CN117510647A - 携带pd-1单链抗体和ccr6趋化因子受体的car-t免疫细胞的制备及其用途 - Google Patents
携带pd-1单链抗体和ccr6趋化因子受体的car-t免疫细胞的制备及其用途 Download PDFInfo
- Publication number
- CN117510647A CN117510647A CN202310199347.2A CN202310199347A CN117510647A CN 117510647 A CN117510647 A CN 117510647A CN 202310199347 A CN202310199347 A CN 202310199347A CN 117510647 A CN117510647 A CN 117510647A
- Authority
- CN
- China
- Prior art keywords
- seq
- signal peptide
- car
- amino acid
- acid sequence
- 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
- 102100025074 C-C chemokine receptor-like 2 Human genes 0.000 title claims abstract description 29
- 101000716068 Homo sapiens C-C chemokine receptor type 6 Proteins 0.000 title claims abstract description 29
- 210000002865 immune cell Anatomy 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 76
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 63
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims abstract description 51
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 47
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 19
- 238000000338 in vitro Methods 0.000 claims abstract description 16
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 12
- 230000008685 targeting Effects 0.000 claims abstract description 7
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims abstract 7
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims abstract 7
- 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 claims abstract 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 44
- 239000013598 vector Substances 0.000 claims description 30
- 238000003776 cleavage reaction Methods 0.000 claims description 22
- 230000007017 scission Effects 0.000 claims description 22
- 108010002350 Interleukin-2 Proteins 0.000 claims description 14
- 241000713666 Lentivirus Species 0.000 claims description 12
- 230000003834 intracellular effect Effects 0.000 claims description 10
- 230000011664 signaling Effects 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 9
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 8
- 201000005202 lung cancer Diseases 0.000 claims description 8
- 208000020816 lung neoplasm Diseases 0.000 claims description 8
- 238000004806 packaging method and process Methods 0.000 claims description 7
- 230000031146 intracellular signal transduction Effects 0.000 claims description 5
- 230000003248 secreting effect Effects 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 208000015181 infectious disease Diseases 0.000 claims description 4
- -1 EGFR-targeting scFv Proteins 0.000 claims description 3
- 230000000259 anti-tumor effect Effects 0.000 claims description 3
- 230000035755 proliferation Effects 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 206010009944 Colon cancer Diseases 0.000 claims description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 2
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 2
- 206010060862 Prostate cancer Diseases 0.000 claims description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 2
- 208000006265 Renal cell carcinoma Diseases 0.000 claims description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 2
- 239000002246 antineoplastic agent Substances 0.000 claims description 2
- 206010017758 gastric cancer Diseases 0.000 claims description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 2
- 210000000822 natural killer cell Anatomy 0.000 claims description 2
- 201000002528 pancreatic cancer Diseases 0.000 claims description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 2
- 201000011549 stomach cancer Diseases 0.000 claims description 2
- 229940034982 antineoplastic agent Drugs 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 10
- 238000001727 in vivo Methods 0.000 abstract description 9
- 108091008036 Immune checkpoint proteins Proteins 0.000 abstract description 6
- 102000037982 Immune checkpoint proteins Human genes 0.000 abstract description 6
- 230000005012 migration Effects 0.000 abstract description 3
- 238000013508 migration Methods 0.000 abstract description 3
- 230000002035 prolonged effect Effects 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 abstract 1
- 230000004913 activation Effects 0.000 abstract 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 abstract 1
- 108010015889 zeta receptor Proteins 0.000 abstract 1
- 102000001301 EGF receptor Human genes 0.000 description 68
- 108060006698 EGF receptor Proteins 0.000 description 68
- 230000002147 killing effect Effects 0.000 description 18
- 108091026890 Coding region Proteins 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 12
- 102000000588 Interleukin-2 Human genes 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 239000000427 antigen Substances 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 7
- 102100036848 C-C motif chemokine 20 Human genes 0.000 description 6
- 101000713099 Homo sapiens C-C motif chemokine 20 Proteins 0.000 description 6
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 description 6
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 5
- 108090000695 Cytokines Proteins 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 4
- 108060001084 Luciferase Proteins 0.000 description 4
- 239000005089 Luciferase Substances 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 3
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 3
- 230000022534 cell killing Effects 0.000 description 3
- 230000005714 functional activity Effects 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 3
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 2
- 108010012236 Chemokines Proteins 0.000 description 2
- 102000019034 Chemokines Human genes 0.000 description 2
- 108010038061 Chymotrypsinogen Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 206010061598 Immunodeficiency Diseases 0.000 description 2
- 208000029462 Immunodeficiency disease Diseases 0.000 description 2
- 208000031671 Large B-Cell Diffuse Lymphoma Diseases 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 101150058049 car gene Proteins 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 206010012818 diffuse large B-cell lymphoma Diseases 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000008629 immune suppression Effects 0.000 description 2
- 230000007813 immunodeficiency Effects 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 201000005249 lung adenocarcinoma Diseases 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 1
- 102100036850 C-C motif chemokine 23 Human genes 0.000 description 1
- 102100025277 C-X-C motif chemokine 13 Human genes 0.000 description 1
- 238000011357 CAR T-cell therapy Methods 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 102000009410 Chemokine receptor Human genes 0.000 description 1
- 108050000299 Chemokine receptor Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010017472 Fumbling Diseases 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 1
- 101000713081 Homo sapiens C-C motif chemokine 23 Proteins 0.000 description 1
- 101000858064 Homo sapiens C-X-C motif chemokine 13 Proteins 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 1
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 1
- 108010009474 Macrophage Inflammatory Proteins Proteins 0.000 description 1
- 102000009571 Macrophage Inflammatory Proteins Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- SAZUGELZHZOXHB-UHFFFAOYSA-N acecarbromal Chemical compound CCC(Br)(CC)C(=O)NC(=O)NC(C)=O SAZUGELZHZOXHB-UHFFFAOYSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229960003852 atezolizumab Drugs 0.000 description 1
- 229950002916 avelumab Drugs 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 229940121420 cemiplimab Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 239000005482 chemotactic factor Substances 0.000 description 1
- 108700010039 chimeric receptor Proteins 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000018732 detection of tumor cell Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 229950009791 durvalumab Drugs 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005746 immune checkpoint blockade Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 230000006028 immune-suppresssive effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229960005386 ipilimumab Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000004705 lumbosacral region Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 229960003301 nivolumab Drugs 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 229960002621 pembrolizumab Drugs 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001103—Receptors for growth factors
- A61K39/001104—Epidermal growth factor receptors [EGFR]
-
- 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
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Toxicology (AREA)
- Public Health (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Virology (AREA)
- Dermatology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
Abstract
本发明公开了携带PD‑1单链抗体和CCR6趋化因子受体的CAR‑T免疫细胞的制备及其用途,属于基因工程和肿瘤免疫领域,提供了一种能阻断免疫检查点,且更易迁移到肿瘤组织的免疫细胞。具体而言,本发明的T细胞表达靶向肿瘤细胞EGFR的嵌合抗原受体CAR,分泌PD‑1单链抗体,且携带CCR6趋化因子受体,CAR包含CD8α信号肽、靶向EGFR的scFv、CD8α铰链域、CD8α跨膜域、4‑1BB共刺激信号域及CD3ζ受体酪氨酸活化域。本发明的CAR‑T细胞在体内外具有更强的迁移作用,并且在体内外对EGFR阳性肿瘤细胞具有更高的清除率,延长生存期。
Description
技术领域
本发明属于基因工程和肿瘤免疫领域,具体地,本发明涉及一种特异性靶向EGFR抗原,分泌PD-1单链抗体,且携带CCR6趋化因子受体的CAR-T免疫细胞的制备及用途。
背景技术
现在,针对肺癌的治疗方法比较传统,即手术治疗、放射治疗和化学治疗等,对肺癌患者来说效果并不是很好。由于肺癌被发现的时候大多已经是晚期,肺癌患者的五年生存率往往低于10%。最近几年,在科研工作者们的研究下,针对癌症的治疗方法得到了拓宽和创新,对于癌症患者来说,治愈肿瘤有了新的希望,对非小细胞肺癌的治疗方法也在探索中得到不断更新。
CAR-T疗法是一种通过提取患者自身的T细胞,体外修饰扩增后再回输到患者体内的一种免疫疗法。CAR结构包括抗原结合域、铰链区、跨膜区和胞内信号域,通过CAR的修饰,T细胞可以不依赖MHC-1,不需要抗原呈递,直接识别靶细胞表面抗原,再通过胞内的信号传导结构域活化T细胞,达到对靶细胞的杀伤作用。
从1993年第一代CAR诞生至今,CAR经历了四代的发展,第一代嵌合抗原受体(CAR)结构由单链可变片段的抗原识别结构域、跨膜结构域和来自CD3 zeta链的细胞内T细胞激活结构域组成。但是第一代CAR的治疗效果十分不理想,于是科研工作者们在第一代嵌合受体的基础上,研发了第二代CAR,第二代CAR增加了一个共刺激信号分子:CD28的胞内区(或者其它共刺激信号分子如4-1BB、OX40、CD27),使改造后的T细胞也能够像正常的T细胞一样,在双信号转导的帮助下更好地靶向杀伤癌细胞。目前商业CAR-T细胞产品均使用第二代CAR设计。第三代CAR结构包含两个不同的共刺激域,如CD28和4-1BB等,第四代CAR是在第二代CAR的基础上引入了新的信号元件,可以让CAR-T在肿瘤抗原刺激下释放大量的细胞因子,通过这种方法来克服肿瘤微环境的免疫抑制作用等。CAR-T细胞疗法已经极大地改变了淋巴细胞恶性肿瘤的治疗格局,特别是弥漫大B细胞淋巴瘤(DLBCL)和急性淋巴母细胞白血病(ALL)。
但是,CAR-T疗法在实体瘤治疗中仍然效果不理想,主要原因有:实体瘤表面的靶抗原大多在正常组织细胞表面也会有低表达,容易产生脱靶效应;许多实体瘤内部会有不同类型的肿瘤细胞即异质性;实体瘤是由肿瘤细胞组成的致密团块,能阻挡CAR-T细胞的浸润;实体瘤还具有不利于免疫杀伤的肿瘤微环境,易形成免疫抑制、免疫逃逸等。
肿瘤微环境的存在极大地限制着CAR-T对实体瘤的治疗效果,其中,免疫检查点的存在为肿瘤细胞的免疫逃逸提供了条件,如程序性死亡受体1(PD-1)、细胞毒性T淋巴细胞相关蛋白(CTLA-4)等,而免疫检查点的调控极大地促进了肿瘤免疫治疗的研究进展。目前已有多款免疫检查点抑制剂上市,2011年,FDA批准了抗CTLA4的抗体药物ipilimumab。此后,针对PD-1 (nivolumab、pembrolizumab和cemiplimab)和PDL1 (atezolizumab、durvalumab和avelumab)的其他免疫检查点阻断抗体被批准用于广泛的晚期癌症,在临床前及临床试验中均取得了良好的疗效。单免疫检查点单抗容易对机体正常组织产生副作用,而相比之下,单链抗体具有可去除非特异性反应的竞争性表面蛋白,使肿瘤显象背景更加清晰性;分子量更小,容易渗透到肿瘤组织中;免疫源性小,可减轻人抗鼠的排异反应;在体内循环的半衰期短,易于清除;易与毒素或酶基因连接,可直接获得免疫毒素或酶标抗体等优点。
针对实体肿瘤致密性的问题,趋化因子发挥一定的辅助功能。趋化因子是由免疫系统不可或缺的小分子蛋白质组成的,除了可以指引免疫细胞的迁移,它在肿瘤细胞的转移,以及肿瘤的病程发展中也起到十分重要的作用。巨噬细胞炎性蛋白(MIP-3α/CCL20)在各种人体组织和免疫细胞中表达,其受体CCR6主要表达于免疫细胞,如Th17、Treg、CD8+T细胞和B细胞等。据美国NCI发起的肿瘤基因组图谱计划(The Cancer Genome Altas,TCGA)显示,趋化因子受体CCL19、CCL20和CXCL13在肺腺癌组织中高表达,可以发挥起到引导CAR-T细胞的趋化作用。
基于现有技术的现状,本申请的发明人拟提供一种特异靶向EGFR抗原,分泌PD-1单链抗体,且携带CCR6趋化因子受体的CAR-T免疫细胞的制备及用途。
发明内容
近几年,出现了许多嵌合抗原受体结构(CAR)、免疫检查点阻断和趋化因子在肿瘤免疫治疗中的应用的研究,但是人们仍然在探索如何更安全更有效地方法实现肿瘤病情的改善。为克服CAR-T疗法在实体瘤治疗中的局限性,探究CAR-T疗法在非小细胞肺癌治疗中更安全有效的应用,我们以在肺癌组织中高表达的表皮生长因子受体(EGFR)为靶点,针对实体瘤的免疫抑制性肿瘤微环境和密致性分别设计了可以分泌PD-1单链抗体以及表达趋化因子受体CCR6的EGFR-PD-1scFv-CCR6-CAR共表达载体,通过慢病毒感染CD3 T细胞,制备EGFR-PD-1scFv-CCR6-CAR-T细胞。
一方面,本发明提供了一种嵌合抗原受体,包含CD8α信号肽、靶向EGFR的scFv、CD8α铰链域、CD8α跨膜域、4-1BB共刺激域及CD3ζ信号传导域。CAR包含胞外域、铰链域、跨膜域及胞内域。所述的剪切信号肽可以使用T2A剪切信号肽、P2A剪切信号肽或IL-2信号肽。较好的,本发明的嵌合抗原受体包括:CD8α信号肽、靶向EGFR的scFv、CD8α铰链域、CD8α跨膜域、4-1BB共刺激域、CD3ζ信号传导域、T2A剪切信号肽、IL-2信号肽、PD-1单链抗体E27的VH区和VL区、P2A剪切信号肽、CCR6趋化因子受体。
较好的,所述的嵌合抗原受体还包括以下元件中的一个或者若干个:4×G4S、3×G4S、HA片段。
更好的,所述的靶向EGFR的scFv氨基酸序列如SEQ ID NO:16所示;所述的4-1BB胞内共刺激域的氨基酸序列如SEQ ID NO:19所示;所述的CD3ζ胞内信号传导域的氨基酸序列如SEQ ID NO:20所示;所述的PD-1单链抗体VH区氨基酸序列如SEQ ID NO:23所示;所述的PD-1单链抗体VL区氨基酸序列如SEQ ID NO:25所示;所述的CCR6趋化因子受体氨基酸序列如SEQ ID NO:28所示;所述的CD8α信号肽的氨基酸序列如SEQ ID NO:15所示;所述的CD8α铰链域的氨基酸序列如SEQ ID NO:17所示;所述的CD8α跨膜域的氨基酸序列如SEQ ID NO:18所示;所述的T2A剪切信号肽的氨基酸序列如SEQ ID NO:21所示;所述的IL-2信号肽的氨基酸序列如SEQ ID NO:22所示;所述3×G4S的氨基酸序列如SEQ ID NO:24所示;所述HA片段的氨基酸序列如SEQ ID NO:26所示;所述的P2A剪切信号肽的氨基酸序列如SEQ ID NO:27所示。
本发明还包括一种载体,所述的载体含有上述嵌合抗原受体的编码序列。
较好的,所述CD8α信号肽的编码序列如SEQ ID NO:1所示;所述靶向EGFR的scFv编码序列如SEQ ID NO:2所示;所述CD8α铰链域的编码序列如SEQ ID NO:3所示;所述CD8α跨膜域的编码序列如SEQ ID NO:4所示;所述4-1BB胞内共刺激域的编码序列如SEQ ID NO:5所示;所述CD3ζ胞内信号传导域的编码序列如SEQ ID NO:6所示;所述T2A剪切信号肽的编码序列如SEQ ID NO:7所示;所述IL-2信号肽的编码序列如SEQ ID NO:8所示;所述分泌型PD-1単链抗体VH区的编码序列如SEQ ID NO:9所示;所述3×G4S的编码序列如SEQ ID NO:10所示;所述分泌型PD-1単链抗体VL区的编码序列如SEQ ID NO:11所示;所述HA片段的编码序列如SEQ ID NO:12所示;所述P2A剪切信号肽的编码序列如SEQ ID NO:13所示;所述CCR6趋化因子受体的编码序列如SEQ ID NO:14所示。
上述嵌合抗原受体或者其编码载体,可以在市售的CAR质粒基础上改良,或者按照目标序列人工合成。例如,使用本领域的常规技术方法获得其基础序列。在EGFR CAR-E27-CCR6载体构建过程中,人工合成anti-EGFR scFv,然后与CD8α信号肽、CD8α铰链域、CD8α跨膜域、4-1BB共刺激域及CD3ζ胞内域连接,构建EGFR CAR载体。将PD-1単链抗体E27和趋化因子受体CCR6片段人工合成后,进行overlap PCR,通过T2A剪切信号肽和P2A剪切肽与EGFRCAR相连。在载体构建的过程中,我们发现,用不同的信号肽会影响PD-1单链抗体的分泌,我们挑选了人类胰凝乳蛋白酶原信号肽、哺乳动物非经典信号肽、人IL-2信号肽和人IgG2信号肽,分别将这些信号肽加载在PD-1单链抗体E27前,再和EGFR CAR结构连接,转染细胞后发现,人IL-2信号肽能较好的让E27表达分泌,出现在细胞上清液中。
本发明还包括一种细胞,所述的细胞是表达靶向肿瘤细胞表面EGFR的嵌合抗原受体CAR,同时能够分泌PD-1单链抗体,并表达CCR6趋化因子受体的免疫细胞。较好的,所述的细胞表达上述嵌合抗原受体。
所述的免疫细胞可以由慢病毒感染后获得。所述的慢病毒包括:CD8α信号肽、靶向EGFR的scFv、CD8α铰链域、CD8α跨膜域、4-1BB共刺激域、CD3ζ信号传导域、T2A剪切信号肽、IL-2信号肽、PD-1单链抗体E27的VH区和VL区、P2A剪切信号肽、CCR6趋化因子受体、4×G4S和HA片段。
在制备EGFR CAR-E27-CCR6-T细胞过程中,本发明选取慢病毒载体作为CAR基因整合的载体,最先使用三质粒的常见包装系统:混合VSV-G和dR8.91病毒包装质粒和CAR载体,共转染293FT-17细胞包被慢病毒,但生产出的慢病毒存在滴度较低的情况,出于意料的无法感染T细胞整合基因组。根据长期的摸索,换用了新的三质粒包装系统,将构建好的CAR载体与pSPAX2、PMD2G包装慢病毒,然后感染293FT-17细胞,得到了滴度相对稳定的慢病毒。
较好的,所述免疫细胞为T淋巴细胞或者NK细胞。
较好的,所述的靶向EGFR的scFv氨基酸序列如SEQ ID NO:16所示;所述的4-1BB胞内共刺激域的氨基酸序列如SEQ ID NO:19所示;所述的CD3ζ胞内信号传导域的氨基酸序列如SEQ ID NO:20所示;所述的PD-1单链抗体VH区氨基酸序列如SEQ ID NO:23所示;所述的PD-1单链抗体VL区氨基酸序列如SEQ ID NO:25所示;所述的CCR6趋化因子受体氨基酸序列如SEQ ID NO:28所示;所述的CD8α信号肽的氨基酸序列如SEQ ID NO:15所示;所述的CD8α铰链域的氨基酸序列如SEQ ID NO:17所示;所述的CD8α跨膜域的氨基酸序列如SEQ ID NO:18所示;所述的T2A剪切信号肽的氨基酸序列如SEQ ID NO:21所示;所述的IL-2信号肽的氨基酸序列如SEQ ID NO:22所示;所述3×G4S的氨基酸序列如SEQ ID NO:24所示;所述HA片段的氨基酸序列如SEQ ID NO:26所示;所述的P2A剪切信号肽的氨基酸序列如SEQ ID NO:27所示。
另一方面,本发明还提供了上述嵌合抗原受体和免疫细胞的应用,即上述嵌合抗原受体或者免疫细胞在制备治疗药物中的应用。
较好的,所述的抗肿瘤药物是抑制肿瘤细胞增殖或者清除肿瘤细胞的药物或者试剂。
在本发明的一个优选实施例中,所述的肿瘤源自非小细胞肺癌。
在一个优选实施例中,所述免疫细胞由表达嵌合抗原受体,PD-1单链抗体和CCR6趋化因子受体的慢病毒载体感染后获得。
在一个优选实施例中,所述的嵌合抗原受体表达序列,PD-1单链抗体表达序列和CCR6趋化因子受体表达序列均整合入所述免疫细胞的基因组中。
在一个优选实施例中,所述肿瘤细胞为EGFR阳性的NSCLC细胞系,本发明所述的免疫细胞使用范围不仅限于此,优选地,所述癌症包含EGFR阳性的肺癌、卵巢癌、胃癌、前列腺癌、肾细胞癌、胰腺癌、结直肠癌等。在一个优选实施例中,所用的是人肺腺癌细胞,具体为A549细胞。
本发明的EGFR CAR-E27-CCR6-T细胞在体外对靶细胞有显著的杀伤效果。在体外将Mock T、EGFR CAR-T、EGFR CAR-E27-T和EGFR CAR-E27-CCR6-T细胞与A549-PDL1-Luc-CCL20细胞的比例不低于5:1,细胞数量比。更好的,效靶比(E:T ratio)为不低于10:1,对细胞杀伤效率存在显著差异性。综合考虑效果、成本和操作的安全和简易程度,效靶比(E:Tratio)可以采用(10-30):1甚至(10-20):1。
本发明提供了能够特异性识别肿瘤细胞的嵌合抗原受体,进而构建了能阻断免疫检查点,且更易迁移到肿瘤组织的免疫细胞。该CAR-T细胞在体内外具有更强的迁移作用,并且在体内外对EGFR阳性肿瘤细胞具有更高的清除率,延长生存期。
附图说明
为了更清楚地说明本申请的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的每一幅附图针对本申请的部分实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1:EGFR CAR-E27-CCR6载体元件示意图及酶切鉴定。
图2:EGFR CAR-E27-CCR6在原代CD3 T细胞的表达效率及PD-1単链抗体、CCR6的检测。
图3:E27在原代CD3 T细胞体外功能活性的检测。
图4:肿瘤细胞靶抗原EGFR的检测。
图5:表达PDL1、CCL20及荧光素酶慢病毒载体的构建及酶切验证。
图6:稳转PDL1、CCL20及荧光素酶肿瘤细胞系的鉴定。
图7:CCR6在原代CD3 T细胞体外功能活性的检测。
图8:EGFR CAR-E27-CCR6-T细胞体外对肿瘤细胞的杀伤作用。
图9:EGFR CAR-E27-CCR6-T细胞促进IL-2、TNF-α、INF-γ细胞因子的分泌。
图10:在NSG小鼠肺癌CDX移植瘤模型中,EGFR CAR-E27-CCR6-T细胞具有更强的抗肿瘤功能。
具体实施方式
下面将通过本申请的实施例对技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分优选实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动所获得的所有其他实施例,都属于本申请的保护范围。
实施例1.构建EGFR CAR-E27-CCR6载体
人工合成anti-EGFR scFv,然后与CD8α信号肽、CD8α铰链域、CD8α跨膜域、4-1BB共刺激域及CD3ζ胞内域连接,构建EGFR CAR载体。将PD-1単链抗体E27和趋化因子受体CCR6片段人工合成后,进行overlap PCR,通过T2A剪切信号肽和P2A剪切肽与EGFR CAR相连。在载体构建的过程中,我们发现,用不同的信号肽会影响PD-1单链抗体的分泌,我们挑选了人类胰凝乳蛋白酶原信号肽(DNA序列为ATGGCTTTCCTCTGGCTCCTCTCCTGCTGGGCCCTCCTGGGTACCACCTTCGGT,氨基酸序列为MAFLWLLSCWALLGTTFG)、哺乳动物非经典信号肽(DNA序列为GAATTCATGGCACAGGAGCTCAGCCTGAACGAGTCTCAG,氨基酸序列为EFAQELSLNESQ)、人IL-2信号肽(DNA序列为ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAAC AGT,氨基酸序列为MYRMQLLSCIALSLALVTNS)和人IgG2信号肽(DNA序列为ATGGGCTGGACCTGCAAGATCCTCTTCTTGGTGGCAGCAGCCACAGGT,氨基酸序列为MGWSCIILFLVATATGVHS),分别将这些信号肽加载在PD-1单链抗体E27前,再和EGFR CAR结构连接,转染细胞后发现,人IL-2信号肽才能让E27表达分泌,出现在细胞上清液中。因此,我们使用IL-2信号肽作为最终复合CAR载体的一部分。载体构建后,EGFR CAR载体用Kpn I和Not I双酶切后得到4449bp,2444bp和1515bp三条片段;EGFR CAR-E27载体用Xba I和Sal I双酶切后得到6899bp、2409bp两条片段;EGFR CAR-E27-CCR6载体用EcoR V和EcoR I双酶切后得到983bp、2232bp、8063bp三条片段,均酶切验证正确,与预期符合。进一步通过sanger测序确认所有载体序列正确无误(图1)。
实施例2.制备EGFR CAR-E27-CCR6-T细胞,检测CAR表达效率
我们选取慢病毒载体作为CAR基因整合的载体,最先使用常见三质粒包装系统:按照4:3:5的摩尔比混合VSV-G和dR8.91病毒包装质粒和CAR载体,共转染293FT-17细胞包被慢病毒,但生产出的慢病毒存在滴度较低的情况,没办法感染T细胞整合基因组,根据长期的摸索,我们换用了新的三质粒包装系统,将构建好的CAR载体与pSPAX2、PMD2G以3:2:1的摩尔比包装慢病毒,然后感染293FT-17细胞,得到了滴度相对稳定的慢病毒,通过定量PCR检测病毒滴度在1.16×106TU/mL左右。利用阴选的方法从PBMC分离人CD3 T细胞,加入CD3/CD28激活剂进行活化,48h后,病毒感染活化后的CD3 T细胞,如图2所示,CAR的表达效率在20-70%(图2A),Western blot表明PD-1単链抗体能够分泌到细胞上清中(图2C),同时,流式检测细胞表面CCR6也能有效表达(图2B)。
实施例3.E27在原代CD3 T细胞功能活性的检测
E27修饰EGFR CAR-T细胞后,为了验证其在体外的功能活性,利用CCK8实验在不同的时相点检测CAR-T细胞增殖能力,在没有外源PDL1刺激下,与Mock T和EGFR CAR-T细胞相比,EGFR CAR-E27-T、EGFR CAR-E27-CCR6-T增殖活性存在显著差异,如图3A所示。为了验证T细胞分泌的E27是否会有效结合自身的PD-1,我们利用流式检测了细胞表面PD-1的表达,结果表明,与Mock T细胞相比,EGFR CAR-E27-T和EGFR CAR-E27-CCR6-T细胞组PD-1的表达效率降低至0(图3B)。
实施例4.制备靶细胞
我们选择了A549作为靶细胞。首先,通过流式细胞检测,A549细胞系表达EGFR抗原,如图4所示。进一步,为了更好地检测PD-1単链抗体和CCR6的作用,我们构建了PDL1和荧光素酶共表达、CCL20的慢病毒载体,荧光素酶是为了方便小鼠活体成像(图5)。利用嘌呤霉素和潮霉素B筛选获得稳转株后,流式细胞检测靶细胞表面PDL1表达在90%左右,同时,利用ELISA检测能检测到CCL20的表达,利用荧光素酶底物也检测到Luc表达,表明肿瘤细胞系制备成功(图6)。
实施例5.CCR6对EGFR CAR-T细胞迁移的影响
首先铺板A549-PDL1-Luc-CCL20细胞系,6孔板中培养24h,吸取分泌有CCL20的上清,离心去杂质后吸取600μL置于transwell的下层小室,上层小室计数105/100μL Mock T、EGFR CAR-T、EGFR CAR-E27-T和EGFR CAR-E27-CCR6-T细胞,培养24h后,通过CCK-8统计穿梭到下层小室的细胞数。结果表明,与其他三组相比,修饰CCR6后,促进了CAR-T细胞向下层小室的迁移,存在显著差异(图7)。
实施例6.EGFR CAR-E27-CCR6-T细胞在体外对靶细胞的杀伤检测
在体外将Mock T、EGFR CAR-T、EGFR CAR-E27-T和EGFR CAR-E27-CCR6-T细胞与A549-PDL1-Luc-CCL20细胞按照5:1,10:1,20:1的效靶比(E:T ratio)分别共孵育6h后,利用荧光素酶检测其杀伤效率,如图8所示,在5:1的浓度比下,6小时共孵育后,Mock T的杀伤效率为22.2%,EGFR CAR-T的杀伤效率为62.4%,EGFR CAR-E27-T的杀伤效率为48.3%,EGFR CAR-E27-CCR6-T的杀伤效率为59.4%;在10:1的浓度比下,6小时共孵育后,Mock T的杀伤效率为37.8%,EGFR CAR-T的杀伤效率为74.4%,EGFR CAR-E27-T的杀伤效率为75.6%,EGFR CAR-E27-CCR6-T的杀伤效率为84.5%;在20:1的浓度比下,6小时共孵育后,Mock T的杀伤效率为42.0%,EGFR CAR-T的杀伤效率为74.9%,EGFR CAR-E27-T的杀伤效率为90.5%,EGFR-E27-CCR6-CAR-T的杀伤效率为87.7%。与Mock T细胞相比,在20:1的情况下,EGFR-CAR-T、EGFR-E27-CAR-T和EGFR CAR-E27-CCR6-T细胞杀伤效率存在显著差异性。
实例7.EGFR CAR-E27-CCR6-T细胞体外细胞因子的释放
将效应细胞和靶细胞共孵育6h后,收集培养基上清,检测细胞因子的表达。CAR-T组IL-2的分泌量最高可达约800pg/ml,IFN-的分泌量最高可达约300pg/ml,TNF-α的分泌量最高可达约1000pg/ml,且各组CAR-T细胞在细胞杀伤过程中分泌的炎性细胞因子水平均和Mock T组之间存在显著差异(图9)。
实施例8.EGFR CAR-E27-CCR6-T细胞体内抗肿瘤作用检测
用免疫缺陷的NOD.PrkdcscidIl2rgem1/Smoc(NSG)小鼠作为体内实验的动物模型。培养靶细胞A549-PDL1-Luc-CCL20,按照3×106个细胞/只小鼠的数量在小鼠后侧腰处皮下种植肿瘤靶细胞。仅观测肿瘤的预实验中我们发现,一些对照组(未种瘤)小鼠也存在不明原因死亡的情况,查阅文献后发现,免疫缺陷的NSG小鼠作为NOD小鼠的变体,可能存在糖尿病的问题,因此,我们在后续实验中换用了低糖鼠粮,一定程度上延长了小鼠的寿命。种瘤后,每7天观测小鼠体内荧光值、小鼠体重和小鼠健康状况,待肿瘤细胞移植成功后随机分为Mock T、EGFR CAR-T、EGFR CAR-E27-T和EGFRCAR-E27-CCR6-T四组,第7、10、13天,分别尾静脉注射5×106效应细胞,每周一次活体成像,监测荧光强度变化。如图10所示,从第二周开始(D13成像),各组NSG小鼠肿瘤荧光值开始有了不同的走向。Mock T组荧光强度不断增加,其他3组的荧光强度不同程度下降。至D32的时候,Mock T组小鼠肿瘤荧光已经全部过曝,而其他3组均有出现肿瘤荧光完全消减的个体,EGFR CAR-T组肿瘤消减率达40%,EGFRCAR-E27-T组肿瘤消减率为80%,而EGFR CAR-E27-CCR6-T组已经几乎全部清除了肿瘤。除了小鼠活体成像的结果,对剥离肿瘤组织进行拍照、称重,更为直观地呈现了不同处理组肿瘤的生长情况,从结果来看,EGFR CAR-T、EGFR CAR-E27-T和EGFR CAR-E27-CCR6-T细胞在体内均能有效地清除肿瘤细胞,EGFR CAR-E27-CCR6-T细胞具有更高的清除率。
本发明涉及的序列如下:
SEQ ID NO:1:CD8αleader
DNA,63
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG
SEQ ID NO:2:EGFR scFv
DNA,747
GAGGTCCAGCTCGTGCAGTCCGGAGCCGAAGTGAAGAAGCCCGGCAGCAGCGTGAAAGTGAGCTGTAAGGCCAGCGGCGGCACATTCTCCAGCTACGCCATTGGATGGGTGAGACAAGCCCCCGGCCAAGGACTGGAATGGATGGGCGGCATCATCCCCATCTTTGGCATCGCCAACTACGCTCAGAAGTTCCAAGGCAGAGTGACCATCACCGCCGACGAGAGCACCAGCTCCGCTTACATGGAGCTCTCCTCTCTGAGGTCCGAAGACACCGCCGTGTACTATTGCGCCAGAGAGGAGGGCCCTTACTGTAGCAGCACCAGCTGTTACGCCGCCTTCGATATTTGGGGCCAAGGCACACTGGTGACAGTGTCCTCCGGCGGCGGCGGATCCGGAGGCGGAGGAAGCGGAGGAGGAGGCTCCCAGAGCGTGCTGACCCAAGACCCCGCCGCTTCCGTGGCTCTGGGCCAAACCGTGAAAATCACATGCCAAGGCGATTCTCTGAGGAGCTACTTCGCTAGCTGGTACCAGCAGAAACCCGGCCAAGCCCCCACACTGGTGATGTACGCTAGAAACGATAGACCCGCCGGCGTGCCCGATAGATTCAGCGGCAGCAAGTCCGGCACATCCGCTTCTCTGGCTATTAGCGGACTGCAGCCCGAGGATGAGGCTGACTACTACTGTGCCGCTTGGGACGATTCTCTGAACGGCTATCTGTTTGGAGCCGGCACAAAGCTGACCGTGCTG
SEQ ID NO:3:CD8α铰链域
DNA,135
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
SEQ ID NO:4:CD8α跨膜域
DNA,72
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC
SEQ ID NO:5:4-1BB共刺激域
DNA,126
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG
SEQ ID NO:6:CD3ζ信号传导域
DNA,336
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
SEQ ID NO:7:T2A剪切信号肽
DNA,66
GGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGG
SEQ ID NO:8:IL-2信号肽
人工序列(artificial sequence)
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT
SEQ ID NO:9:PD-1单链抗体VH
DNA,351
GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCAACTACATCTCTATGTTCGATTCTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEQ ID NO:10:4×G4S
DNA,
GGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCGGCGGCGGCGGCTCT
SEQ ID NO:11:PD-1单链抗体VL
DNA,人工序列(artificial sequence)
CAGTCTGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAGGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATTATGTCTTCGGAATTGGGACCAAGGTCACCGTCCTAGGT
SEQ ID NO:12:HA片段
DNA,27
TACCCGTACGACGTTCCGGACTACGCT
SEQ ID NO:13:P2A剪切信号肽
DNA,57
GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT
SEQ ID NO:14:CCR6趋化因子受体
DNA,1125
ATGAGCGGGGAATCAATGAATTTCAGCGATGTTTTCGACTCCAGTGAAGATTATTTTGTGTCAGTCAATACTTCATATTACTCAGTTGATTCTGAGATGTTACTGTGCTCCTTGCAGGAGGTCAGGCAGTTCTCCAGGCTATTTGTACCGATTGCCTACTCCTTGATCTGTGTCTTTGGCCTCCTGGGGAATATTCTGGTGGTGATCACCTTTGCTTTTTATAAGAAGGCCAGGTCTATGACAGACGTCTATCTCTTGAACATGGCCATTGCAGACATCCTCTTTGTTCTTACTCTCCCATTCTGGGCAGTGAGTCATGCCACCGGTGCGTGGGTTTTCAGCAATGCCACGTGCAAGTTGCTAAAAGGCATCTATGCCATCAACTTTAACTGCGGGATGCTGCTCCTGACTTGCATTAGCATGGACCGGTACATCGCCATTGTACAGGCGACTAAGTCATTCCGGCTCCGATCCAGAACACTACCGCGCAGCAAAATCATCTGCCTTGTTGTGTGGGGGCTGTCAGTCATCATCTCCAGCTCAACTTTTGTCTTCAACCAAAAATACAACACCCAAGGCAGCGATGTCTGTGAACCCAAGTACCAGACTGTCTCGGAGCCCATCAGGTGGAAGCTGCTGATGTTGGGGCTTGAGCTACTCTTTGGTTTCTTTATCCCTTTGATGTTCATGATATTTTGTTACACGTTCATTGTCAAAACCTTGGTGCAAGCTCAGAATTCTAAAAGGCACAAAGCCATCCGTGTAATCATAGCTGTGGTGCTTGTGTTTCTGGCTTGTCAGATTCCTCATAACATGGTCCTGCTTGTGACGGCTGCAAATTTGGGTAAAATGAACCGATCCTGCCAGAGCGAAAAGCTAATTGGCTATACGAAAACTGTCACAGAAGTCCTGGCTTTCCTGCACTGCTGCCTGAACCCTGTGCTCTACGCTTTTATTGGGCAGAAGTTCAGAAACTACTTTCTGAAGATCTTGAAGGACCTGTGGTGTGTGAGAAGGAAGTACAAGTCCTCAGGCTTCTCCTGTGCCGGGAGGTACTCAGAAAACATTTCTCGGCAGACCAGTGAGACCGCAGATAACGACAATGCGTCGTCCTTCACTATGTGA
SEQ ID NO:15:CD8αleader
PRT,21
MALPVTALLLPLALLLHAARP
SEQ ID NO:16:EGFR scFv
PRT,249
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIGWVRQAPGQGLEWMGGIIPIFGIANYAQKFQGRVTITADESTSSAYMELSSLRSEDTAVYYCAREEGPYCSSTSCYAAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQDPAASVALGQTVKITCQGDSLRSYFASWYQQKPGQAPTLVMYARNDRPAGVPDRFSGSKSGTSASLAISGLQPEDEADYYCAAWDDSLNGYLFGAGTKLTVL
SEQ ID NO:17:CD8α铰链域
PRT,45
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
SEQ ID NO:18:CD8α跨膜域
PRT,24
IYIWAPLAGTCGVLLLSLVITLYC
SEQ ID NO:19:4-1BB共刺激域
PRT,42
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
SEQ ID NO:20:CD3ζ信号传导域
PRT,112
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO:21:T2A剪切信号肽
PRT,22
GSGEGRGSLLTCGDVEENPGPR
SEQ ID NO:22:IL-2信号肽
PRT,20
MYRMQLLSCIALSLALVTNS
SEQ ID NO:23:PD-1単链抗体VH
PRT,117
EVQLVESGGGLIQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNYISMFDSWGQGTLVTVSS
SEQ ID NO:24:4×G4S
PRT,15
GGGGSGGGGSGGGGSGGGGS
SEQ ID NO:25:PD-1単链抗体VL
PRT,108
QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQRPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVFGIGTKVTVLG
SEQ ID NO:26:HA片段
PRT,9
YPYDVPDYA
SEQ ID NO:27:P2A剪切信号肽
PRT,19
ATNFSLLKQAGDVEENPGP
SEQ ID NO:28:CCR6趋化因子受体
PRT,374
MSGESMNFSDVFDSSEDYFVSVNTSYYSVDSEMLLCSLQEVRQFSRLFVPIAYSLICVFGLLGNILVVITFAFYKKARSMTDVYLLNMAIADILFVLTLPFWAVSHATGAWVFSNATCKLLKGIYAINFNCGMLLLTCISMDRYIAIVQATKSFRLRSRTLPRSKIICLVVWGLSVIISSSTFVFNQKYNTQGSDVCEPKYQTVSEPIRWKLLMLGLELLFGFFIPLMFMIFCYTFIVKTLVQAQNSKRHKAIRVIIAVVLVFLACQIPHNMVLLVTAANLGKMNRSCQSEKLIGYTKTVTEVLAFLHCCLNPVLYAFIGQKFRNYFLKILKDLWCVRRKYKSSGFSCAGRYSENISRQTSETADNDNASSFTM*
以上所述的实施例仅为本申请的具体实施方式,但本申请的保护范围并不局限于此,任何熟悉本领域技术的技术人员在本申请公开的技术范围内,可以不通过创造性劳动即能够联想到的变化或替换,都应涵盖在本申请的保护范围之内。因此,本申请的保护范围应以本申请中权利要求的保护范围为准。
Claims (10)
1.一种嵌合抗原受体,其特征在于,所述的嵌合抗原受体包括:CD8α信号肽、靶向EGFR的scFv、CD8α铰链域、CD8α跨膜域、4-1BB共刺激域、CD3ζ信号传导域、PD-1单链抗体E27的VH区和VL区、CCR6趋化因子受体和剪切信号肽。
2.如权利要求1所述的嵌合抗原受体,其特征在于,所述的嵌合抗原受体还包括以下元件中的一个或者若干个:3个或者4个G4S、HA片段;或者
所述的剪切信号肽是T2A剪切信号肽、P2A剪切信号肽和/或IL-2信号肽。
3.如权利要求2所述的嵌合抗原受体,其特征在于,所述的靶向EGFR的scFv氨基酸序列如SEQ ID NO:16所示;所述的4-1BB胞内共刺激域的氨基酸序列如SEQ ID NO:19所示;所述的CD3ζ胞内信号传导域的氨基酸序列如SEQ ID NO:20所示;所述的PD-1单链抗体VH区氨基酸序列如SEQ ID NO:23所示;所述的PD-1单链抗体VL区氨基酸序列如SEQ ID NO:25所示;所述的CCR6趋化因子受体氨基酸序列如SEQ ID NO:28所示;所述的CD8α信号肽的氨基酸序列如SEQ ID NO:15所示;所述的CD8α铰链域的氨基酸序列如SEQ ID NO:17所示;所述的CD8α跨膜域的氨基酸序列如SEQ ID NO:18所示;所述的T2A剪切信号肽的氨基酸序列如SEQ IDNO:21所示;所述的IL-2信号肽的氨基酸序列如SEQ ID NO:22所示;或者,所述的P2A剪切信号肽的氨基酸序列如SEQ ID NO:27所示。
4.一种载体系统,其特征在于,所述的载体系统含有权利要求1所述的嵌合抗原受体的基因序列。
5.一种细胞,其特征在于,所述的细胞是表达靶向肿瘤细胞EGFR的CAR,同时能够分泌PD-1单链抗体,并表达CCR6趋化因子受体的免疫细胞;或者,所述的细胞表达权利要求1所述的嵌合抗原受体。
6.如权利要求5所述的细胞,其特征在于,所述的免疫细胞是体外培养的T淋巴细胞或者NK细胞,由慢病毒感染后获得,
所述的慢病毒包括:CD8α信号肽、靶向EGFR的scFv、CD8α铰链域、CD8α跨膜域、4-1BB共刺激域、CD3ζ信号传导域、T2A剪切信号肽、IL-2信号肽、PD-1单链抗体E27的VH区和VL区、P2A剪切信号肽、CCR6趋化因子受体、4×G4S和HA片段。
7.权利要求1-4中任意一种嵌合抗原受体的应用,其特征在于,所述的应用是权利要求1-4中任意一种嵌合抗原受体在制备抗肿瘤药剂中的应用。
8.如权利要求7所述的应用,其特征在于,所述的抗肿瘤药物是抑制肿瘤细胞增殖或者清除肿瘤细胞的药物或者试剂。
9.如权利要求7所述的应用,其特征在于,使用表达权利要求1所述的嵌合抗原受体的CAT载体、pSPAX2载体、PMD2G载体包装慢病毒,通过体外感染的方式构建制备EGFR CAR-E27-CCR6-T细胞。
10.如权利要求7所述的应用,其特征在于,所述肿瘤源自肺癌、卵巢癌、胃癌、前列腺癌、肾细胞癌、胰腺癌、结直肠癌。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310199347.2A CN117510647A (zh) | 2023-03-03 | 2023-03-03 | 携带pd-1单链抗体和ccr6趋化因子受体的car-t免疫细胞的制备及其用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310199347.2A CN117510647A (zh) | 2023-03-03 | 2023-03-03 | 携带pd-1单链抗体和ccr6趋化因子受体的car-t免疫细胞的制备及其用途 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117510647A true CN117510647A (zh) | 2024-02-06 |
Family
ID=89744485
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310199347.2A Pending CN117510647A (zh) | 2023-03-03 | 2023-03-03 | 携带pd-1单链抗体和ccr6趋化因子受体的car-t免疫细胞的制备及其用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117510647A (zh) |
-
2023
- 2023-03-03 CN CN202310199347.2A patent/CN117510647A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3674328B1 (en) | Chimeric antigen receptor (car) binding to bcma, and uses thereof | |
EP3313874B1 (en) | Masking chimeric antigen receptor t cells for tumor-specific activation | |
CN110818802B (zh) | 一种嵌合t细胞受体star及其应用 | |
CN109862912A (zh) | 携带双特异性T细胞衔接器(BiTE)的腺病毒 | |
CN110872577B (zh) | 修饰的免疫细胞及其应用 | |
CN108064252A (zh) | 嵌合抗原受体及其使用方法 | |
CN109562126A (zh) | 嵌合抗原受体(car)、组合物及其使用方法 | |
CN114774444A (zh) | 通用嵌合抗原受体表达性免疫细胞及其制造方法和治疗用途 | |
TW201619377A (zh) | Car表現載體及car表現t細胞 | |
JP2015513394A (ja) | 二重特異性キメラ抗原受容体およびその治療的使用 | |
CN111944054B (zh) | 抗bcma的car及其表达载体和应用 | |
WO2022199125A1 (zh) | 一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞 | |
TWI811278B (zh) | 表現特異性辨識人類間皮素之細胞表面分子、il-7、及ccl19之免疫活性細胞 | |
CN108064251A (zh) | 碳酸酐酶ix特异性嵌合抗原受体及其使用方法 | |
CN108085340A (zh) | 一种同时表达靶向cd19和cd20的car与pd1-cd28嵌合受体的慢病毒载体 | |
CN113913379A (zh) | T淋巴细胞及其应用 | |
Lo et al. | Regression of established renal cell carcinoma in nude mice using lentivirus-transduced human T cells expressing a human anti-CAIX chimeric antigen receptor | |
CN115003700A (zh) | 促实体瘤浸润的增强型cart细胞及其制备方法和细胞药物 | |
CN110511912A (zh) | 免疫细胞的功能调节 | |
CN111944053B (zh) | 抗bcma的car及其表达载体和应用 | |
CN110194803B (zh) | 一种靶向EpCAM的嵌合抗原受体及其应用 | |
EP4190820A1 (en) | Chimeric antigen receptor and use thereof | |
CN117510647A (zh) | 携带pd-1单链抗体和ccr6趋化因子受体的car-t免疫细胞的制备及其用途 | |
CN113493520A (zh) | 靶向双特异性位点的双car及其应用 | |
CN112048021A (zh) | 一种靶向ror2的嵌合抗原受体、表达基因、表达载体、t细胞及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication |