CN112105650A - Chimeric single-chain molecule and application thereof - Google Patents
Chimeric single-chain molecule and application thereof Download PDFInfo
- Publication number
- CN112105650A CN112105650A CN201980031497.4A CN201980031497A CN112105650A CN 112105650 A CN112105650 A CN 112105650A CN 201980031497 A CN201980031497 A CN 201980031497A CN 112105650 A CN112105650 A CN 112105650A
- Authority
- CN
- China
- Prior art keywords
- cell
- cells
- chimeric single
- hla
- molecule
- 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
- 210000000822 natural killer cell Anatomy 0.000 claims abstract description 62
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 52
- 102100028970 HLA class I histocompatibility antigen, alpha chain E Human genes 0.000 claims abstract description 45
- 101000986085 Homo sapiens HLA class I histocompatibility antigen, alpha chain E Proteins 0.000 claims abstract description 45
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 45
- 108010033276 Peptide Fragments Proteins 0.000 claims abstract description 10
- 102000007079 Peptide Fragments Human genes 0.000 claims abstract description 10
- 101000937544 Homo sapiens Beta-2-microglobulin Proteins 0.000 claims abstract description 3
- 102100027314 Beta-2-microglobulin Human genes 0.000 claims abstract 2
- 210000004027 cell Anatomy 0.000 claims description 146
- 108090000623 proteins and genes Proteins 0.000 claims description 34
- 102000004169 proteins and genes Human genes 0.000 claims description 25
- 230000022534 cell killing Effects 0.000 claims description 23
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 claims description 22
- 108091008874 T cell receptors Proteins 0.000 claims description 20
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims description 20
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 17
- 230000002401 inhibitory effect Effects 0.000 claims description 16
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 9
- 230000002950 deficient Effects 0.000 claims description 7
- 150000007523 nucleic acids Chemical group 0.000 claims description 7
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- 102100028976 HLA class I histocompatibility antigen, B alpha chain Human genes 0.000 claims description 3
- 210000002540 macrophage Anatomy 0.000 claims description 3
- 101710117995 B-lymphocyte antigen CD19 Proteins 0.000 claims description 2
- 230000002147 killing effect Effects 0.000 abstract description 26
- 230000000735 allogeneic effect Effects 0.000 abstract description 21
- 238000012217 deletion Methods 0.000 abstract description 13
- 230000037430 deletion Effects 0.000 abstract description 13
- 239000000427 antigen Substances 0.000 abstract description 6
- 102000036639 antigens Human genes 0.000 abstract description 6
- 108091007433 antigens Proteins 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 4
- 238000009169 immunotherapy Methods 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 230000001900 immune effect Effects 0.000 abstract 1
- 102000015736 beta 2-Microglobulin Human genes 0.000 description 39
- 108010081355 beta 2-Microglobulin Proteins 0.000 description 39
- 102000043129 MHC class I family Human genes 0.000 description 30
- 108091054437 MHC class I family Proteins 0.000 description 30
- 108091033409 CRISPR Proteins 0.000 description 23
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 20
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 18
- 238000010362 genome editing Methods 0.000 description 15
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 11
- 210000004986 primary T-cell Anatomy 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 241000700605 Viruses Species 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- 229920001184 polypeptide Polymers 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 9
- 238000001890 transfection Methods 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 241000713666 Lentivirus Species 0.000 description 7
- 108091027544 Subgenomic mRNA Proteins 0.000 description 7
- 230000004913 activation Effects 0.000 description 7
- 210000000170 cell membrane Anatomy 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 238000000684 flow cytometry Methods 0.000 description 7
- 108091008042 inhibitory receptors Proteins 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 101710163270 Nuclease Proteins 0.000 description 6
- 241000193996 Streptococcus pyogenes Species 0.000 description 6
- 230000006044 T cell activation Effects 0.000 description 6
- 230000003213 activating effect Effects 0.000 description 6
- 101150063416 add gene Proteins 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 230000004807 localization Effects 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 102100028967 HLA class I histocompatibility antigen, alpha chain G Human genes 0.000 description 4
- 108010024164 HLA-G Antigens Proteins 0.000 description 4
- 239000012124 Opti-MEM Substances 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000002062 proliferating effect Effects 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 102000006306 Antigen Receptors Human genes 0.000 description 3
- 108010083359 Antigen Receptors Proteins 0.000 description 3
- 238000010453 CRISPR/Cas method Methods 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 108010042407 Endonucleases Proteins 0.000 description 3
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 3
- 108010075704 HLA-A Antigens Proteins 0.000 description 3
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 description 3
- 101150069255 KLRC1 gene Proteins 0.000 description 3
- 101100404845 Macaca mulatta NKG2A gene Proteins 0.000 description 3
- 102100022682 NKG2-A/NKG2-B type II integral membrane protein Human genes 0.000 description 3
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 241000194020 Streptococcus thermophilus Species 0.000 description 3
- 238000003501 co-culture Methods 0.000 description 3
- 230000000139 costimulatory effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 230000035939 shock Effects 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 101150076800 B2M gene Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102100028966 HLA class I histocompatibility antigen, alpha chain F Human genes 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101000986080 Homo sapiens HLA class I histocompatibility antigen, alpha chain F Proteins 0.000 description 2
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 2
- 241000186805 Listeria innocua Species 0.000 description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 2
- 230000006051 NK cell activation Effects 0.000 description 2
- 241000588650 Neisseria meningitidis Species 0.000 description 2
- 101710153660 Nuclear receptor corepressor 2 Proteins 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 108091028113 Trans-activating crRNA Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 101150100265 cif-1 gene Proteins 0.000 description 2
- 238000011198 co-culture assay Methods 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 108010026638 endodeoxyribonuclease FokI Proteins 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 238000003197 gene knockdown Methods 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 238000003151 transfection method Methods 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- RXACEEPNTRHYBQ-UHFFFAOYSA-N 2-[[2-[[2-[(2-sulfanylacetyl)amino]acetyl]amino]acetyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)CNC(=O)CNC(=O)CS RXACEEPNTRHYBQ-UHFFFAOYSA-N 0.000 description 1
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 description 1
- 101150033839 4 gene Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 101001005269 Arabidopsis thaliana Ceramide synthase 1 LOH3 Proteins 0.000 description 1
- 101001005312 Arabidopsis thaliana Ceramide synthase LOH1 Proteins 0.000 description 1
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 1
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 description 1
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- -1 C-Met Proteins 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 102100028801 Calsyntenin-1 Human genes 0.000 description 1
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101100438439 Escherichia coli (strain K12) ygbT gene Proteins 0.000 description 1
- 101150106478 GPS1 gene Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102100032530 Glypican-3 Human genes 0.000 description 1
- 102100028971 HLA class I histocompatibility antigen, C alpha chain Human genes 0.000 description 1
- 108010074032 HLA-A2 Antigen Proteins 0.000 description 1
- 102000025850 HLA-A2 Antigen Human genes 0.000 description 1
- 108010058607 HLA-B Antigens Proteins 0.000 description 1
- 108010021736 HLA-B15 Antigen Proteins 0.000 description 1
- 108010091938 HLA-B7 Antigen Proteins 0.000 description 1
- 108010052199 HLA-C Antigens Proteins 0.000 description 1
- 108010070768 HLA-C*03 antigen Proteins 0.000 description 1
- 108010007655 HLA-C*70 antigen Proteins 0.000 description 1
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 1
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 1
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 description 1
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 description 1
- 101000998120 Homo sapiens Interleukin-3 receptor subunit alpha Proteins 0.000 description 1
- 101000991061 Homo sapiens MHC class I polypeptide-related sequence B Proteins 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 description 1
- 101000633784 Homo sapiens SLAM family member 7 Proteins 0.000 description 1
- 101000874179 Homo sapiens Syndecan-1 Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 1
- 102100033493 Interleukin-3 receptor subunit alpha Human genes 0.000 description 1
- 102000002698 KIR Receptors Human genes 0.000 description 1
- 108010043610 KIR Receptors Proteins 0.000 description 1
- 102100030300 MHC class I polypeptide-related sequence B Human genes 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 108091008043 NK cell inhibitory receptors Proteins 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 1
- 101150030723 RIR2 gene Proteins 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 101800000684 Ribonuclease H Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101000668858 Spinacia oleracea 30S ribosomal protein S1, chloroplastic Proteins 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 101100166147 Streptococcus thermophilus cas9 gene Proteins 0.000 description 1
- 101000898746 Streptomyces clavuligerus Clavaminate synthase 1 Proteins 0.000 description 1
- 102100035721 Syndecan-1 Human genes 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 101100329497 Thermoproteus tenax (strain ATCC 35583 / DSM 2078 / JCM 9277 / NBRC 100435 / Kra 1) cas2 gene Proteins 0.000 description 1
- 241001313536 Thermothelomyces thermophila Species 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 108010073062 Transcription Activator-Like Effectors Proteins 0.000 description 1
- 102100037116 Transcription elongation factor 1 homolog Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 1
- 101150100826 UL40 gene Proteins 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 108091093126 WHP Posttrascriptional Response Element Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 101710185494 Zinc finger protein Proteins 0.000 description 1
- 102100023597 Zinc finger protein 816 Human genes 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 101150058049 car gene Proteins 0.000 description 1
- 101150000705 cas1 gene Proteins 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000006690 co-activation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 108010087914 epidermal growth factor receptor VIII Proteins 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 150000002333 glycines Chemical class 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 102000047279 human B2M Human genes 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000011502 immune monitoring Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 239000010445 mica Substances 0.000 description 1
- 229910052618 mica group Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229910052754 neon Inorganic materials 0.000 description 1
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000005316 response function Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 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
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- 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
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
-
- 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
- A61K39/464411—Immunoglobulin superfamily
- A61K39/464412—CD19 or B4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- 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
-
- 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
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Crystallography & Structural Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a chimeric single-chain molecule, a lymphocyte containing the chimeric single-chain molecule and application of the lymphocyte as an immunotherapy medicament. The chimeric single-chain molecule comprises a chimeric molecule presenting a peptide fragment, a B2M protein and an HLA-E heavy chain. When the chimeric single-chain molecule is used in the genetic engineering lymphocyte with inactivated I-class major histocompatibility antigen (MHC) expression, the immunological rejection of allogeneic plants can be avoided; the chimeric single-chain molecule of the invention is expressed in T cells, and can avoid killing host NK cells caused by I-type major histocompatibility antigen deletion.
Description
The invention relates to a chimeric single-chain molecule, in particular to a chimeric molecule containing a presenting peptide segment, B2M protein and HLA-E heavy chain, a lymphocyte containing the chimeric single-chain molecule and application of the lymphocyte as an immunotherapy drug.
Adoptive cellular immunotherapy is the isolation of primary T cells from the body, ex vivo stimulation, expansion and culture of these cells in vitro, and finally the transfusion of these cells back to the patient for disease treatment. Chimeric antigen receptor T cell therapy (CAR-T) is one of the current representatives of the success of adoptive cellular immunotherapy. CAR-T therapy refers to the isolation of T cells that activate donors in vitro, and the transfer of CAR genes, including antigen recognition, co-activation and activation regions, into T cells, further large scale ex vivo expansion and then back infusion to patients to treat disease. The existing CAR-T technology is individualized and uses patient autologous T cells to produce CAR-T cells. The CAR-T cells prepared from autologous T cells have many defects, such as long production period, high preparation cost, poor T cell state of patients in many cases and the like. CAR-T technology therefore requires the break of individualized limitations, i.e. the production of CAR-T cells using allogeneic T cells from healthy donors as a cell source, such cells being without recipient limitations and therefore being called universal CAR-T.
The universal CAR-T cells are derived from healthy donors and can be prepared in advance for use by any patient. The preparation of the universal CAR-T takes into account two issues. In one aspect, removal of expression of class I MHC molecules from the donor cell can abrogate donor cell antigen presentation, escape recognition and killing by host T cells, and CAR-T cells survive. On the other hand, removal of expression of the TCR complex of the donor cell prevents recognition of killer host cells by the donor T cells. MHC class I molecules comprise two subunits, termed the heavy and light chains. Because of the differences in heavy chains, class I MHC is classified into several different classes A/B/C/E/F/D. The light chain is a universal subunit (B2M) and can form an MHC class I complex with either heavy chain. The major intracellular role of the class I MHC complex is to present degraded polypeptide fragments of intracellular proteins, which are bound to the polypeptide fragments in the cell to form a polypeptide-class I MHC complex, which is transported to the cell surface. The polypeptide-class I MHC complex can be recognized by the TCR of a specific T cell clone, resulting in killing of the T cell. The TCR is composed of two subunits, an alpha (α) subunit and a beta (β) subunit. Both subunits have many subtypes, and different subtypes combine to form a large number of different TCR clones. The TCR is specifically expressed on the surface of T cells, and binds to a variety of different CD3 molecules to form a TCR complex, which recognizes the polypeptide-MHC class I complex, and which, upon recognition, causes activation of T cells and killing of the recognized cells.
The methods for removing intracellular protein expression are mainly two: gene knockdown by RNA silencing and nuclease-mediated gene editing techniques. Gene knockdown and gene editing techniques resulting from RNA silencing can achieve simultaneous removal of MHC class I and TCR complexes within donor T cells. The gene editing technology is to use specific rare-cutting endonuclease to cut the intracellular genome, after the cutting is finished, the inherent non-homologous recombination repair mechanism in the cell occurs, the repair is non-specific repair, and the frame shift gene mutation is easy to generate, thereby leading to gene knockout.
Although gene knockout can achieve the removal of class I MHC complex expression and avoid the attack of receptor T cells, the deletion of class I MHC can cause the activation and killing of receptor NK cells. NK cells are a natural immune monitoring cell. There are many receptors on the surface of NK cells, including activating and inhibitory receptors. The activating receptor mainly comprises NKG2D and the like, and the ligand of NKG2D comprises MICA, MICB, ULBP1-6 and the like. Inhibitory receptors are mainly KIRs, the ligands of which are MHC class I molecules. In addition, the inhibitory receptors also include NKG2A, the ligand of which is the class I MHC family of molecules analogue HLA-E (Morvan and Lanier, Nat Rev Cancer 2016.16, 7-19).
Whether NK cells are activated depends on the balance of activating and inhibitory signals. Normal cells express both activating and inhibitory NK cell activating ligands. MHC class I complexes expressed by normal cells bind to NK cell inhibitory receptors, thereby avoiding activation and killing of NK cells. Cells lacking MHC class I complexes are unable to bind inhibitory receptors, resulting in the inhibitory receptors being unable to be activated, and the balance tends to activate NK cells, and thus cells lacking MHC class I complexes can be recognized and killed by NK cells. Many mechanisms can avoid killing of NK cells. Cells infected with the virus express virus-derived analogues of class I MHC family proteins, which bind to the inhibitory receptor NKG2A, inhibiting NK cell activation. Introducing HLA-E molecule of MHC class I family member into cell, wherein the molecule can bind to inhibitory receptor NKG2A to inhibit NK cell activation (CN 106103475A). In addition, in HLA class Inull721.221 cells, which lack expression of MHC class I molecules, are recognized and killed by NK cells. The artificial introduction of HLA-E or HLA-G can inhibit the activation of NK cells, thereby avoiding the killing by NK cells. Introduction of immunosuppressive polypeptides such as viral MHC class I homologues or ligands of NKG2D can also be resistant to NK cell killing.
Since cell membrane expression of HLA-E is dependent on B2M to form a complex, HLA-E does not localize to the cell membrane after general CAR-T inhibits or knockouts B2M protein. In addition, the membrane localization of HLA-E is also dependent on the presenting polypeptide. HLA-E typically presents specifically the signal peptides of HLA-A, -B, -C and-G and polypeptides of certain viruses. The expression of the HLA-E/B2M complex on cells can be increased by artificially constructing the HLA-E chimeric single-chain molecule. It has been shown that HLA-E chimeric single chain molecules expressing a presented HLA-G signal peptide in tumor cell lines or pluripotent stem cells are effective against killing of allogeneic NK cells (WO 2004/103149A 2).
Although class I MHC molecule-deficient cells expressing HLA-E chimeric single chain molecules can effectively inhibit NK cell killing, much of the data is focused on model cell lines, virus-infected cells, pluripotent stem cells or tumor cells. The expression of such HLA-E chimeric single chain molecules in immune lymphocytes deficient in MHC class I molecules has not been reported. The expression pattern (expression or not, expression amount) and function of the same protein in different tissue cells are completely different, and it is not clear whether the HLA-E chimeric single-chain molecule can be efficiently expressed in immune lymphocytes and can exert the NK cell inhibitory function. In addition, different subtypes of HLA-E heavy chain and different signal peptides presented by HLA-E/B2M complex are important for whether HLA-E chimeric single-chain molecules are expressed on cell membranes, the expression level is high or low, and the immune function is important. The optimal combination is screened from different HLA-E heavy chain subtypes and a plurality of presenting peptides and is expressed in engineered immune lymphocytes, and the optimal combination has great significance for allogeneic universal immune cell therapy.
Brief summary of the invention
The invention relates to a chimeric single-chain molecule containing a presenting peptide segment, B2M and HLA-E, which can realize the cell membrane expression of the HLA-E in B2M-deleted lymphocytes. The chimeric single-chain molecule containing the presenting peptide segment, B2M and HLA-E can be further applied to universal CAR-T lacking TCR and B2M, and can avoid killing NK cells.
The invention provides the following technical scheme:
1. a chimeric single-chain molecule comprising: (a) presenting the peptide fragment; (b) B2M protein; (c) HLA-E heavy chain; and (d) a linker sequence for linking the aforementioned (a) and (b), and (b) and (c) fragments; wherein the HLA-E heavy chain is HLA-E01: 01 or HLA-E01: 03 subtype.
2. The chimeric single-chain molecule described in technical scheme 1, which comprises from the N-terminus to the C-terminus in sequence: presentation peptide-linker-B2M protein-linker-HLA-E heavy chain.
3. The chimeric single-chain molecule of claim 1 or 2, wherein the presented peptide fragment is a signal peptide of an MHC class I molecule.
4. The chimeric single-chain molecule of claim 3, wherein the presented peptide fragment comprises or is selected from one of the following sequences: 2, 4, 5, 6 and 7.
5. The chimeric single-chain molecule according to claim 4, wherein the presented peptide fragment is represented by SEQ ID NO. 5.
6. The chimeric single-chain molecule of any of the preceding claims, wherein the linker sequence is (EAAAK) n or (GGGGS) n, wherein n is 3 or 4 or 5.
7. The chimeric single-chain molecule of claim 6, wherein the linker sequence is selected from one or more of the following sequences: SEQ ID NO 8 and SEQ ID NO 9.
8. The chimeric single-chain molecule of any of the preceding claims, wherein the HLA-E x 01:01 subtype is SEQ ID NO 12 or a variant thereof.
9. The chimeric single-chain molecule of any of the preceding claims, wherein the HLA-E01: 03 subtype is SEQ ID NO 13 or a variant thereof.
10. The chimeric single-chain molecule according to any one of the preceding technical schemes is SEQ ID NO: 18. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: shown at 25.
11. A nucleic acid sequence expressing the chimeric single-stranded molecule of any one of the preceding claims.
12. The nucleic acid sequence shown in technical scheme 11 is SEQ ID NO: 19 to
13. An expression vector comprising the nucleic acid sequence set forth in claim 11 or 12.
14. A host cell comprising the expression vector of claim 13.
15. A lymphocyte of a cell surface expressing a chimeric molecule of any one of claims 1-10, wherein said lymphocyte is deficient in an MHC class I molecule.
16. The lymphocyte of claim 15, wherein said lymphocyte is a T cell, an NK cell, a B cell or a macrophage.
17. The lymphocyte of claim 16, further characterized in that at least one gene encoding a component of a T Cell Receptor (TCR) is inactivated.
18. The lymphocyte of any of claims 15-17, wherein the T cell is a CAR-T cell.
19. The lymphocyte of claim 18, wherein said CAR is directed to the B lymphocyte antigen CD 19.
20. The lymphocyte of claim 19, wherein the protein sequence that simultaneously expresses the CAR molecule and the chimeric multimeric single-chain molecule in the lymphocyte is represented by SEQ ID NO: shown at 20.
21. The use of a chimeric single chain molecule according to any one of claims 1 to 10 for inhibiting NK cell killing.
22. The use of a chimeric single chain molecule according to any one of claims 1 to 10 in the preparation of a medicament for inhibiting NK cell killing.
23. The use of a lymphocyte of any of claims 15-20 in inhibiting NK cell killing.
24. The lymphocyte of any of technical schemes 15-20, in the preparation of inhibition of NK cell killing medicine application.
25. A method of inhibiting NK cells comprising providing to NK cells or a patient lymphocytes according to any one of claims 15-20.
26. The method of claim 25, wherein the lymphocyte is preferably a T cell, more preferably a CAR-T cell.
27. A pharmaceutical composition for inhibiting NK cell killing comprising the chimeric single chain molecule of any one of claims 1-10.
28. A pharmaceutical composition for inhibiting NK cell killing comprising the lymphocytes of any one of claims 15-20.
The chimeric single-chain molecule can make I-class major histocompatibility antigen (MHC) express inactivated genetically engineered lymphocyte, and avoid killing host NK cells caused by I-class MHC molecule deletion. The chimeric single-chain molecule of the invention enables the lymphocyte with gene modification to be suitable for allogeneic use, and reduces the reaction of host rejection of transplant. The chimeric single-chain molecule described in the invention can further inactivate the expression of a T Cell Receptor (TCR) when used in a genetically engineered T cell, and reduce graft-versus-host reaction, so that the chimeric single-chain molecule can be used as a universal T cell, is more suitable for allogeneic use, and can be further applied to a chimeric antigen receptor T cell (CAR-T) or a T cell receptor T cell (TCR-T).
Detailed description of the invention
Defining:
class I MHC molecules
Class I MHC molecules, also known as class I major histocompatibility complex, heterodimeric glycoproteins consisting of two peptide chains joined by a non-covalent bond; one of them is called heavy chain, the structure is polymorphic, and the other is light chain or called beta 2 microglobulin (B2M). Functionally, MHC class I molecules present polypeptides that are degraded intracellularly and are not self-proteins, thereby activating the immune system. Human MHC molecules are called HLA molecules, and human MHC class I molecules are classified into classical HLA molecules (HLA-A, HLA-B, HLA-C) and non-classical HLA molecules (HLA-E, HLA-G, HLA-F).
In a specific embodiment, the cell surface deletion of MHC class I molecules refers to the deletion of HLA-A/B/C/E/F/G human lymphocytes, which can be achieved by editing B2M or the corresponding heavy chain gene to inactivate it.
B2M
Beta-2 microglobulin, also known as B2M protein, is the light chain of MHC class I molecules and is an integral part of MHC class I molecules. The human B2M protein consists of 119 amino acids (SEQ ID NO: 16) and has a molecular weight of 11.800 daltons. The absence of the B2M molecule renders the cell unable to normally express MHC class I molecules on the cell membrane. Such cells are recognized and killed by NK cells.
The absence of the B2M molecule renders the cell incapable of expressing class I MHC molecules, also known as HLA-I molecules, on the cell membrane.
HLA-E chimeric single-chain molecules
The chimeric single-chain molecule of the invention comprises the following components from the N end to the C end in sequence: presentation peptide fragment-linker-B2M protein-linker-HLA-E heavy chain molecule. The structure is shown in fig. 2.
In a specific embodiment, the chimeric single-chain molecule is: membrane localization signal peptide-presenting peptide fragment- (G)4S) 3-B2M mature protein- (G)4S) 4-HLA-E heavy chain molecule wherein the membrane localization signal peptide is removed prior to localization of the chimeric molecule to the cell membrane.
Wherein the membrane localization signal peptide includes, for example, signal peptides of membrane localization proteins such as B2M and CD 8.
Signal peptide: about 20-80 amino acids in length, located at the N-terminus of the mature protein, directs the protein to cross the membrane and is later cleaved off. Such as B2M, CD8, etc.
Wherein, the HLA-E heavy chain comprises HLA-E01: 01 and HLA-E01: 03 subtypes; or variants of HLA-E01: 01 and HLA-E01: 03 subtypes.
The variant refers to a molecule which retains the function of the original protein through insertion, substitution and/or deletion of one or more amino acids in the original protein sequence; for example, molecules that retain HLA-E heavy chain function by one or more amino acid insertions, substitutions and/or deletions in the HLA-E01: 01 or HLA-E01: 03 sequence.
Presentation of peptide fragments: a section of polypeptide presented by the HLA-E complex can stabilize the stability and the structural specificity of the HLA-E complex, and the polypeptide is derived from a signal peptide of an MHC class I molecule, such as HLA-A2, HLA-B7, HLA-B15, HLA-Cw3, HLA-Cw7, HLA-G, HLA-F and the like; viral protein polypeptides such as CMV (UL40), EBV, HIV and the like.
Ligation sequences
The connecting sequence of the invention is used for connecting a presenting peptide segment in the chimeric single-chain molecule with the B2M protein and connecting the B2M protein with an HLA-E heavy chain. The linker sequence may be (EAAAK) n, (GGGGS) n, n-3 or 4 or 5, etc.
In particular embodiments, the linker sequence is a 3-4 repeats of 4 glycines and one serine, e.g., (G)4S) 3The sequences are (SEQ ID NO: 2), (G)4S) 4The sequence is (SEQ ID NO: 3)
Lymphocytes
Lymphocytes are a diverse group of cells that produce and mature in the lymphatic system and have an immune response function. This patent includes T cells, B cells, Natural Killer (NK) cells, macrophages, and the like.
Base editing (base editing)
Adenine (a), guanine (G), cytosine (C), thymine (T) or uracil (U) in a nucleic acid (DNA or RNA) are subjected to a precise site-specific change, which switches between them.
ZFN
Zinc-finger nucleotides are composed of a DNA recognition domain and a non-specific endonuclease. The DNA recognition domain consists of a series of Cys2-His2 zinc finger protein cascades, each zinc finger unit comprising approximately 30 amino acids, for specific binding to DNA. The non-specific endonuclease is a FokI endonuclease, which forms dimers to cleave DNA.
TALEN
TALENs (Transcription Activator-Like Effector nuclei) are Transcription Activator Effector Nucleases. The TALE protein is a core component of a DNA binding domain, generally consists of a plurality of basic repeating units which are connected in series, can specifically recognize a DNA sequence through the designed and combined serial units, and can realize the cutting of the specific DNA sequence after being coupled with FokI endonuclease.
Chimeric Antigen Receptor (CAR)
Car (chimeric Antigen receptor) refers to a specific Antigen receptor that expresses some intracellular activation signals simultaneously, and is composed of 3 parts: antigen receptors expressed extracellularly, mainly from the variable domains (scFv) of single chain antibodies, transmembrane and intracellular signaling domains connecting extracellular and intracellular structures, mainly contain T cell activation and costimulatory signals, where costimulatory molecules may comprise one or two, common costimulatory molecules are CD28, CD137(4-1BB), CD27, OX40, CD30, CD40, etc. The extracellular antigen receptor is derived mainly from the relevant tumor antigens and can be selected from the following antigens: CD19, CD20, CD22, CD123, CD33/IL3Ra, CD138, CD33, BCMA, CS1, C-Met, EGFRvIII, CEA, Her2, GD2, MAG3, GPC3, NY-ESO-1, and the like.
CRISPR/Cas system
The CRISPR/Cas system is a nuclease system consisting of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and a CRISPR-binding protein (i.e., Cas protein), and can cut almost all genome sequences adjacent to a protospacer-adjacent motif (PAM) in eukaryotic cells (Cong et al. science 2013.339: 819. 823). The "CRISPR/Cas system" is used to collectively refer to transcripts involved in CRISPR-associated ("Cas") genes, as well as other elements involved in their expression or directing their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portions of tracrRNA), tracr mate sequences (encompassing "direct repeats" and processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences, or other sequences and transcripts from CRISPR loci. In general, CRISPR systems are characterized by elements that facilitate the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in endogenous CRISPR systems). .
Formation of a CRISPR complex (comprising a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 50, or more base pairs). The tracr sequence (which may comprise or consist of all or part of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence)) may also form part of a CRISPR complex, such as by hybridizing along at least a portion of the tracr sequence to all or part of a tracr mate sequence operably linked to the guide sequence. In some embodiments, the tracr sequence is sufficiently complementary to a tracr mate sequence to hybridize and participate in the formation of a CRISPR complex. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more vectors that drive expression of one or more elements of the CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system directs formation of a CRISPR complex at one or more target sites.
Generally, the tracr mate sequence comprises a tracr mate sequence having sufficient complementarity to a tracr sequence to facilitate formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence. Typically, the degree of complementarity is in terms of the best alignment of the tracr mate sequence with the tracr sequence along the length of the shorter of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm, and the effects of secondary structure can be further taken into account, such as self-complementarity within the tracr sequence or tracr mate sequence. When optimally aligned, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two is about or greater than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. The tracr sequence is about or more than about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
Non-limiting examples of Cas proteins include: cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7 (also known as Csn 7 and Csx 7), Cas7, Csy 7, Cse 7, Csc 7, Csa 7, Csn 7, Csm 7, Cmr 7, Csb 7, Csx 36x 7, Csx 36f 7, csxf 7, Csx 36f 7, Csx 36x 7, Csx 36x 7, Csx 36x. In some embodiments, the Cas protein is a Cas9 protein.
Cas9, also known as Csn1, is a giant protein that is involved in both crRNA biosynthesis and in destroying invading DNA. Cas9 has been described in different bacterial species such as streptococcus thermophilus (s.thermophiles), Listeria innocua (Listeria innocua) (gasinuas, Barrangou et al 2012; Jinek, chylinki et al 2012) and streptococcus pyogenes (s.pyogenes) (Deltcheva, chynski et al 2011). The giant Cas9 protein (>1200 amino acids) contains two predicted nuclease domains, an HNH (McrA-like) nuclease domain located in the middle of the protein and a split RuvC-like nuclease domain (RNAase H fold) (Makarova, Grishin et al (2006)). Cas9 variants may be Cas9 endonucleases that do not occur naturally in nature and are obtained from protein engineering or by random mutagenesis. For example, a Cas9 variant according to the present invention may be obtained by mutation, i.e. deletion or insertion or substitution of at least one residue in the amino acid sequence of the streptococcus pyogenes Cas9 endonuclease (COG 3513). In some embodiments, the Cas9 protein is a streptococcus pneumoniae, streptococcus pyogenes, or streptococcus thermophilus Cas9, and may include mutated Cas9 derived from these organisms, or variants with other amino acid sequences attached to Cas9, such as fokl enzymes attached to Cas 9. These Cas9 are known; for example, the amino acid sequence of the Streptococcus pyogenes (Streptococcus pyogenes) Cas9 protein can be found under SwissProt database accession No. Q99ZW2, the amino acid sequence of the Neisseria meningitidis (Neisseria meningitidis) Cas9 protein can be found under UniProt database No. A1IQ68, the amino acid sequence of the Streptococcus thermophilus (Streptococcus thermophilus) Cas9 protein can be found under UniProt database No. Q03LF7, and the amino acid sequence of the Staphylococcus aureus (Staphylococcus aureus) Cas9 protein can be found under UniProt database No. J7RUA 5.
Method for preparing engineered lymphocytes
The method of preparing the engineered lymphocytes comprises: (i) preparing lymphocytes with surface lacking class I MHC molecules; (ii) expressing the chimeric single-chain molecule on the surface of the lymphocyte; the chimeric single-chain molecule body comprises: (a) presenting the peptide fragment; (b) B2M protein; c) HLA-E heavy chain; and (d) a linker sequence for linking the aforementioned (a) and (b) and (c) fragments. Wherein the order of steps (i) and (ii) may be interchanged.
The lentivirus preparation method comprises the following steps: using a three-plasmid system, the expression plasmid for lentivirus (Addgene ID: #12252), the helper plasmids psPAX2(Addgene ID: #12260) and pMD2.G (Addgene ID: #12259) were packaged. Viral packaging was performed in HEK293T cells (shanghai cell institute of department of sciences, origin). The preparation process comprises the following steps: the HEK293T cells in the cryopreserved working cells were thawed, and cultured in DMEM medium (+ 10% FBS + 1% P/S) (Cellgro 10-013-CMR) in a 10cm dish, and the medium was changed after 2 days of thawing. After the cells are full, passage is started (usually 1 dish is full and then 5 dishes are obtained), and after 4 passages, the cells are transfected with plasmid. Our system prefers transfection using PEI as the transfection reagent, PEI: plasmid (mass ratio) 2: 1. The mixture of plasmid and PEI was added to Opti-MEM medium (Gibco, cat # 31985-. After 6 hours of transfection, the medium was replaced with fresh medium containing 2% FBS, and then the culture was continued for 72 hours, and the supernatant of HEK293T cells was collected. The collected virus supernatant was concentrated by ultracentrifugation (82200g, centrifugation at 4-8 ℃ for 2 hours), and the concentrated virus was sterilized by filtration through a 0.22um filter membrane and then resuspended for use.
Vector construction: a target gene (e.g., an HLA-E chimeric single-stranded molecule DNA sequence) is constructed into a lentiviral vector. Lentiviral vectors contain a long terminal repeat 5 'LTR and a truncated 3' LTR, RRE, rev response element (cPPT), Central Termination Sequence (CTS) and post-translational regulatory element (WPRE). The HLA-E chimeric single-chain molecule is constitutively expressed by an EF-1a (elongation factor-1 a) promoter, and is constructed on a lentiviral vector by digestion with BamHI and SalI.
Primary T cell activation Primary T cells were derived from Peripheral Blood (PBMC) of healthy human volunteers. The culture medium is complete culture medium, ImmunoCultTMXF T Cell Expansion Medium (Stem Cell Technology, cat #10981) +300IU/ml IL2(Cayan, cat # HEILP-0201 c). T cells were activated using Dynabeads (Thermo, cat #11141D), which: cell 3: 1. t cells are activated for 24 hours and then are apparently clustered and enlarged.
T cell lentivirus transfection method: after primary T cells are activated, the cells are obviously clustered and enlarged to enter the division period for 24-72 hours, and lentivirus transfection can be carried out. The virus dosage is as follows: cell number 10: 1, MOI ═ 10 transfected cells. Protein expression assays can be performed 48 hours after transfection.
The gene editing method comprises the following steps: 4 days after T cell activation, or 2 days after virus transfection, cells were collected, washed 3 times with electrotransfer buffer, buffer T, T4, Opti-MEM (Gibco, cat #31985-8And/ml. In vitro, the required sgRNA (total amount 300-. The shock voltage conditions were as follows: 1200 and 1600v, 10ms (3 times). Designing the sgRNA candidate sequence according to the predictive analysis of related websites; cas9 protein is derived from Alt-R s.p. case 9 nuclear 3NLS protein from IDT DNA technology.
Preparation of engineered CAR-T cells expressing chimeric single-chain molecules: t cell activation, viral transfection, gene editing methods as above. The process is as follows: on day 0, primary PBMC activation; day 2 virus transfection, day 4 gene editing (TCR and B2M knockout), and day 6-7 flow cytometry to detect individual protein expression.
Desired antibody and cell dye information: anti-CD3-APC (BD,555335), anti-CD3-FITC (BD,555916), anti-B2M-PE (BD,551337), anti-MHC-I-APC (R)&D,FAB7098A),anti-HLA-E-PE(Biolegend,342604),anti-HLA-E-APC(Biolegend,342606),anti-Fab-Biotin(Jackson ImmunoResearch,115-065-072),Streptavidin-PE(BD,554061),Dye eFluor TM 670(eBioscience,65-0840-90)
Fig. 1. editing of the B2M gene in primary T cells using electroporation transfection methods targeting the sgRNA of B2M and the Cas9 protein.
A. Control group, no gene editing; cells showed double positive clustering of MHC-I and B2M.
B.B2M gene edited group; the cells were both MHC-I and B2M depleted, in a double negative population.
FIG. 2 is a schematic diagram of the structure of an HLA-E chimeric single-chain molecule.
FIG. 3 flow cytometry detection of primary T cell chimeric single chain molecule expression.
A. Expression of the chimeric single-chain molecule in T cells.
B. Expression of chimeric single chain molecules in T cells depleted of MHC class I molecules. The arrows indicate that the cells in which B2M was edited were able to efficiently express the chimeric single-chain molecule.
Figure 4. T cells expressing the chimeric single chain molecule are effective against NK cell killing in vitro.
A. T cells lacking MHC class I molecules express chimeric single chain molecules, and the proportion of the cell population increases after 24 hours of coculture with NK-92 (left) control group and (right).
And B, comparing killing and cracking rates of NK cells.
Figure 5 is a schematic representation of the CAR and chimeric single-chain molecule constructed on the same vector.
FIG. 6 immunophenotypic test.
Car/chimeric single-chain molecule is co-expressed efficiently in T cells.
CAR-T cell CD3/B2M double gene editing efficiency is more than 75%.
C. The CAR-T cell CD3/B2M double gene editing efficiency of the expression chimeric single-chain molecule is more than 75%.
Figure 7 engineered CAR-T cells expressing chimeric single-chain molecules can be effective against NK cell killing.
Nk92 cell killing assay.
B.1# donor primary NK cell killing assay.
Primary NK cell killing assay of c.2# donor.
FIG. 8 proliferation assay of CAR-T cells in resisting NK cell killing; CAR-edi-CM exhibits stronger proliferative activity because it can effectively resist killing of NK cells.
FIG. 9 proliferation assay of CAR-T cells in allogeneic PBMC environment; CAR-edi-CM exhibits stronger proliferative activity due to its potent resistance to NK cell killing in PBMC, maintaining efficient proliferative activity even in a 40-fold allogeneic PBMC environment.
The invention is further illustrated by the following examples. These examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention in any way.
The abbreviations have the following meanings: "h" refers to hours, "min" refers to minutes, "s" refers to seconds, "ms" refers to milliseconds, "d" refers to days, "μ L" refers to microliters, "ml" refers to milliliters, "L" refers to liters, "bp" refers to base pairs, "mM" refers to millimoles, and "μ M" refers to micromoles.
Example 1: engineered T cells producing surface-deleted MHC class I molecules
sgRNA candidate sequences
Based on the information of the relevant websites, we screened and designed sgRNA SEQ ID NO: 17, SEQ ID NO: 21.
Acquisition of Cas9 protein
Cas9 Nuclear 3NLS protein is selected as Cas9 protein in this example, Alt-R s.p.Cas9 NLS protein from IDT DNA technology.
Primary T cell activation
Primary T cells were derived from Peripheral Blood (PBMC) of healthy human volunteers. The culture medium is complete culture medium, ImmunoCultTM-XF T Cell Expansion Medium(Stem Cell Technology，cat#10981)+300
IU/ml IL2(Cayan, cat # HEILP-0201 c). T cells were activated using Dynabeads (Thermo, cat #11141D), which: cell 3: 1. t cells are activated for 24 hours and then are apparently clustered and enlarged.
Electric shock conversion method
4 days after T cell activation, cells were collected, washed 3 times with electrotransfer buffer, buffer T, T4, Opti-MEM (Gibco, cat #31985-8And/ml. In vitro, the two groups of sgrnas (150 ng each) and 1ug of Cas9 protein were mixed uniformly, incubated at room temperature for 10min, and the mixture was added to the resuspended cells for electric shock to a total volume of 10ul, and then subjected to electric transfer using a Neon electrotransfer. The shock voltage conditions were as follows: 1200v, 10ms (3 times).
Gene editing assay
The efficiency of primary T cell editing was examined 3 days after completion of the shock. Take out about 1X106The cells were washed 1 times with 1ml PBS (Gibco, cat # C10010500BT), resuspended in 100ul PBS, added 20ul B2M-PE antibody to the cells, mixed well and incubated at 4 ℃ for 30 min. And detecting by using a flow cytometer after cleaning. As shown in fig. 1, B2M negative cells were genetically edited and these cells also lacked expression of MHC class I molecules, indicating that the lack of B2M affected expression of MHC class I molecules.
Example 2: production of engineered T cells expressing chimeric single chain molecules
Method for preparing lentivirus
Three plasmid systems were used: the lentiviral expression plasmid (Addgene ID: #12252), the packaging helper plasmids psPAX2(Addgene ID: #12260) and pMD2.G (Addgene ID: # 12259). Viral packaging was performed in HEK293T cells (purchased from shanghai cell institute of chinese academy of sciences). The preparation process comprises the following steps: the HEK293T cells in the cryopreserved working cells were thawed, and cultured in DMEM medium (+ 10% FBS + 1% P/S) (Cellgro 10-013-CMR) in a 10cm dish, and the medium was changed after 2 days of thawing. After the cells are full, passage is started (usually 1 dish is full and then 5 dishes are obtained), and after 4 passages, the cells are transfected with plasmid. Our system prefers transfection using PEI as the transfection reagent, PEI: plasmid (mass ratio) 2: 1. The mixture of plasmid and PEI was added to Opti-MEM medium (Gibco, cat # 31985-. After 6 hours of transfection, the medium was replaced with fresh medium containing 2% FBS, and then the culture was continued for 72 hours, and the supernatant of HEK293T cells was collected. The collected virus supernatant was concentrated by ultracentrifugation (82200g, centrifugation at 4-8 ℃ for 2 hours), and the concentrated virus was sterilized by filtration through a 0.22 μm filter and resuspended for use.
Detection of T cell expression of chimeric single chain molecules
T cells were activated (as in example 1), 48 hours later, activated T cells were transfected with lentiviruses prepared as described above, 48 hours later, gene editing (knock-out B2M) was performed, and 72 hours later, positive expression after infection was detected by flow cytometry. As shown in fig. 3, in primary T cells, the chimeric single-chain molecule was well expressed, with an efficiency of about 25% in this example, while the cells lacking B2M (or MHC class I molecules) still expressed HLA-E chimeric single-chain molecule well after B2M was edited (arrow). It is demonstrated that our designed sgRNA editing B2M can specifically recognize and edit B2M in the genome, but not the B2M subunit in the HLA-E chimeric single-stranded molecule.
Example 3: the I MHC molecule-deleted engineered T cell expressing the chimeric single-chain molecule can effectively inhibit the killing of NK cells
A flow cytometry detection method for NK-92 cell killing. Culture medium ImmunoCultTMFrom STEMCELL (same as example 1). NK-92 cells were purchased from the Shanghai cell institute of Chinese academy of sciences. The target cells were: primary T cells derived from peripheral blood of healthy human volunteers, MHC class I molecule-deficient T cells prepared in example 1, and MHC class I molecule-deficient T cells expressing chimeric single-chain molecules prepared in example 2. The final concentration of target cells was 2X 104/ml。
Effector cells NK-92 cells: engineered T-cells ═ 1:1 mixing, culturing in 96-well culture plate at 37 deg.C for 24 hr with 100ul per well. Flow cytometry detection, CD56 protein was used to distinguish NK cells from T cells, and MHC-I protein was used to identify edited T cells.
The expression of the HLA-E chimeric single-chain molecule can effectively inhibit the killing of NK-92 cells. As shown in FIG. 4, the ratio of cells expressing the HLA-E chimeric single-chain molecule after killing by NK-92 cells was significantly larger than the initial ratio (from 39.85% to 65.64% as indicated by the arrow), while the ratio of cells not expressing the HLA-E chimeric single-chain molecule was significantly decreased. It is demonstrated that NK-92 cells are more likely to kill MHC class I molecule-depleted cells, and expression of HLA-E chimeric single chain molecules is effective in inhibiting NK cell-mediated killing. Statistical analysis revealed that cells lacking MHC class I molecules were lysed at about 70%, whereas cells expressing HLA-E chimeric single-stranded body molecules under the same conditions were lysed at about 7.5%.
Example 4: various chimeric single-chain molecules can effectively resist killing of NK cells
Various chimeric single-chain molecules were tested for their effect against NK cell killing. The procedure is as in example 3, the target cells are genetically edited to delete class I MHC molecules using Jurkat cell line (T cell-derived) respectively (procedure of example 2); also, to more fully confirm the function of the chimeric multi-single chain molecule, the K562 cell line was used for the target cells (K562 is a cell with a deletion of native MHC class I molecules). As shown in Table 1, various chimeric multi-single chain molecules can be highly effective against NK cell killing to various degrees in MHC class I molecule-deficient cells.
Table 1: killing rate of NK cells against target cells
Wherein, the sequence of the chimeric single-chain molecule A in the table 1 is SEQ ID NO: 22; the sequence of the chimeric single-chain molecule B is SEQ ID NO: 23; the sequence of the chimeric single-chain molecule C is SEQ ID NO: 24; the sequence of the chimeric single-chain molecule D is SEQ ID NO: 25; the sequence of the chimeric single-chain molecule E is SEQ ID NO: 18.
example 5: generating engineered CAR-T cells with surface deletion of MHC class I molecules and CD3/TCR Complex
Methods for lentivirus preparation, gene editing, etc. were the same as in examples 1 and 2. The deletion of TCR/CD3 complex adopts gene editing TRAC gene, according to the predictive analysis of related websites, we screen and involved sgRNA of editing TRAC gene, the sequence is SEQ ID NO: 26. The CAR gene and the chimeric single-chain molecule gene are connected by 2A (self-cutting polypeptide 2A), and the sequence is SEQ ID NO:20 as shown in FIG. 5. Flow cytometry detection of CAR and chimeric single-chain molecule can efficiently co-express on T cell surface, as shown in fig. 6, expression efficiency is above 20%, CAR expression detection utilizes anti-murine Fab antibody; the double-gene editing efficiency of CD3/B2M is over 75 percent (the editing efficiency, namely the efficiency of knocking out the corresponding gene by gene editing is reflected in the proportion of cells with negative expression of the gene to the total cells).
Wherein, for nomenclature: a gene-edited CAR-T cell, i.e., CAR-edi (CAR-eating, CAR-T cell lacking MHC class I molecules and TCR/CD3 complex); a genetically edited CAR-T cell expressing a Chimeric single chain Molecule, i.e., a CAR-edi-CM (CAR-editing-Chimeric Molecule, CAR-T cell lacking MHC class I molecules and TCR/CD3 complex, while expressing a Chimeric single chain Molecule).
Example 6: engineered CAR-T cells expressing the chimeric single-chain molecule can be effective against killing of primary NK cells.
In vitro NK killing experiments were the same as in example 3. Specifically, the target cells are:
the gene-edited CAR-T cells prepared in example 5, i.e., CAR-edi, and the gene-edited CAR-T cells expressing the chimeric single-chain molecule, i.e., CAR-edi-CM;
primary NK cells were derived from healthy human PBMCs. And respectively detecting the reduction number of the CAR-T cells with the deletion of the MHC class I molecules and the reduction number of the CAR-T cells with the deletion of the MHC class I molecules expressing the chimeric single chains by flow cytometry, and performing killing comparison.
FIG. 6 shows that both NK92 and primary NK cells kill CAR-edi-CM significantly less, indicating that CAR-T cells expressing chimeric single chains are effective against killing of a variety of NK cells.
Example 7: engineered CAR-T cells expressing chimeric single-chain molecules have efficient cell expansion activity while resisting NK cell killing.
Establishment of edited CAR-T cells, tumor Raji cells, NK-92 cells co-culture assay.
The assay simulates an in vivo environment, i.e., the co-presence of tumor cells, CAR-T cells, NK cells, and detects whether the edited CAR-T cells still have tumor cell-induced proliferative capacity while being killed by NK cells. The killing experiment was the same as in example 6. Specifically, cell co-culture, edited CAR-T cells: raji: NK92 ═ 1:5:5, where NK-92 cell number was 1X105Perml, other cells were scaled up, cultured in 24-well plates at 37 ℃ and the number of edited CAR-T cells was recorded at different time points (FIG. 8).
Figure 7 shows that edited CAR-T cells expressing chimeric single-chain molecules (CAR-edi-CM) showed efficient expansion capacity, suggesting that this group of cells can resist killing by NK cells; in contrast, under the same conditions, CAR-edi still did not expand normally even in the presence of Raji tumor cell activation, as NK cells killed it.
Example 6: engineered CAR-T cells expressing the chimeric single-chain molecule have efficient cell expansion activity in the environment of allogeneic PBMC.
Establishment of edited CAR-T cells co-culture assay with allogeneic PBMC cells.
The test scientifically simulates the in vivo environment further, and because the in vivo immune cells are complex and not only contain NK cells and target cells, the real allogeneic PBMC environment is established to be more close to the in vivo environment. The edited CAR-T cells were tested for their ability to expand in an allogeneic PBMC environment, i.e. there was allogeneic immune rejection (not just the originating NK cells), as well as the target cells (CD19 positive B cells).
Cell co-culture, edited CAR-T cells: allogeneic PBMC as 1:10 or 1: 40, wherein the allogeneic PBMCs are PBMCs of volunteers of a different origin than the CAR-T cells prepared. Edited CAR-T cells, cell number 5X 104Perml, other cells were scaled up, cultured in 24-well plates at 37 ℃ and the number of edited CAR-T cells was recorded at different time points (FIG. 8).
Dye eFluor was used before co-culture of allogeneic PBMCTM670 staining to distinguish CAR-T cells from allogeneic PBMC cells: adjusting cell density to 1X107And/ml, adding e670 dye with the final concentration of 10 mu M, incubating for 5min at room temperature in a dark place, and washing the culture medium for three times for experiments.
Figure 8 shows that edited CAR-T cells expressing chimeric single-chain molecules (CAR-edi-CM) showed efficient expansion capacity, suggesting that this group of cells could resist killing by allogeneic immune cells; in contrast, CAR-edi did not expand normally under the same conditions, suggesting that allogeneic PBMC cells killed or somewhat inhibited it. In particular, the data show that the more allogeneic PBMCs, the more significant inhibition of CAR-edi is, however, the CAR-edi-CM group is unaffected and is able to respond to B cells in allogeneic PBMCs with efficient expansion.
Claims (26)
- A chimeric single-chain molecule comprising: (a) presenting the peptide fragment; (b) B2M protein; (c) HLA-E heavy chain; and (d) a linker sequence for linking the aforementioned (a) and (b), and (b) and (c) fragments; wherein the HLA-E heavy chain is HLA-E01: 01 or HLA-E01: 03 subtype.
- The chimeric single-chain molecule of claim 1, comprising, in order from N-terminus to C-terminus: presentation peptide-linker-B2M protein-linker-HLA-E heavy chain.
- The chimeric single-chain molecule of claim 1 or 2, wherein the presenting peptide fragment is a signal peptide of an MHC class I molecule.
- The chimeric single-chain molecule of claim 3, wherein the presenting peptide segment comprises or is selected from one of the following sequences: 2, 4, 5, 6 and 7.
- The chimeric single-chain molecule of claim 4, wherein said presenting peptide fragment is represented by SEQ ID NO 5.
- The chimeric single-chain molecule of any one of the preceding claims, wherein the linker sequence is (EAAAK) n or (GGGGS) n, wherein n-3 or 4 or 5.
- The chimeric single-chain molecule of claim 6, wherein the linker sequence is selected from one or more of the following sequences: SEQ ID NO 8 and SEQ ID NO 9.
- The chimeric single-chain molecule of any one of the preceding claims, wherein the HLA-E01: 01 subtype is SEQ ID NO 12 or a variant thereof.
- The chimeric single-chain molecule of any one of the preceding claims, wherein the HLA-E01: 03 subtype is SEQ ID NO 13 or a variant thereof.
- The chimeric single-chain molecule of any one of the preceding claims is SEQ ID NO: 18. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: shown at 25.
- A nucleic acid sequence expressing the chimeric single-stranded molecule of any one of the preceding claims.
- The nucleic acid sequence of claim 11 as set forth in SEQ ID NO: 19 to
- An expression vector comprising the nucleic acid sequence set forth in claim 11 or 12.
- A host cell comprising the expression vector of claim 13.
- A lymphocyte that expresses on the surface of a cell a chimeric molecule of any one of claims 1-10, wherein said lymphocyte is deficient in an MHC class I molecule.
- The lymphocyte of claim 15, wherein the lymphocyte is a T cell, an NK cell, a B cell, or a macrophage.
- The lymphocyte of claim 16, further characterized in that at least one gene encoding a component of a T Cell Receptor (TCR) is inactivated.
- The lymphocyte of any of claims 15-17, wherein said T cell is a CAR-T cell.
- The lymphocyte of claim 18, wherein said CAR is directed against the B lymphocyte antigen CD 19.
- The lymphocyte of claim 19, wherein the protein sequence that simultaneously expresses the CAR molecule and the chimeric multimeric single-chain molecule in the lymphocyte is as set forth in sequence SEQ ID NO: shown at 20.
- Use of the chimeric single chain molecule of any one of claims 1 to 10 for inhibiting NK cell killing.
- The lymphocyte of any of claims 15-20, for use in inhibiting NK cell killing.
- A method of inhibiting NK cells comprising providing the lymphocytes of any one of claims 15-20 to an NK cell or a patient.
- The method of claim 23, wherein the lymphocyte is preferably a T cell, more preferably a CAR-T cell.
- A pharmaceutical composition for inhibiting NK cell killing comprising the chimeric single chain molecule of any one of claims 1-10.
- A pharmaceutical composition for inhibiting NK cell killing comprising the lymphocytes of any one of claims 15-20.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810595346 | 2018-06-11 | ||
| CN2018105953469 | 2018-06-11 | ||
| PCT/CN2019/090605 WO2019238016A1 (en) | 2018-06-11 | 2019-06-10 | Chimeric single-stranded molecule and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112105650A true CN112105650A (en) | 2020-12-18 |
Family
ID=68842697
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201980031427.9A Pending CN112105720A (en) | 2018-06-11 | 2019-06-10 | Genetically engineered immune lymphocyte and preparation method thereof |
| CN201980031497.4A Pending CN112105650A (en) | 2018-06-11 | 2019-06-10 | Chimeric single-chain molecule and application thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201980031427.9A Pending CN112105720A (en) | 2018-06-11 | 2019-06-10 | Genetically engineered immune lymphocyte and preparation method thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN112105720A (en) |
| WO (2) | WO2019238015A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112830973B (en) * | 2020-04-08 | 2022-11-01 | 苏州克睿基因生物科技有限公司 | Chimeric polypeptides and uses thereof |
| EP4386010A1 (en) * | 2021-08-12 | 2024-06-19 | Cure Genetics Co., Ltd. | Modified cell, and preparation method therefor and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107723275A (en) * | 2017-10-20 | 2018-02-23 | 重庆精准生物技术有限公司 | Universal CAR T cells and its preparation method and application |
| WO2018064594A2 (en) * | 2016-09-29 | 2018-04-05 | Nantkwest, Inc. | Hla class i-deficient nk-92 cells with decreased immunogenicity |
-
2019
- 2019-06-10 CN CN201980031427.9A patent/CN112105720A/en active Pending
- 2019-06-10 WO PCT/CN2019/090604 patent/WO2019238015A1/en active Application Filing
- 2019-06-10 CN CN201980031497.4A patent/CN112105650A/en active Pending
- 2019-06-10 WO PCT/CN2019/090605 patent/WO2019238016A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018064594A2 (en) * | 2016-09-29 | 2018-04-05 | Nantkwest, Inc. | Hla class i-deficient nk-92 cells with decreased immunogenicity |
| CN107723275A (en) * | 2017-10-20 | 2018-02-23 | 重庆精准生物技术有限公司 | Universal CAR T cells and its preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| CREW MD等: "An HLA-E single chain trimer inhibits human NK cell reactivity towards porcine cells", 《MOLECULAR IMMUNOLOGY》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019238016A1 (en) | 2019-12-19 |
| WO2019238015A1 (en) | 2019-12-19 |
| CN112105720A (en) | 2020-12-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7203873B2 (en) | An engineered meganuclease with a recognition sequence found in the human β-2 microglobulin gene | |
| JP6681837B2 (en) | Method for making T cells compatible with allogeneic transplantation | |
| EP3692057B9 (en) | Modified epidermal growth factor receptor peptides for use in genetically-modified cells | |
| JP2021506243A (en) | Immortalized CAR-T cells genetically modified to eliminate T cell receptor and β2 microglobulin expression | |
| KR20210148293A (en) | Gene-modified immune cells containing microRNA-adapted shRNAs (shRNAmiRs) | |
| AU2018202976A1 (en) | Polyclonal gamma delta t cells for immunotherapy | |
| CN109415687A (en) | Chimeric antigen receptor T cell composition | |
| JP2023175699A (en) | Genetically modified t cells comprising modified intron of t cell receptor alpha gene | |
| KR102617818B1 (en) | Optimized engineered nuclease with specificity for human T cell receptor alpha constant region gene | |
| CN108531457A (en) | A kind of method that Cas9/RNP knocks out T cell PD-1 and LAG3 gene and prepares CAR-T cells | |
| Tal et al. | An NCR1-based chimeric receptor endows T-cells with multiple anti-tumor specificities | |
| JP2020519255A (en) | Artificially engineered immune cells | |
| CN111875708B (en) | Chimeric antigen receptor T lymphocyte and application thereof in preparation of product for treating solid tumor | |
| CN112105650A (en) | Chimeric single-chain molecule and application thereof | |
| US20230133564A1 (en) | Cd3-fusion protein and uses thereof | |
| US20220411479A1 (en) | Cd20 chimeric antigen receptors and methods of use for immunotherapy | |
| WO2024047561A1 (en) | Biomaterials and processes for immune synapse modulation of hypoimmunogenicity |
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 | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40034315 Country of ref document: HK |
|
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20201218 |
|
| WD01 | Invention patent application deemed withdrawn after publication |