CN108484775A - 靶向pd-l1的t和nkt混合细胞、制备方法和应用 - Google Patents
靶向pd-l1的t和nkt混合细胞、制备方法和应用 Download PDFInfo
- Publication number
- CN108484775A CN108484775A CN201810203095.5A CN201810203095A CN108484775A CN 108484775 A CN108484775 A CN 108484775A CN 201810203095 A CN201810203095 A CN 201810203095A CN 108484775 A CN108484775 A CN 108484775A
- Authority
- CN
- China
- Prior art keywords
- cell
- pdl1
- culture
- nkt
- expression
- 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
- 238000002156 mixing Methods 0.000 title claims abstract description 55
- 210000000581 natural killer T-cell Anatomy 0.000 title claims abstract description 44
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 36
- 108010074708 B7-H1 Antigen Proteins 0.000 title abstract description 34
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 102000008096 B7-H1 Antigen Human genes 0.000 title abstract 4
- 210000004027 cell Anatomy 0.000 claims abstract description 93
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 claims abstract description 62
- 230000014509 gene expression Effects 0.000 claims abstract description 35
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 claims abstract description 30
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims abstract description 23
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 24
- 239000001963 growth medium Substances 0.000 claims description 11
- 208000015181 infectious disease Diseases 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 6
- 241000124008 Mammalia Species 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 108020003175 receptors Proteins 0.000 claims description 3
- 102000005962 receptors Human genes 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 241000700605 Viruses Species 0.000 abstract description 13
- 230000008685 targeting Effects 0.000 abstract description 12
- 230000002147 killing effect Effects 0.000 abstract description 11
- 238000002474 experimental method Methods 0.000 abstract description 5
- 230000022534 cell killing Effects 0.000 abstract description 3
- 239000002245 particle Substances 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 25
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 10
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 10
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 10
- 230000006907 apoptotic process Effects 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 102100037850 Interferon gamma Human genes 0.000 description 7
- 108010074328 Interferon-gamma Proteins 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 7
- 238000013467 fragmentation Methods 0.000 description 6
- 238000006062 fragmentation reaction Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000034994 death Effects 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 108090000672 Annexin A5 Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 102000004121 Annexin A5 Human genes 0.000 description 3
- 206010041047 Slow virus infection Diseases 0.000 description 3
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 230000001665 lethal effect Effects 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 2
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 2
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 101710094000 Programmed cell death 1 ligand 1 Proteins 0.000 description 2
- 108700005078 Synthetic Genes Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 230000000139 costimulatory effect Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 1
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 1
- 102100034283 Annexin A5 Human genes 0.000 description 1
- 101150091609 CD274 gene Proteins 0.000 description 1
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 1
- 101100407305 Homo sapiens CD274 gene Proteins 0.000 description 1
- 101000603882 Homo sapiens Nuclear receptor subfamily 1 group I member 3 Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000011577 humanized mouse model Methods 0.000 description 1
- 230000005746 immune checkpoint blockade Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- 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
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4613—Natural-killer cells [NK or NK-T]
-
- 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
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4631—Chimeric Antigen Receptors [CAR]
-
- 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
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
- A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
-
- 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
- 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
- C07K16/2827—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 B7 molecules, e.g. CD80, CD86
-
- 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
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
-
- 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
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- 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
- C07K2319/00—Fusion polypeptide
-
- 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
- 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
Abstract
本发明涉及靶向PD‑L1的T和NKT混合细胞、制备方法和应用。本发明合成了嵌合抗原受体anti‑PDL1‑CD8‑41BB‑CD3ξ,其序列如SEQ ID NO.1所示,将其病毒质粒感染PMBC细胞,培养得到表达anti‑PDL1‑CD8‑41BB‑CD3ξ的T和NKT混合细胞,实验表明,表达anti‑PD‑L1‑CD8‑41BB‑CD3ξ的T+NKT混合细胞对PDL1+细胞具有较强的杀伤能力,且比单独的表达anti‑PD‑L1‑CD8‑41BB‑CD3ξ的T细胞对PDL1+细胞杀伤能力要强。
Description
技术领域
本发明涉及生物医药领域,特别是涉及靶向PD-L1的T和NKT混合细胞、制备方法和应用。
背景技术
程序性细胞死亡-配体1(programmed cell death protein-ligant-1),也称为表面抗原分化簇274(cluster of differentiation 274,CD274)或B7同源体(B7homolog 1,B7-H1),是人类体内的一种蛋白质,由CD274基因编码。PD-L1是一种分子量为40kD的一型跨膜蛋白,与T细胞表面的程序性细胞死亡蛋白PD-1(programmed cell death protein-1)或B7-H1 结合,传递抑制信号,减少T细胞增殖,诱发凋亡,从而抑制T细胞的功能。很多肿瘤细胞通过高表达PD-L1,使得T细胞在遇到肿瘤细胞的时候被抑制,造成免疫耐受的结果,无法有效识别肿瘤细胞及在肿瘤微环境内有效累积是造成免疫系统失灵的重要原因,因此围绕肿瘤微环境的组成成分进行研究,对PD-1/PD-L1抑制性通路有效封闭,对于临床实践及药物应用都有重要的意义。
嵌合抗原受体(Chimeric Antigen Receptor,简称CAR)是由胞外的抗原结合区、跨膜区以及胞内信号区组成的融合蛋白。第一代CAR的胞内区主要是CD3ξ,第二代CAR的胞内区由一个共刺激分子和CD3ξ组成,第三代CAR的胞内区包含多个共刺激分子和CD3ξ。嵌合抗原受体T细胞(Chimeric Antigen Receptor T-Cell,简称CAR-T)和嵌合抗原受体NKT 细胞(Chimeric Antigen Receptor Natural Killer T-Cell,简称CAR-NKT)是将CAR表达在T细胞或NK细胞上。一旦表达了CAR,CAR-T细胞或CAR-NK细胞通过单链抗体识别肿瘤上的抗原而被激活,最终使肿瘤细胞裂解死亡。
目前针对PD-L1(或PD-1)及其抗体在细胞表达的应用主要有三种:
1.在CAR-T细胞上表达分泌型anti-PD-L1抗体:分泌性抗体可以封闭PD-L1位点(Suarea ER, Chan De-K,Sun J,Sui J,Freeman GJ,Signoretti S,Zhu Q,MarascoWA.Chimeric antigen receptor T cells secreting anti-PD-La antibodies moreeffectively regress renal cell carcinoma in a humanized mousemodel.Oncotarget,2016,7(23):34341-55);
2.在CAR-T细胞上表达PD-1显性抑制受体:PD-1的胞外区可以与PD-L1结合,但又不传递抑制性信号(Cherkassky L,Morello A,Villena-Vargas J,Feng Y,Dimitrov DS,Jones DR, Sadelain M,Adusumilli PS.Human CAR T cells with cell-intrinsic PD-1checkpoint blockade resist tumor-mediated inhibition.J Clin Invest,2016,126(8): 3130-44);
3.信号转换器:T细胞表达PD1-CD28,PD-1与PD-L1结合后,激活CD28,将抑制性信号转换为刺激性信号(Kobold S,Grassmann S,Chaloupka M,Lampert C,Wenk S,Kraus F,Rapp M,Duwell P,Zeng Y,Schmollinger JC,Schnurr M,Endres S,Simon R.Impact ofNew Fusion Receptor on PD-1-Mediated Immunosupppression in Adoptive T cellTherapy. Journal of the National Cancer Institute,2015,107(8))。
与已有的抗体封闭和单一的CAR-T/CAR-NK技术相比,PD-L1靶向性T细胞或NKT细胞,在对PD-1/PD-L1免疫检查进行封闭保护T细胞或NKT细胞的同时,可以激活T细胞和NKT细胞,释放IFN-γ等细胞因子,达到消灭肿瘤细胞的目的。但是单纯的T细胞和NKT细胞是不容易分离得到的。
发明内容
针对上述领域的不足,本发明提供一种嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ,能特异性结合靶标PD-L1,杀死过量表达PD-L1的肿瘤细胞。
同时本发明还提供表达该嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ的表达载体以及表达的T和NKT混合细胞。本发明混合细胞不仅能结合靶标PD-L1,且能激活T细胞和NKT细胞,达到消灭肿瘤细胞的目的。
嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ,其核苷酸序列如SEQ ID NO.1所示。
嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ的表达载体,由上述嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ连接于哺乳动物的慢病毒表达载体而得到。
所述慢病毒表达载体为pCDH-MSCV-EF1-GFP或pLVX-EF1。
嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ的表达细胞,为T和NKT混合细胞,由上述嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ的表达载体感染PBMC细胞后经培养而得到。
所述混合细胞中99.6%的细胞为T细胞,17.5%的细胞为NKT细胞。
所述培养的顺序方法为:
1)以PBS将培养基OKM25稀释100倍备用,6孔板中加入2mL稀释过的OKM25,室温6h后,弃上清,备用;
2)将感染第二天的PBMC转入OKM25预包板的6孔板,补2mL培养基OKM100+12%FBS,37℃ 5%CO2培养;
3)5天后,将细胞全部转移至培养瓶中,以培养基OKM100+12%FBS调整细胞密度在1×106 cells/mL;
4)3天后,将细胞转移至培养瓶中,培养基为OKM200+5%FBS,再培养4-8天即可得T+NKT混合细胞。
上述嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ的表达细胞在制备杀死PDL1+细胞的药物中的应用。
本发明合成了嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ,将其病毒质粒感染PMBC细胞,培养得到表达anti-PDL1-CD8-41BB-CD3ξ的T和NKT混合细胞,实验表明,表达anti-PD-L1-CD8-41BB-CD3ξ的T+NKT混合细胞对PDL1+细胞具有较强的杀伤能力,且比单独的表达anti-PD-L1-CD8-41BB-CD3ξ的T细胞对PDL1+细胞杀伤能力要强。
附图说明
图1荧光显微镜观察转染情况,
图2流式细胞仪检测anti-PD-L1-CD8-41BB-CD3ξ的表达,
图3流式检测PD-L1的表达情况,
图4流式检测淋巴细胞分型,
图5A流式检测PBMC对H358的杀伤能力(第一次独立重复),
图5B流式检测PBMC对H358的杀伤能力(第二次独立重复),
图6靶向PDL1的T+NKT混合细胞的杀伤效果
图7A靶向PDL1的T+NKT混合细胞对A549的杀伤作用,
图7B靶向PDL1的T+NKT细胞对A549的杀伤作用的统计结果,
图8ELISA检测IFN-γ结果,
图9靶向PD-L1的混合T+NKT细胞与T细胞的杀伤活性比较。
具体实施方式
下面结合实施例对本发明做进一步的详细说明。
下述实验材料均为市售。
一、实验材料
1、细胞:
H358、A549、K562PD-L1+、K562、PBMC
2、培养基:
PBMC:X-Vivo Lonza公司,货号:04-418Q
T+NKT:OKM25、OKM100+12%FBS、OKM200+5%FBS
H358、K562、K562-PD-L1:1640+10%FBS
3、流式抗体:
Protein L488:ACROBiosystems China
PD-L1抗体:PE-PD-L1antibody(Biolegend)
CD3抗体:PerCP-CD3(BD公司)
CD4抗体:PerCP-CD4(BD公司)、FITC-CD4(BD公司)
CD8抗体:PE-CD8(BD公司)
CD56抗体:FITC-CD56(BD公司)
4、细胞因子检测试剂盒:
IFN-γ:Human IFN-γELISA Kit(DKW12-1000-096)
5、其它主要试剂:
Dynabeads Human T-activator CD3/CD28for T-Cell Expansion andActivation,Thermo Fisher公司,货号:11131D
凋亡试剂盒:APC Annexin V Apoptosis Detection Kit with 7-AAD(Biolegend公司)
二、实验方法
1、合成基因:
合成基因anti-PDL1-CD8-41BB-CD3ξ并连接于哺乳动物的慢病毒表达载体 pCDH-MSCV-EF1-GFP或pLVX-EF1系列载体上,优选pLVX-EF1系列载体;
Anti-PDL1-CD8-41BB-CD3ξ序列如SEQ ID NO.1所示。
2、质粒大提
天根生物质粒大提试剂盒,按说明书操作;
3、慢病毒包装
1)转染前一天,将4×106的293T(加拿大)细胞接种10cm2平皿上(10mL DMEM+10%FBS);
2)取15mL离心管标记为A,加入以下试剂并混匀:
慢病毒质粒 12μg
慢病毒包装质粒 6μg+6μg
1640 500μL
3)取15mL离心管标记为B,分别加入500μL 1640(无FBS)培养基及72μLlipofectamine 2000,混匀,室温静置5min;
4)将A管液体转入B管内,混匀,室温静置25min,
5)293T细胞,从培养箱中取出,弃培养液,再加入10mL 1640(无FBS)培养液;
6)将1mL混合物逐滴均匀加入到293T细胞培养液中,轻微混匀,置于37℃,5%CO2培养箱中培养6h;
7)换正常培养液DEME+10%FBS培养
8)48h后,收病毒液,将培养液转移至50mL离心管中4000g,离心5min,4℃收集上清病毒液,过0.45μm滤膜,然后4℃保存;
4、慢病毒浓缩
1)按5:1的比例加浓缩液(合生基因),混匀后,4℃过夜。
2)2)4℃、4000g,离心30min浓缩慢病毒
3)弃上清,用移液器吸走全部液体
4)每管用150μL PBS重悬,混匀后,分装,留10μL进行滴度测定。
5、慢病毒感染PBMC
1)PBMC复苏:约1×107,稀释冻存液,复苏到25cm2细胞培养瓶中,X-VIVO 15+10%FBS 培养基;
2)培养4天有生长迹象,混匀所有PBMC,计数,分别取出1×106进行病毒感染。12孔板,总体积1mL,每孔Polybrene 1μL。本次实验病毒质量和数量受限,MOI=5:1。
3)感染第二天开始,进行T+NKT混合细胞的培养;
6、T+NKT混合细胞的培养
5)以PBS将OKM25稀释100倍备用,6孔板中加入2mL稀释的OKM25,室温6h后,弃上清,备用;
6)将感染第二天的PBMC转入OKM25预包板的6孔板,补2mL OKM100+12%FBS,37℃5% CO2培养;
7)5天后,将细胞全部转移至T75cm2培养瓶中,以OKM100+12%FBS调整细胞密度在1×106 cells/mL;
8)3天后,将细胞转移至T175cm2培养瓶中,培养基为OKM200+5%FBS,再培养4~8天即可得T+NKT混合细胞;
7、Anti-PDL1-CD8-41BB-CD3ξ表达情况的测定
1)感染后6天取1×106细胞,1000rpm离心5min收集细胞,并以PBS洗一次;
2)加入5μL Protein L,避光反应30min;
3)PBS洗两次,每次1000rpm,离心5min;
4)最后以500μL PBS重悬细胞,进行流式检测,PD-L1抗体在膜上表达的情况;
8、流式检测细胞分型
1)PBMC在培养第14天,取细胞进行分型分析;
2)取5×106细胞,1000rpm离心5min收集细胞,并以PBS洗一次;最后以PBS重悬,
分为100μL/tube;
3)分别加入5μL CD4、5μL CD3ξ、5μL CD56、5μL CD8进行单染以及同时加入
各抗体进行细胞分型检测;
4)室温避光,孵育30min;
5)PBS洗两次,每次1000rpm,离心5min;最后以500μL PBS重悬,进行流式检测; 9、构建高表达PD-L1的K562细胞系(K562-PDL1+)
1)利用PCR反应,以cDNA序列为模版,扩增PD-L1的全长及胞外区(去除信号肽);
GCATTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAATTCCCAGT AGAAAAACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTATGCCTTGGTGTAGCACTGACATTCATCTTCCGTTTAAGAAAAGGGAGAATGATGGATGTGAAAAAATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTAA
PCR体系如下:
PCR条件如下:
1)目的片段和载体的双酶切
利用NcoI和XhoI对目的片段进行双酶切,反应体系如下:
利用NcoI和XhoI对pLV-EF1载体进行双酶切,反应体系如下:
37℃处理30min后,切胶纯化
2)连接转化
将双酶切纯化后的目的片段和载体按照下面体系进行连接,室温连接30min;
将5μL连接产物转入Transtbl3感受态细胞中,冰浴30min,42℃热击30s,冰浴5min,加入950μL LB培养基37℃200rpm培养1h,取100μL涂板,37℃培养过夜。
3)按照方法5包装表达PD-L1的慢病毒载体
4)PD-L1慢病毒感染K562细胞:K562复苏24h后,进行慢病毒的感染;12孔板,总体积 1mL,每孔Polybrene 1μL。MOI=5:1。感染后第三天起细胞增多,转移至25cm2培养瓶;视细胞增长速度情况转移至75cm2培养瓶中。
10、A549、H358、K562细胞PD-L1表达情况的检测
1)直接取K562细胞和K562-PDL1+细胞,各取106个细胞,2000rpm离心5min;
2)胰酶消化A549和H358细胞,并用0.5mL PBS洗一次;取106个细胞,1000rpm离心5min,以200μL PBS重选;
3)分别加入10μL PE-PD-L1抗体和PE-IgG1抗体,避光孵育30min;
4)再加入800μL PBS,2000rpm离心10min,再以800μL PBS洗一次;
5)最后重悬于500μL PBS中,进行流式检测;
11、流式细胞仪检测细胞凋亡
1)靶细胞处理:H358以胰酶消化,加入1640+10%FBS终止反应,1000rpm离心5min;以1640重悬细胞,细胞数<107个/mL,加入5μL 2mM CFSE,避光静止20min;加入 1640+10%FBS终止反应,1000rpm离心5min,再加入少量1640+10%FBS静止10min, 1000rpm离心5min,以PBS洗一次,1000rpm离心5min;最后以杀伤培养基(1640+2%FBS) 重悬细胞,计数,调整至1×106个/mL,每孔100μL(24空板),以杀伤培养基补足至 200μL;
2)PBMC处理与LDH法一致,最后将细胞调整至4×107个/mL,每孔加入100μL(24孔板),最终体积为300μL,37℃5%CO2培养箱继续培养至3.5h;
3)以1.5mL EP管收集细胞,2000rpm离心10min,以PBS洗一次;
4)弃上清,以100μL PBS重悬细胞,分别加入5μL Annexin V或7AAD进行单染,或同时加入5μL Annexin V和5μL 7AAD,避光30min;
5)2000rpm离心10min,再以PBS洗一次,最后以500μL PBS重悬细胞,流式细胞仪检测;
12、细胞因子的检测——ELISA检测法
按照厂家提供的说明书进行测定。
三、实验结果
1.荧光显微镜观察感染anti-PDL1-CD8-41BB-CD3ξ的PBMC
将合成的anti-PDL1-CD8-41BB-CD3ξ(PD-L1抗体+信号分子)pCDH-MSCV-EF1-GFP载体进行慢病毒的包装并感染PBMC,荧光显微镜观察GFP表达情况如图1所示,PBMC在感染第3 天时,可见微小绿色克隆团,在感染后第5天时,绿色克隆团较多且清晰可见。
2.流式检测anti-PD-L1-CD8-41BB-CD3ξ的表达情况
慢病毒感染后的PBMC,在感染后第10天和14天时,分别以Protein L 488检测PD-L1抗体是否表达,结果如图2所示,第10天时,PBMC中anti-PD-L1-CD8-41BB-CD3ξ的表达效率为34.4%;第14天时,PBMC中anti-PD-L1-CD8-41BB-CD3ξ的表达效率为48.3%;
3.流式检测靶细胞上PD-L1的表达情况
以流式检测靶细胞上PD-L1的表达情况,结果如图3所示,A549和K562不表达PD-L1, H358中82.5%的细胞表达PD-L1,K562-PDL1+细胞99.8%的细胞表达PD-L1;因此,A549和 K562可作为PDL1-的靶细胞,H358和K562-PDL1+作为PDL1+靶细胞,进行杀伤效果比较分析;
4.流式检测淋巴细胞分型
培养14天的细胞,进行流式检测分型,其中,99.6%的细胞为T细胞(CD3+),17.5%的细胞为NKT细胞(CD56+CD3+),T细胞中,CD4+T细胞(CD4+CD3+)占19.34%,,CD8+T细胞(CD8+CD3+)占79.9%;说明,此方法培养PBMC,第14天时,为混合的T和NKT混合细胞;
5.靶向PDL1的T+NKT混合细胞的杀伤效果
以流式检测细胞凋亡的方法,分析了靶向PDL1的T+NKT混合细胞 (anti-PD-L1-CD8-41BB-CD3ξ-T+NKT混合细胞)对H358的杀伤能力,效靶比为10:1(E: T),先后进行了两次独立重复实验,每次设置两组重复结果如图5A和5B所示,杀伤效率如表2所示:第一次独立重复,对照组(无表达anti-PD-L1-CD8-41BB-CD3ξ-T+NKT混合细胞) 的平均杀伤效率为6.7%,转入anti-PD-L1-CD8-41BB-CD3ξ后的平均杀伤效率为14.5%;第二次独立重复对照组的平均杀伤效率为4.9%,靶向PDL1的T+NKT混合细胞平均杀伤效率为 18.4%;两次独立重复实验,靶向PDL1的T+NKT混合细胞的杀伤效率均明显高于对照组P<0.01 (图6)。
表2 PBMC对H358的杀伤作用
a:死亡率=Q1+Q2+Q3
b:杀伤效率=处理组死亡率-自发死亡率
同样以流式检测细胞凋亡的方法,分析了靶向PDL1的混合细胞(anti-PD-L1-CD8-41BB-CD3ξ-T+NKT)对A549(PDL1)的杀伤能力,效靶比为10:1(E:T) 三次独立重复实验结果如图7A和7B所示:对照组的杀伤效率为11.63%,转入 anti-PD-L1-CD8-41BB-CD3ξ后的T+NKT混合细胞的杀伤效率为8.83%;两组处理无统计学差异。
此结果说明转入anti-PD-L1-CD8-41BB-CD3ξ的T+NKT混合细胞对PDL1低表达对照细胞没有明显的杀伤效果,对PDL1+的细胞具有特异性杀伤作用。
6.靶向PDL1的T+NKT混合细胞对K562和K562-PDL1+的杀伤效果
构建了高表达PDL1的K562细胞系,并以流式检测细胞凋亡的方法,分析了 anti-PD-L1-CD8-41BB-CD3-T+NKT混合细胞对K562及K562-PDL1+靶细胞的杀伤能力,杀伤结果如表3所示:效靶比(E:T)为10:1时,anti-PD-L1-CD8-41BB-CD3ξ-T+NKT混合细胞与对照组相比,对K562-PDL1+细胞的杀伤效率由21.0%提高至76.6%;而两组细胞对K562细胞的杀伤效率仅20%左右;说明,表达anti-PD-L1-CD8-41BB-CD3ξ的T+NKT混合细胞对PDL1+ 细胞具有较强的杀伤能力;
表3 T+NKT混合细胞对K562及K562PD-L1+的杀伤效率
a:杀伤效率=处理组死亡率-自发死亡率
7、ELISA检测共培养上清中细胞因子水平
以ELISA检测试剂盒检测了IFN-γ在T+NKT混合细胞与H358和A549共培养上清中的含量,结果如图8所示,表达anti-PD-L1-CD8-41BB-CD3ξ的T+NKT混合细胞与H358的共培养上清中能检测到IFN-γ的分泌。高水平的IFN-γ的表达证明膜表面表达 anti-PD-L1-CD8-41BB-CD3ξ对于T细胞杀伤肿瘤细胞具有积极意义;而表达 anti-PD-L1-CD8-41BB-CD3ξ的T+NKT混合细胞与A549共培养上清中均检测不到IFN-γ明显的变化,这一结果与对A549细胞无杀伤效果是一致的,更进一步说明表达anti-PD-L1-CD8-41BB-CD3ξ的T+NKT混合细胞对PDL1靶点的特异性。
1:H358;2:H358与无表达anti-PD-L1-CD8-41BB-CD3ξ的混合细胞;3:H358与表达anti-PD-L1-CD8-41BB-CD3ξ的靶向PDL1的混合细胞;4:A549;5:A549与无表达 anti-PD-L1-CD8-41BB-CD3ξ的混合细胞;6:A549与anti-PD-L1-CD8-41BB-CD3ξ-T+NKT;
8.靶向PDL1的混合细胞与靶向PDL1的单T细胞杀伤效率的比较
PBMC中,T细胞比例为15%-30%,而NKT的比例仅为1%,但如果先分离T细胞再进行培养,那起始细胞数大大减少,增加了后期培养难度,而直接以PBMC培养,可以经过一次培养,同时获得T细胞和NKT细胞的混合细胞,且这种靶向PDL1的混合细胞对PDL1+的H358的杀伤效率高于单独表达anti-PD-L1-CD8-41BB-CD3ξT细胞(图9) 。
序列表
<110> 北京鼎成肽源生物技术有限公司
<120> 靶向PD-L1的T和NKT混合细胞、制备方法和应用
<130> PP18003-DCT
<141> 2018-03-13
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1889
<212> DNA
<213> 嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ序列(嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ序列)
<400> 2
gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60
ctccacgccg ccaggccgga catccagatg acccagtctc caagcagcct gtctgccagc 120
gtgggcgaca gagtgaccat cacatgtaga gccagccagg acgtgtcaac agccgtggct 180
tggtatcagc agaagcctgg caaggcccct aagctgctga tctacagcgc cagctttctg 240
tactccggcg tgcccagcag attttctggc agcggaagcg gcaccgactt caccctgaca 300
atatccagcc tccagcctga ggacttcgcc acctactatt gccagcagta cctgtatcac 360
cccgccacct ttggccaggg caccaaagtg gaaatcaagc ggacagtggc cggctccact 420
agcggttccg gcaaacctgg cagcggagaa ggcagcacca aaggggaagt gcagctggtg 480
gaatctggcg gaggactggt tcaacctggc ggctctctga gactgtcttg tgccgccagc 540
ggcttcacct tcagcgactc ttggattcac tgggtccgac aggcccctgg caaaggcctt 600
gaatgggttg cctggatcag cccttacggc ggcagcacct actacgccga ttctgtgaag 660
ggcagattca ccatcagcgc cgacaccagc aagaacaccg cctacctcca gatgaacagc 720
ctgagagccg aggacaccgc cgtgtactat tgtgccagaa ggcattggcc aggcggcttc 780
gattattggg gccagggaac actggtcacc gtgtctagcg cctctacaaa gggcttcgtg 840
ccagtattcc tgcccgcgaa gcccactact actcccgccc ctaggccacc tacaccggcc 900
cccacaattg catcacaacc actcagcctc cggcccgaag cttgccgccc cgctgctggg 960
ggcgccgtgc acactcgggg cctggacttt gcctgcgata tttatatttg ggcaccactt 1020
gcagggacgt gcggcgtact gcttctgtct ctcgtgatta ctctttattg taatcatagg 1080
aaccgatagg cggccgcaca ccggccccca caattgcatc acaaccactc agcctccggc 1140
ccgaagcttg ccgccccgct gctgggggcg ccgtgcacac tcggggcctg gactttgcct 1200
gcgatattta tatttgggca ccacttgcag ggacgtgcgg cgtactgctt ctgtctctcg 1260
tgattactct ttattgtaat cataggaacc gatctaagcg gtccagactg ctccacagtg 1320
attacatgaa catgacccca cgcaggcctg ggccaacacg aaaacactat cagccttacg 1380
cacctccccg cgactttgct gcctaccgct ctagattcag cgtggtgaag cgaggaagaa 1440
aaaaactcct ttatatattc aagcagcctt tcatgcgacc tgtccagaca actcaggaag 1500
aagacggctg ttcttgtagg ttcccagaag aggaagaggg aggctgcgag ctgcgcgtca 1560
aattctcccg atcagcagat gcacccgcat accaacaggg acagaaccag ctgtacaacg 1620
aactgaatct tggaagaagg gaggagtacg atgtgcttga taaaaggcgc ggccgggacc 1680
ccgaaatggg cggtaagcct agacgaaaaa atcctcaaga gggcctgtat aatgagcttc 1740
agaaagacaa gatggctgag gcgtactctg aaatagggat gaaaggagaa agaaggcggg 1800
gaaaaggcca tgatggcctg taccagggcc tcagcaccgc taccaaggac acatacgatg 1860
cccttcatat gcaggccctc cccccacgg 1889
Claims (7)
1.嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ,其核苷酸序列如SEQ ID NO.1所示。
2.嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ的表达载体,由权利要求1所述的嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ连接于哺乳动物的慢病毒表达载体而得到。
3.根据权利要求1所述的表达载体,所述慢病毒表达载体为pCDH-MSCV-EF1-GFP或pLVX-EF1。
4.嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ的表达细胞,为T和NKT混合细胞,由权利要求2或3所述的嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ的表达载体感染PBMC细胞后经培养而得到。
5.根据权利要求4所述的的表达细胞,所述混合细胞中99.6%的细胞为T细胞,17.5%的细胞为NKT细胞。
6.根据权利要求4所述的的表达细胞,所述培养的顺序方法为:
1)以PBS将培养基OKM25稀释100倍备用,6孔板中加入2mL稀释过的OKM25,室温6h后,弃上清,备用;
2)将感染第二天的PBMC转入OKM25预包板的6孔板,补2mL培养基OKM100+12%FBS,37℃5%CO2培养;
3)5天后,将细胞全部转移至培养瓶中,以培养基OKM100+12%FBS调整细胞密度在1×106cells/mL;
5)3天后,将细胞转移至培养瓶中,培养基为OKM200+5%FBS,再培养4-8天即可得T+NKT混合细胞。
7.权利要求4-6任一所述嵌合抗原受体anti-PDL1-CD8-41BB-CD3ξ的表达细胞在制备杀死PDL1+细胞的药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810203095.5A CN108484775A (zh) | 2018-03-13 | 2018-03-13 | 靶向pd-l1的t和nkt混合细胞、制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810203095.5A CN108484775A (zh) | 2018-03-13 | 2018-03-13 | 靶向pd-l1的t和nkt混合细胞、制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN108484775A true CN108484775A (zh) | 2018-09-04 |
Family
ID=63338572
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810203095.5A Pending CN108484775A (zh) | 2018-03-13 | 2018-03-13 | 靶向pd-l1的t和nkt混合细胞、制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108484775A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111944850A (zh) * | 2020-08-28 | 2020-11-17 | 澳门大学 | 表达抗cd22嵌合抗原受体和pd-l1阻断蛋白的细胞的制备方法、表达载体及应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105331585A (zh) * | 2015-11-13 | 2016-02-17 | 科济生物医药(上海)有限公司 | 携带pd-l1阻断剂的嵌合抗原受体修饰的免疫效应细胞 |
CN106350533A (zh) * | 2015-10-09 | 2017-01-25 | 上海宇研生物技术有限公司 | Anti‑PD‑L1‑CAR‑T及其制备方法和应用 |
CN106399242A (zh) * | 2016-09-13 | 2017-02-15 | 北京多赢时代转化医学研究院 | 联合制备CAR‑Vγ9Vδ2T细胞和CAR‑NKT细胞的方法 |
CN107022525A (zh) * | 2017-04-28 | 2017-08-08 | 中卫华医(北京)医院管理有限公司 | 用于肿瘤治疗的nk细胞培养方法 |
CN107384870A (zh) * | 2017-07-31 | 2017-11-24 | 时力生物科技(北京)有限公司 | 一种靶向pd‑l1嵌合抗原受体修饰的t淋巴细胞及其制备方法和应用 |
US20200102534A1 (en) * | 2018-09-30 | 2020-04-02 | Beijing Dcty Biotech Co., Ltd. | Afft2 cell |
-
2018
- 2018-03-13 CN CN201810203095.5A patent/CN108484775A/zh active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106350533A (zh) * | 2015-10-09 | 2017-01-25 | 上海宇研生物技术有限公司 | Anti‑PD‑L1‑CAR‑T及其制备方法和应用 |
CN105331585A (zh) * | 2015-11-13 | 2016-02-17 | 科济生物医药(上海)有限公司 | 携带pd-l1阻断剂的嵌合抗原受体修饰的免疫效应细胞 |
CN106399242A (zh) * | 2016-09-13 | 2017-02-15 | 北京多赢时代转化医学研究院 | 联合制备CAR‑Vγ9Vδ2T细胞和CAR‑NKT细胞的方法 |
CN107022525A (zh) * | 2017-04-28 | 2017-08-08 | 中卫华医(北京)医院管理有限公司 | 用于肿瘤治疗的nk细胞培养方法 |
CN107384870A (zh) * | 2017-07-31 | 2017-11-24 | 时力生物科技(北京)有限公司 | 一种靶向pd‑l1嵌合抗原受体修饰的t淋巴细胞及其制备方法和应用 |
US20200102534A1 (en) * | 2018-09-30 | 2020-04-02 | Beijing Dcty Biotech Co., Ltd. | Afft2 cell |
Non-Patent Citations (2)
Title |
---|
SHOU-HUI DU 等: "Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy", 《PLOS ONE》 * |
贺庆 等: "PD-1/PD-L1 抗体临床药效标志物和 CAR-T 细胞临床前药效评价的特点及检测方法", 《药物分析杂志》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111944850A (zh) * | 2020-08-28 | 2020-11-17 | 澳门大学 | 表达抗cd22嵌合抗原受体和pd-l1阻断蛋白的细胞的制备方法、表达载体及应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN105112370B (zh) | 一种体外刺激外周血γδT细胞高效增殖的方法及其应用 | |
AU2016250211B2 (en) | Modified gamma delta cells and uses thereof | |
Ptáčková et al. | A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21 | |
CN105624107B (zh) | 一种多种淋巴细胞亚群的扩增方法及其应用 | |
CN105924533B (zh) | Ror1特异性嵌合抗原受体及其应用 | |
EP3728612B1 (en) | Method for nk cell transduction | |
CN108531457A (zh) | 一种Cas9/RNP敲除T细胞PD-1和LAG3基因及制备CAR-T细胞的方法 | |
Shimasaki et al. | Natural killer cell reprogramming with chimeric immune receptors | |
Chicaybam et al. | CAR T cells generated using sleeping beauty transposon vectors and expanded with an EBV-transformed lymphoblastoid cell line display antitumor activity in vitro and in vivo | |
US20220064599A1 (en) | Car nk cells | |
Zhang et al. | Natural killer cells: of-the-shelf cytotherapy for cancer immunosurveillance | |
EP4188397A1 (en) | Universal antigen-specific t cell banks and methods of making and using the same therapeutically | |
Omer et al. | Chimeric antigen receptor signaling domains differentially regulate proliferation and native T cell receptor function in virus-specific T cells | |
Parente-Pereira et al. | Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells | |
Taştan et al. | Preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B-cells for the first Turkish academic clinical trial with relapsed/refractory ALL and NHL patients | |
CN109824783A (zh) | 表达于t淋巴细胞表面的嵌合抗原受体及其应用 | |
CN108484775A (zh) | 靶向pd-l1的t和nkt混合细胞、制备方法和应用 | |
CN114573710A (zh) | 一种靶向抗原同时外泌cd47抗体的免疫细胞及其应用 | |
CN114045309A (zh) | 一种通用的嵌合抗原受体调节t细胞制备方法及应用 | |
CN114671957A (zh) | 一种靶向实体瘤的car-t细胞的构建方法和应用 | |
Basingab et al. | ICAM-1 overexpression counteracts immune-suppress cell-derived PGE2 to restore CTL function | |
CN114269902A (zh) | 一种工程化免疫杀伤细胞、其制备方法及应用 | |
WO2023125860A1 (zh) | 通用型car-t细胞的制备技术及其应用 | |
CN117265009B (zh) | 一种gpc3-car-nkt细胞的制备方法及其应用 | |
CN108743617A (zh) | miR-15a-5p在提高CAR-T细胞抗肿瘤能力中的应用 |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180904 |
|
RJ01 | Rejection of invention patent application after publication |