽;
2)将步骤1)得到的第一批候选的高亲和力表位肽,采用构象酶联免疫的实验方法评价候选表位肽与HLA-A0201型MHC复合,进一步筛选到具有较强亲和力的第二批候选的高亲和力表位肽;
3)使用HLA-A0201型别的肿瘤患者PBMC样本,采用酶联免疫斑点法,检测步骤2)筛选到的第二批候选的高亲和力表位肽是否具有免疫原性,刺激PBMC分泌免疫效应因子IFNγ,筛选出的具有免疫原性的新生抗原表位肽,获得肿瘤新生抗原表位肽。
3.一种多聚体,其特征在于,所述多聚体的抗原肽为权利要求1所述的肿瘤新生抗原表位肽Pep2。
4.根据权利要求3所述的多聚体,其特征在于,所述多聚体为四聚体、五聚体、六聚体,或通过其他载体聚合pMHC制备的多聚体。
5.根据权利要求4所述的多聚体,其特征在于,所述多聚体为四聚体,所述四聚体包括链酶亲和素,链酶亲和素与四组连接在MHCα3重链上的生物素结合,每个MHC上特异性连接肿瘤新生抗原表位肽Pep2。
6.一种权利要求5所述多聚体的制备方法,包括以下步骤:
1)pMHC制备及肽置换:
采用pMHC单体制备法制备携带生物素的敏感肽psenseMHC,以制备得到的敏感肽psenseMHC为基础,通过紫外照射后与作为靶肽的肿瘤新生抗原表位肽Pep2孵育,制备得到靶肽pMHC单体复合物;
2)将步骤1)制得的靶肽pMHC单体复合物与链酶亲和素共孵育,使链霉亲和素与连在MHCα3重链上的生物素结合,制得四聚体。
7.根据权利要求6所述的制备方法,其特征在于,所述敏感肽序列为KILGFVFJV。
8.根据权利要求6所述的制备方法,其特征在于,步骤1)添加亲和素之前,先将靶肽pMHC通过与包被有β2M抗体的ELISA板孵育,使靶肽pMHC被捕获到ELISA板上,通过ELISA方法检测制备得到的靶肽pMHC,确认靶肽pMHC制备成功后再进一步制备四聚体;
步骤2)所述链酶亲和素为带有荧光的链酶亲和素。
9.权利要求1所述肿瘤新生抗原表位肽Pep2在检测DC-CTL或TCR-T的抗原特异性方面的应用。
10.权利要求3-5任一所述多聚体在检测DC-CTL或TCR-T的抗原特异性方面的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111202851.0A CN114106136A (zh) | 2021-10-15 | 2021-10-15 | 一种肿瘤新生抗原表位肽Pep2及其多聚体和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111202851.0A CN114106136A (zh) | 2021-10-15 | 2021-10-15 | 一种肿瘤新生抗原表位肽Pep2及其多聚体和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114106136A true CN114106136A (zh) | 2022-03-01 |
Family
ID=80375746
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111202851.0A Pending CN114106136A (zh) | 2021-10-15 | 2021-10-15 | 一种肿瘤新生抗原表位肽Pep2及其多聚体和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114106136A (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2649091A1 (en) * | 2010-12-09 | 2013-10-16 | Stichting Het Nederlands Kanker Instituut | Immune restricted peptides with increased efficacy |
US20180134804A1 (en) * | 2015-03-20 | 2018-05-17 | Memorial Sloan-Kettering Cancer Center | Monoclonal antigen-binding proteins to intracellular oncogene products |
CN112110995A (zh) * | 2019-06-19 | 2020-12-22 | 上海交通大学医学院 | 肿瘤新抗原多肽及其用途 |
CN112876542A (zh) * | 2021-02-08 | 2021-06-01 | 暨南大学 | 一种新型冠状病毒t细胞的抗原表位肽及其应用 |
-
2021
- 2021-10-15 CN CN202111202851.0A patent/CN114106136A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2649091A1 (en) * | 2010-12-09 | 2013-10-16 | Stichting Het Nederlands Kanker Instituut | Immune restricted peptides with increased efficacy |
US20180134804A1 (en) * | 2015-03-20 | 2018-05-17 | Memorial Sloan-Kettering Cancer Center | Monoclonal antigen-binding proteins to intracellular oncogene products |
CN112110995A (zh) * | 2019-06-19 | 2020-12-22 | 上海交通大学医学院 | 肿瘤新抗原多肽及其用途 |
WO2020253643A1 (zh) * | 2019-06-19 | 2020-12-24 | 上海交通大学医学院 | 肿瘤新抗原多肽及其用途 |
CN112876542A (zh) * | 2021-02-08 | 2021-06-01 | 暨南大学 | 一种新型冠状病毒t细胞的抗原表位肽及其应用 |
Non-Patent Citations (1)
Title |
---|
JINGCHENG WU;WENYI ZHAO;BINBIN ZHOU;ZHIXI SU;XUN GU;ZHAN ZHOU;SHUQING CHEN;: "TSNAdb: A Database for Tumor-specific Neoantigens from Immunogenomics Data Analysis", GENOMICS,PROTEOMICS & BIOINFORMATICS, no. 04, pages 126 - 70 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Chen et al. | Neoantigen identification strategies enable personalized immunotherapy in refractory solid tumors | |
Hoekstra et al. | Long-distance modulation of bystander tumor cells by CD8+ T-cell-secreted IFN-γ | |
WO2020253643A1 (zh) | 肿瘤新抗原多肽及其用途 | |
Poropatich et al. | OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity | |
Robbins et al. | Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells | |
CN112534045A (zh) | 获得肿瘤特异性t细胞受体的方法 | |
CN109485721A (zh) | 一种获得肿瘤特异性t细胞受体的方法 | |
EA029831B1 (ru) | Пептид, индуцирующий цитотоксические т-лимфоциты и стимулирующий антиопухолевые иммунные ответы | |
CN114085281A (zh) | 一种肿瘤抗原表位肽及其多聚体和应用 | |
KR20220030208A (ko) | T 세포 조성물의 제조를 위한 조성물 및 방법, 및 그의 용도 | |
CN117486999A (zh) | 一种肿瘤T细胞抗原表位肽、pMHC及其制备和应用 | |
US9720000B2 (en) | Targeted identification of immunogenic peptides | |
JP2021502110A (ja) | がん特異的抗原に対するtリンパ球のスクリーニング | |
WO2019036043A2 (en) | METHOD FOR GENERATING A COCKTAIL OF PERSONALIZED ANTICANCER VACCINES FROM TUMOR DERIVED GENETIC MODIFICATIONS FOR THE TREATMENT OF CANCER | |
CN113611362A (zh) | 一种点突变birc5抗原表位肽筛选的方法 | |
CN106589106B (zh) | Hla-a24限制性ecm1特异性的ctl表位肽及其应用 | |
CN114106136A (zh) | 一种肿瘤新生抗原表位肽Pep2及其多聚体和应用 | |
CN114106135A (zh) | 一种肿瘤新生抗原表位肽Pep3及其多聚体和应用 | |
CN114085282A (zh) | 一种肿瘤新生抗原表位肽Pep6及其多聚体和应用 | |
CN114085286A (zh) | 一种肿瘤新生抗原表位肽Pep5及其多聚体和应用 | |
CN114106137A (zh) | 一种肿瘤新生抗原表位肽Pep1及其多聚体和应用 | |
CN114106138A (zh) | 一种肿瘤新生抗原表位肽Pep4及其多聚体和应用 | |
CN115975943A (zh) | 基于acp5阳性t细胞的用于杀伤肿瘤的tcr-t细胞及其制备方法和应用 | |
JP2006521093A (ja) | Mhc分子と結合する腫瘍関連ペプチド | |
Tang et al. | A lncRNA Dleu2-encoded peptide relieves autoimmunity by facilitating Smad3-mediated Treg induction |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20220301 |
|
WD01 | Invention patent application deemed withdrawn after publication |