CN116003628A - Fully human specific chimeric antigen receptor targeting human DLL3 antigen and application thereof - Google Patents
Fully human specific chimeric antigen receptor targeting human DLL3 antigen and application thereof Download PDFInfo
- Publication number
- CN116003628A CN116003628A CN202211314320.5A CN202211314320A CN116003628A CN 116003628 A CN116003628 A CN 116003628A CN 202211314320 A CN202211314320 A CN 202211314320A CN 116003628 A CN116003628 A CN 116003628A
- Authority
- CN
- China
- Prior art keywords
- seq
- amino acid
- car
- acid sequence
- dll3
- 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
- 101000928513 Homo sapiens Delta-like protein 3 Proteins 0.000 title claims abstract description 201
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 title claims abstract description 177
- 239000000427 antigen Substances 0.000 title claims abstract description 49
- 102000036639 antigens Human genes 0.000 title claims abstract description 49
- 108091007433 antigens Proteins 0.000 title claims abstract description 49
- 230000008685 targeting Effects 0.000 title claims description 15
- 102000053255 human DLL3 Human genes 0.000 title abstract description 46
- 210000004027 cell Anatomy 0.000 claims abstract description 186
- 102100036466 Delta-like protein 3 Human genes 0.000 claims abstract description 154
- 229940127276 delta-like ligand 3 Drugs 0.000 claims abstract description 153
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 45
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 claims abstract description 29
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims abstract description 29
- 208000000587 small cell lung carcinoma Diseases 0.000 claims abstract description 29
- 206010041067 Small cell lung cancer Diseases 0.000 claims abstract description 28
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 19
- 210000002865 immune cell Anatomy 0.000 claims abstract description 16
- 230000004068 intracellular signaling Effects 0.000 claims abstract description 9
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 88
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 85
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 56
- 230000014509 gene expression Effects 0.000 claims description 50
- 239000013598 vector Substances 0.000 claims description 29
- 108020004707 nucleic acids Proteins 0.000 claims description 27
- 102000039446 nucleic acids Human genes 0.000 claims description 27
- 150000007523 nucleic acids Chemical class 0.000 claims description 27
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 20
- 208000020816 lung neoplasm Diseases 0.000 claims description 20
- 201000005202 lung cancer Diseases 0.000 claims description 19
- 201000010099 disease Diseases 0.000 claims description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 13
- 239000008194 pharmaceutical composition Substances 0.000 claims description 13
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 11
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 11
- 201000011510 cancer Diseases 0.000 claims description 10
- 230000000139 costimulatory effect Effects 0.000 claims description 9
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 claims description 4
- 210000004986 primary T-cell Anatomy 0.000 claims description 4
- 208000009018 Medullary thyroid cancer Diseases 0.000 claims description 3
- 206010060862 Prostate cancer Diseases 0.000 claims description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 3
- 208000005017 glioblastoma Diseases 0.000 claims description 3
- 230000003834 intracellular effect Effects 0.000 claims description 3
- 208000023356 medullary thyroid gland carcinoma Diseases 0.000 claims description 3
- 201000001441 melanoma Diseases 0.000 claims description 3
- 201000002120 neuroendocrine carcinoma Diseases 0.000 claims description 3
- 230000011664 signaling Effects 0.000 claims description 3
- 230000002147 killing effect Effects 0.000 abstract description 44
- 238000000338 in vitro Methods 0.000 abstract description 28
- 239000003814 drug Substances 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 11
- 230000005764 inhibitory process Effects 0.000 abstract description 8
- 238000002474 experimental method Methods 0.000 abstract description 5
- 238000010171 animal model Methods 0.000 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 description 26
- 210000004881 tumor cell Anatomy 0.000 description 21
- 108090000623 proteins and genes Proteins 0.000 description 18
- 239000013612 plasmid Substances 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 230000008859 change Effects 0.000 description 12
- 238000012258 culturing Methods 0.000 description 9
- 238000003501 co-culture Methods 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000012636 effector Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 6
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 6
- 241001529936 Murinae Species 0.000 description 6
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 6
- 230000000259 anti-tumor effect Effects 0.000 description 6
- 230000007774 longterm Effects 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 108010074328 Interferon-gamma Proteins 0.000 description 5
- 239000006285 cell suspension Substances 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000010361 transduction Methods 0.000 description 5
- 230000026683 transduction Effects 0.000 description 5
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 4
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 4
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 4
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000009260 cross reactivity Effects 0.000 description 4
- 230000000857 drug effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 4
- 230000003285 pharmacodynamic effect Effects 0.000 description 4
- 238000002331 protein detection Methods 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 3
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 101100499378 Mus musculus Dll3 gene Proteins 0.000 description 3
- 101100439689 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) chs-4 gene Proteins 0.000 description 3
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 102000023732 binding proteins Human genes 0.000 description 3
- 108091008324 binding proteins Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000002538 fungal effect Effects 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 3
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 2
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 2
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 102100021244 Integral membrane protein GPR180 Human genes 0.000 description 2
- 102000008579 Transposases Human genes 0.000 description 2
- 108010020764 Transposases Proteins 0.000 description 2
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 101150058049 car gene Proteins 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000005206 flow analysis Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000012212 insulator Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000010534 mechanism of action Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- 244000247812 Amorphophallus rivieri Species 0.000 description 1
- 235000001206 Amorphophallus rivieri Nutrition 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 1
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 1
- -1 CD86 Proteins 0.000 description 1
- 102100037904 CD9 antigen Human genes 0.000 description 1
- 102100025466 Carcinoembryonic antigen-related cell adhesion molecule 3 Human genes 0.000 description 1
- 101100118093 Drosophila melanogaster eEF1alpha2 gene Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 1
- 101000914337 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 3 Proteins 0.000 description 1
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 1
- 101000599940 Homo sapiens Interferon gamma Proteins 0.000 description 1
- 101000777628 Homo sapiens Leukocyte antigen CD37 Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000934346 Homo sapiens T-cell surface antigen CD2 Proteins 0.000 description 1
- 101000934341 Homo sapiens T-cell surface glycoprotein CD5 Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 102100025390 Integrin beta-2 Human genes 0.000 description 1
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 1
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 description 1
- 229920002752 Konjac Polymers 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102100031586 Leukocyte antigen CD37 Human genes 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 108010064548 Lymphocyte Function-Associated Antigen-1 Proteins 0.000 description 1
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 206010041047 Slow virus infection Diseases 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 description 1
- 102100025244 T-cell surface glycoprotein CD5 Human genes 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 208000002458 carcinoid tumor Diseases 0.000 description 1
- 210000002390 cell membrane structure Anatomy 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 235000010485 konjac Nutrition 0.000 description 1
- 239000000252 konjac Substances 0.000 description 1
- 238000002843 lactate dehydrogenase assay Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 101150003509 tag gene Proteins 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
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
-
- 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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- 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
-
- 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
-
- 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
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- 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
- C12N15/11—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
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Abstract
The invention relates to a specific Chimeric Antigen Receptor (CAR) based on a fully human anti-DLL 3 single chain antibody and application thereof. The CAR comprises a CD8 signal peptide, a fully human single chain antibody specific for human DLL3 antigen and a CD8 transmembrane region, 41bb, cd3ζ intracellular signaling domain, in tandem. The present invention provides human DLL3 chimeric antigen receptor modified immune cells that are capable of specifically binding to human DLL3 antigens by constructing and screening for different DLL3 antigen specific humanized single chain antibodies. In-vitro killing experiments on human DLL3 antigen positive tumor models, the human DLL3 antigen-targeted CAR-T cells can generate remarkable specific killing on human DLL3 positive Small Cell Lung Cancer (SCLC) cell strains. Animal model experiments show that after the preferable DLL3CAR-T is dosed, each medicine group has obvious curative effect and the tumor inhibition rate is 25-97%.
Description
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to a chimeric antigen receptor targeting human DLL3, a preparation method thereof and application thereof in preparing medicines, treating and preventing diseases.
Background
Small Cell Lung Cancer (SCLC) is one of lung cancer, and according to NCCN guidelines, accounts for about 15% of all lung cancers, has the characteristics of rapid progression, high recurrence rate, early metastasis, poor prognosis, 5-year survival rate of less than 5%, and limited therapeutic approaches. The traditional radiotherapy and chemotherapy can only benefit in a short period, and an effective means for obviously prolonging the survival time of a patient is not available.
Human DLL3 protein (Delta-Like Ligand, DLL 3) is a single transmembrane protein consisting of 619 amino acids, belonging to the Notch Ligand family. It is a highly tumor-selective cell surface target that is highly expressed in most Small Cell Lung Carcinoma (SCLC) and carcinoid subgroups (Lung Cancer;135:73-79; 2019), but not in normal Lung carcinoma tissues and paracancestor tissues. In one study, independent tumor specimens of 1073 SLCL patients showed that DLL3 positive expression (. Gtoreq.25%) reached 85% and DLL3 high expression (. Gtoreq.75%) reached 68% (Lung Cancer;147:237-243; 2020). Chimeric antigen receptor T cells targeting human DLL3 can stimulate T cells to specifically kill tumor cells by recognizing tumor surface DLL3 antigen.
The AMG757 project developed by american security corporation (Amgen) validated DLL3 targets in the clinic. AMG757 is a bispecific antibody that binds DLL3 and CD 3. In the mid-stage 1 dose discovery study for treatment of SCLC, the disease control rate (disease stabilization/CR/PR) was 51%, median PFS was 3.5 months, median OS was 12.3 months, showing preliminary efficacy of AMG757 and acceptable safety evidence (NCT 03319940). Meanwhile, the DLL3 CAR-T project AMG 119 that it developed was also in a clinical phase I study (NCT 03392064) that assessed safety, tolerability and efficacy for SCLC treatment. The OBT-620 of the Harpoon Therapeutics company HPN-328, boringer John, is also in clinical phase I/II. DLL3 is used as an important Notch ligand and is closely related to the occurrence and development of various tumors, and the development of various treatment methods with DLL3 as a target point is expected to bring more treatment options for clinical tumor treatment.
Aiming at the above, the CAR-T cell targeting DLL3 has great significance as an anticancer cell therapy, especially in cancers with high expression of DLL 3. The invention provides the chimeric antigen receptor T cells of the target human DLL3 with good anti-human DLL3 positive tumor effect, and brings new hope for patients with advanced DLL3 positive tumor.
Disclosure of Invention
In order to solve the technical problem of providing more anticancer drugs, in particular providing drugs for cancers with high expression of DLL3, such as lung cancer, in particular small cell lung cancer, the present invention provides a Chimeric Antigen Receptor (CAR) targeting DLL3, immune cells, such as T cells, comprising said CAR. The invention also provides a nucleic acid encoding the CAR; expression cassettes, vectors, cells containing said nucleic acids; a pharmaceutical composition comprising the CAR, the nucleic acid, the expression cassette, the vector, the cell; a kit comprising the CAR, the nucleic acid, the expression cassette, the vector, the cell, the pharmaceutical composition; the CAR, immune cells comprising the CAR, the nucleic acid, the expression cassette, the vector, the cell, the use of the pharmaceutical composition for preventing, treating, detecting or diagnosing a disease associated with DLL3, or for the manufacture of a medicament or formulation for preventing and/or treating a cancer or tumor, the disease associated with DLL3 being a DLL3 high expression disease, further the disease being a DLL3 high expression cancer or tumor, still further the cancer or tumor being selected from one or more of lung cancer, melanoma, medullary thyroid cancer, glioblastoma, prostate cancer, neuroendocrine cancer; still further the cancer or tumor is lung cancer, in particular small cell lung cancer. The invention also provides a method for preparing the engineering immune cells.
A first aspect of the invention provides a Chimeric Antigen Receptor (CAR) targeting DLL3 antigen, characterized in that the CAR comprises an scFv of an anti-DLL 3 antibody, the scFv comprising 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, wherein:
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 93, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 58, HCDR2 shown in SEQ ID NO. 59 and HCDR3 shown in SEQ ID NO. 60; preferably, the CAR number is 654;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 62, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 38, HCDR2 shown in SEQ ID NO. 64 and HCDR3 shown in SEQ ID NO. 65; preferably, the CAR number is 108;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 73, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 74, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 76, HCDR2 shown in SEQ ID NO. 71 and HCDR3 shown in SEQ ID NO. 77; preferably, the CAR number is 131;
The 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 19, LCDR2 shown in SEQ ID NO. 20 and LCDR3 shown in SEQ ID NO. 21, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 23, HCDR2 shown in SEQ ID NO. 24 and HCDR3 shown in SEQ ID NO. 25; preferably, the CAR number is 332;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 27, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 29, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 31, HCDR2 shown in SEQ ID NO. 32 and HCDR3 shown in SEQ ID NO. 33; preferably, the CAR number 244;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 36, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 38, HCDR2 shown in SEQ ID NO. 39 and HCDR3 shown in SEQ ID NO. 40; preferably, the CAR number is 306;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 42, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 29, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 44, HCDR2 shown in SEQ ID NO. 45 and HCDR3 shown in SEQ ID NO. 46; preferably, the CAR number is 304;
The 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 48, LCDR2 shown in SEQ ID NO. 49 and LCDR3 shown in SEQ ID NO. 50, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 52, HCDR2 shown in SEQ ID NO. 53 and HCDR3 shown in SEQ ID NO. 54; preferably, the CAR number is 229;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 56, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 58, HCDR2 shown in SEQ ID NO. 59 and HCDR3 shown in SEQ ID NO. 60; preferably, the CAR number 564;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 67, LCDR2 shown in SEQ ID NO. 68 and LCDR3 shown in SEQ ID NO. 69, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 44, HCDR2 shown in SEQ ID NO. 71 and HCDR3 shown in SEQ ID NO. 46; preferably, the CAR number is 83;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 48, LCDR2 shown in SEQ ID NO. 49 and LCDR3 shown in SEQ ID NO. 79, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 81, HCDR2 shown in SEQ ID NO. 71 and HCDR3 shown in SEQ ID NO. 82; preferably, the CAR number is 136;
The 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 84, LCDR2 shown in SEQ ID NO. 49 and LCDR3 shown in SEQ ID NO. 79, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 86, HCDR2 shown in SEQ ID NO. 71 and HCDR3 shown in SEQ ID NO. 82; preferably, the CAR number is 412; or alternatively, the process may be performed,
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 67, LCDR2 shown in SEQ ID NO. 68 and LCDR3 shown in SEQ ID NO. 88, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 90, HCDR2 shown in SEQ ID NO. 91 and HCDR3 shown in SEQ ID NO. 92; preferably, the CAR number is 303.
The invention further provides said chimeric antigen receptor targeting DLL3 antigen, characterized in that,
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 61 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 94; preferably, the CAR number is 654;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 95 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 63; preferably, the CAR number is 108;
The scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 72 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 75; preferably, the CAR number is 131;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 18 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 22; preferably, the CAR number is 332;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 26 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 30; preferably, the CAR number 244;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 34 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 37; preferably, the CAR number is 306;
The scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 41 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 43; preferably, the CAR number is 304;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 47 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 51; preferably, the CAR number is 229;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 55 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 57; preferably, the CAR number 564;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 66 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 70; preferably, the CAR number is 83;
The scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 78 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 80; preferably, the CAR number is 136;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 83 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 85; preferably, the CAR number is 412; or alternatively, the process may be performed,
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 87 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 89; preferably, the CAR number is 303.
Further, the CAR further comprises a transmembrane domain, preferably the transmembrane domain is a CD8 transmembrane region, which may also be selected from the transmembrane regions of the following proteins: the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, PD1, or a combination thereof.
Further, the CAR further comprises an intracellular signaling domain, preferably the intracellular signaling domain is a cd3ζ intracellular signaling domain, which may also be selected from signaling domains of the following proteins: fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CDs, CD22, CD79a, CD79b, CD66d, or a combination thereof.
Further, the CAR further comprises a hinge region, preferably the hinge region is a CD8 hinge region, which hinge region may also be selected from the hinge regions of the following proteins: CD28, GM-CSF, CD4, CD137, or a combination thereof.
Further, the CAR further comprises a costimulatory signal domain, preferably the costimulatory signal domain is a 4-1BB costimulatory signal, which can also be selected from the costimulatory signals of the following proteins: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), HVEM, or combinations thereof.
Further, the CAR further comprises a signal peptide, preferably the signal peptide is a CD8 a signal peptide, which signal peptide may also be selected from the signal peptides of the following proteins: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.
Further, the CD8 a signal peptide comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 1; the CD8 hinge region and the transmembrane region comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 2; the 4-1BB costimulatory signal domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 3; alternatively, the CD3ζ intracellular signaling domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence depicted in SEQ ID NO. 4.
Further, the CAR comprises a CD8 alpha signal peptide, a DLL3 antibody scFv VH-linker-DLL3 antibody scFv VL, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB costimulatory signal and a CD3 zeta intracellular signal domain from the N-terminal to the C-terminal in sequence; optionally, the CAR does not comprise the amino acid sequence of the CD8 a signal peptide.
Further, the amino acid sequence of the CAR is shown in any one of SEQ ID NOs 5-17; more preferably, the CAR removes the amino acid sequence of the CD8 alpha signal peptide based on any of the amino acid sequences shown in SEQ ID NOs 5-17.
In a second aspect the invention provides a nucleic acid encoding the CAR.
In a third aspect the invention provides an expression cassette comprising said nucleic acid.
In a fourth aspect the invention provides a vector comprising a nucleic acid encoding said CAR or said expression cassette. The vector may be used to express the CAR. Preferably, the vector may be a viral vector; preferably, the viral vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, or the like; preferably, the vector may be a non-viral vector; preferably, the vector may be a mammalian cell expression vector; preferably, the expression vector may be a bacterial expression vector; preferably, the expression vector may be a fungal expression vector.
In a fifth aspect the invention provides a cell comprising said nucleic acid, or said expression cassette, or said vector, said cell expressing said CAR. Preferably, the cell is a bacterial cell; preferably, the bacterial cells are E.coli cells or the like; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast cells are pichia cells and the like; preferably, the cell is a mammalian cell; preferably, the mammalian cells are chinese hamster ovary Cells (CHO), human embryonic kidney cells (293), B cells, T cells, DC cells, NK cells or the like. Preferably, the cell is an engineered immune cell; more preferably, the engineered immune cell is a T cell; most preferably, the T cell is a primary T cell, a gamma delta T cell or an NK T cell. A sixth aspect of the invention provides a pharmaceutical composition comprising the CAR, nucleic acid, expression cassette, vector or cell, preferably the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, preferably the pharmaceutically acceptable carrier comprises one or more of: pharmaceutically acceptable solvents, dispersing agents, additives, shaping agents and pharmaceutical excipients.
In a seventh aspect the invention provides a kit comprising a CAR of the invention, or a nucleic acid encoding a CAR, or the expression cassette.
In an eighth aspect the invention provides the use of the CAR, nucleic acid, expression cassette, vector or cell in the manufacture of a pharmaceutical composition for the treatment or prophylaxis of a disease.
The ninth aspect of the invention provides the use of the CAR or nucleic acid or expression cassette in the preparation of a diagnostic and detection kit.
In a tenth aspect the invention provides a method of treating or preventing a disease comprising administering the CAR, nucleic acid, expression cassette, vector, cell or pharmaceutical composition of the invention to a subject in need thereof.
An eleventh aspect of the invention provides a method of diagnosis, detection comprising administering the CAR, nucleic acid, expression cassette, kit or pharmaceutical composition of the invention to a subject or sample in need thereof.
A twelfth aspect of the invention provides the use of the CAR, nucleic acid, expression cassette, vector, cell or pharmaceutical composition for the treatment or prevention of a disease.
A thirteenth aspect of the invention provides the use of the CAR, nucleic acid, expression cassette, kit, or pharmaceutical composition for detection, diagnosis.
In a fourteenth aspect the invention provides the use of said CAR, said nucleic acid, or said expression cassette, or said vector, or said pharmaceutical composition for the prevention, treatment, detection or diagnosis of a disease associated with DLL 3.
In the scheme of the invention, the DLL3 related disease is a DLL3 high expression disease; preferably, the disease is a DLL3 high expressing cancer or tumor; more preferably, the cancer or tumor is selected from one or more of lung cancer, melanoma, medullary thyroid cancer, glioblastoma, prostate cancer, neuroendocrine cancer; most preferably, the cancer is lung cancer, particularly small cell lung cancer.
A fifteenth aspect of the present invention provides a method of preparing an engineered immune cell, comprising the steps of:
(1) Providing an immune cell to be modified; and
(2) Introducing the nucleic acid, or the expression cassette, or the vector into the immune cell.
Preferably, the immune cells are T cells; more preferably, the T cell is a primary T cell, a gamma delta T cell or an NK T cell.
The fully human specific chimeric antigen receptor targeting human DLL3 antigen provided by the invention has one or more of the following advantages:
1. The human DLL3 chimeric antigen receptor modified T cells (DLL 3 CAR-T) of the present invention are capable of specifically binding to human DLL3 antigen.
2. The human DLL3 chimeric antigen receptor modified T cell (DLL 3 CAR-T) adopts a full human anti-DLL 3 antibody scfv structure, and has the advantage of low immunogenicity in theory.
3. The 13 DLL3 CAR-T provided by the invention has basically stable CAR expression conditions under different days of culture, and no abnormal phenomena such as drop and the like occur.
4. The in vitro toxicity test of 13 DLL3 CAR-T provided by the invention shows that under the condition of fixed number of tumor cells, the killing efficiency of 12 DLL3 CAR-T and tumor cells under co-incubation with an effective target ratio of 5:1 is 18% -48% after 24 hours for SHP-77 cell strains expressed in human DLL3 antigen; for SHP-77-hDLL3 cell strain with high expression of human DLL3 antigen, the killing efficiency after 24 hours is 21% -56%; while the cell strain NCI-H460 with negative human DLL expression has no obvious killing effect
5. The long-term antitumor effect of different scfv DLL3 CAR-T cells in vitro was evaluated in effector cells: target cells were at 1:10, the present invention 108, 131 and 654scfv DLL3 CAR-T were able to completely clear tumor cells.
6. Animal model experiments show that after the optimized 6 DLL3 CAR-T (332, 304, 229, 654, 108, 131scfv DLL3 CAR-T) are administrated for 17 days, each medicine group has obvious curative effect and the tumor inhibition rate is 25-97%.
Drawings
Fig. 1 shows a schematic structure of recombinant plasmid pRRLSIN-DLL3 CAR-EGFP (PB DLL3 CAR).
FIGS. 2A-2N show the passage of anti-human IgG (Fab) 2 Antibody detection CAR positive flow charts of T lymphocytes expressing different targeted DLL3 chimeric antigen receptors, wherein fig. 2A shows T cell results of the untransduced CAR, fig. 2B shows results of the 332DLL3 CAR-T, fig. 2C shows results of the 244DLL3 CAR-T, fig. 2D shows results of 3067 DLL3 CAR-T, fig. 2E shows results of the 304DLL3 CAR-T, fig. 2F shows results of the 229DLL3 CAR-T, fig. 2G shows results of the 564DLL3 CAR-T, fig. 2H shows results of the 654DLL3 CAR-T, fig. 2I shows results of the 108DLL3 CAR-T, fig. 2J shows results of the 83DLL3 CAR-T, fig. 2K shows results of the 131DLL3 CAR-T, fig. 2L shows results of the 136DLL3 CAR-T, fig. 2M shows results of 412L 3 CAR-T, and fig. 2N shows results of the 303n 3 CAR-T.
3A-3N, a schematic of a CAR positive flow diagram showing detection of T cells expressing different targeted DLL3 chimeric antigen receptors by DLL antigen, wherein FIG. 3A shows T cell results of non-transduced CAR, FIG. 3B shows results of 332DLL3 CAR-T, FIG. 3C shows 244DLL3 CAR-T results, FIG. 3D shows 306DLL3 CAR-T results, FIG. 3E shows 304DLL3 CAR-T results, FIG. 3F shows 229DLL3 CAR-T results, FIG. 3G shows 564DLL3 CAR-T results, FIG. 3H shows 654DLL3 CAR-T results, FIG. 3I shows 108DLL3 CAR-T results, FIG. 3J shows 83DLL3 CAR-T results, FIG. 3K shows 131DLL3 CAR-T results, FIG. 3L shows 136L 3-T results, FIG. 3M shows 413C shows 303DLL3 CAR-T results, and FIG. 3L 3N shows 303L 3.
FIGS. 4A-4B, wherein FIG. 4A shows an anti-human IgG (Fab) 2 CAR positive rate results of 13 DLL3 chimeric antigen receptor-targeted T cells detected by the antibody detection method; fig. 4B shows 13 targeted DLLs 3 detected by the human DLL3 antigen protein detection methodCAR positive rate results for T cells of chimeric antigen receptor.
FIGS. 5A-5B, FIG. 5A shows an anti-human IgG (Fab) 2 The change result of the CAR positive rate of 13T cells targeting the DLL3 chimeric antigen receptor in the antibody detection method under different days of culture; fig. 5B shows the results of the change in CAR positive rate of 13 DLL3 chimeric antigen receptor targeted T cells of the human DLL3 antigen protein detection method at different days of culture.
FIG. 6 shows the results of flow assays showing the expression of DLL3 antigen from human NCI-H460, SHP-77-hDLL3 lung cancer cell lines.
Fig. 7A to 7C, fig. 7A shows the in vitro specific killing result of 13 DLL3 CAR-T cells against NCI-H460 (non-small cell lung cancer) cell line, fig. 7B shows the in vitro specific killing result of 13 DLL3 CAR-T cells against SHP-77 (small cell lung cancer) cell line, and fig. 7C shows the in vitro specific killing result of 13 DLL3 CAR-T cells against human DLL3 positive SHP-77-hll 3 (small cell lung cancer).
Fig. 8A to 8C, fig. 8A shows IFN- γ release after co-culturing 13 DLL3 CAR-T with NCI-H460 cell lines, fig. 8B shows IFN- γ release after co-culturing 13 DLL3 CAR-T with SHP-77 cell lines, and fig. 8C shows IFN- γ release after co-culturing 13 DLL3 CAR-T with SHP-77-hDLL3 cell lines.
Fig. 9A to 9C, fig. 9A shows the in vitro killing result of 13 DLL3 CAR-T cells against a 293T cell line, fig. 9B shows the in vitro killing result of 13 DLL3 CAR-T cells against a human DLL3 protein over-expressed 293T cell line (293T-hll 3), and fig. 9C shows the in vitro killing result of 13 DLL3 CAR-T cells against a murine DLL3 protein over-expressed 293T cell line (293T-mll 3).
Fig. 10A to 10C, fig. 10A shows IFN- γ release results after in vitro killing of 293T cell lines by 13 DLL3 CAR-T cells, fig. 10B shows IFN- γ release results after in vitro killing of 293T cell lines (293T-hll 3) over-expressed by human DLL3 protein by 13 DLL3 CAR-T cells, and fig. 10C shows IFN- γ release results after in vitro killing of 293T cell lines (293T-mll 3) over-expressed by murine DLL3 protein by 13 DLL3 CAR-T cells.
Fig. 11A to 11B show the long-term antitumor effect results of 13 DLL3 CAR-T cells on DLL3 expression positive cells, wherein fig. 11A shows the change in the number of tumor cells of 13 DLL3 CAR-T cells on DLL3 expression positive cells, and fig. 11B shows the change in the number of CAR positive T cells of 13 DLL3 CAR-T cells on DLL3 expression positive cells.
Fig. 12A-12G show different DLL3 CAR-T cell positive rate detection results for in vivo efficacy studies, with fig. 12A showing T cell results for untransduced CAR, fig. 12B showing 332dl l3 CAR-T results, fig. 12C showing 3049 dl l3 CAR-T results, fig. 12D showing 229dl l3 CAR-T results, fig. 12E showing 264 dl l3 CAR-T results, fig. 12F showing 108dl l3 CAR-T results, and fig. 12G showing 131dl l3 CAR-T results.
Fig. 13 shows a pharmacodynamic study of the weight change of 6 DLL3 CAR-T cells in small cell lung cancer SHP-77 tumor-bearing NCG mice.
Fig. 14 shows a pharmacodynamic study of the mouse tumor size change of 6 DLL3 CAR-T cells in small cell lung cancer SHP-77 tumor-bearing NCG mice.
Fig. 15 shows a pharmacodynamic study of the mouse survival of 6 DLL3 CAR-T cells in small cell lung cancer SHP-77 tumor-bearing NCG mice.
Detailed Description
The invention will be further illustrated with reference to specific examples. The described embodiments are some, but not all, embodiments of the invention. It is to be understood that the following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compositions of the present invention may be utilized and are not intended to limit the scope of what the present invention may be used. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 preparation of a targeting DLL3 Chimeric Antigen Receptor (CAR) Gene fragment
The invention designs fusion gene segments according to the sequence of the following coding genes: CD8 alpha signal peptide, DLL3 anti-body scFv VH-linker-DLL3 anti-body scFv VL, CD8 hinge The fusion gene is directly synthesized by a gene synthesis technology through the region, a CD8 transmembrane region, a 4-1BB co-stimulatory signal and a CD3 zeta intracellular signal domain, so that the expressed chimeric antigen receptor has the amino acid structure of scFv VH-linker-scFv VL-CD8 hinge-CD8TM-4-IBB-CD3 zeta. Wherein the linker (linker) has the amino acid sequence of (G) 4 S) 3 Namely, the GGGGSGGGGSGGGGS, CD alpha signal peptide has the amino acid sequence of SEQ ID NO. 1, the CD8 hinge region (CD 8 hinge) has the amino acid sequence of SEQ ID NO. 2, the 4-1BB has the amino acid sequence of SEQ ID NO. 3, the CD3 zeta has the amino acid sequence of SEQ ID NO. 4, and the specific sequences of SEQ ID NO. 1-4 are shown in tables 1-1. The targeted DLL3 chimeric antigen receptor gene fragment is prepared by a gene synthesis technology, the amino acid sequences of the gene fragment are shown in SEQ ID NO. 5-17, and the specific sequences are shown in tables 1-2. Wherein the amino acid sequences of the scFv VH and VL, LCDR1-3 and HCDR1-3 of the different active clone anti-DLL 3 antibodies used in the CAR are shown in tables 1-3, and the analysis system is an IMGT system.
TABLE 1-1 CAR partial element amino acid sequence
TABLE 1-2 DLL3 CAR amino acid sequence
TABLE 1-3 cloning of variable region and CDR amino acid sequences for anti-DLL 3 antibody Activity
Note that: the underlined parts of VL and VH are CDRs.
EXAMPLE 2 preparation of DLL3 specific chimeric antigen receptor modified T cells
2.1 construction of non-viral Piggybac (PB) transposon vector
The pBluescirpt vector (general biosynthesis) is taken as an initial framework, a gene insulator sequence cHS4 is found, the gene insulator sequence cHS4 is arranged at two ends of a polyclonal site, 5'ITR and 3' ITR sequences of PB transperson are found, the inner side of the cHS4 sequence entering the vector is constructed, an EF1a promoter is inserted at the 5 end and a polyA signal is inserted at the 3 end, a polyclonal sequence is reserved in the middle, a CD8 alpha-DLL 3 anti-quality ScFv HV-linker-ScFv VL-CD8 hinge-CD8TM-4-1BB-CD3 zeta (DLL 3 CAR) sequence gene is accessed in the polyclonal sequence, and an EGFP tag gene sequence is accessed behind the DLL3 CAR sequence to be connected with P2A. The above sequences form pRRLSIN-DLL3 CAR-EGFP plasmid structure (plasmid is called PB DLL3 CAR for short), and the schematic diagram is shown in FIG. 1.
PB DLL3 CAR vectors constructed from 13 different DLL3 antibodies ( active clone numbers 332, 244, 306, 304, 229, 564, 654, 108, 83, 131, 136, 412, 303) scfv were extracted (completed by nanking kunstagain) to give PB DLL3 CAR transfection grade plasmids.
2.2 Preparation of T lymphocytes
Magnetic bead labeling was performed on Peripheral Blood Mononuclear Cells (PBMC) (Shanghai Australian organisms) by the CD3 microblades human-lyophilized Kit (Miltenyi Biotech), and high-purity CD3 positive T lymphocytes were positively sorted, and the proportion of the sorted CD3 positive T cells was over 95%. The purified T cells were then used for T lymphocyte activation and proliferation using Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher, 11132D).
2.3 CAR gene transduction T cells
The PB DLL3 CAR plasmids constructed using 13 DLL3 antibodies (332, 244, 306, 304, 229, 564, 654, 108, 83, 131, 136, 412, 303) scfv obtained in example 2.1 were used for electrotransformation, respectively, 72-96 hours after T cell stimulation activation. T cells for electrotransformation were first resuspended, T cell pellet was blown off using a pipette tip or pipette, T cell resuspension was counted using a 5x10 6 T cells were used for a single electrotransformation experiment. Using DPBS (GIBCO, 14190-144) 5X10 6 Cells were diluted to 5mL, centrifuged for 10 min at 300x g at room temperature, supernatant was removed as much as possible, T cell pellet was avoided by resuspension washing T cells with 5mL DPBS, supernatant was removed as much as possible by centrifugation for 10 min at 300x g at room temperature, T cell pellet was avoided by resuspension with 100 μl electrotransfer buffer en-E (Engreen, 98668-20), and cell suspension was transferred to a 1.5mL centrifuge tube. The components in Table 2 were added to a centrifuge tube and mixed well.
TABLE 2 electric rotating system for each group
| Component (A) | Volume (mu L) |
| PB DLL3 CAR plasmid (1 ug/uL) | 5 |
| Transposase mRNA(1ug/uL) | 5 |
| |
100 |
| Total volume of | 110 |
The cell/plasmid suspension was rapidly transferred to the cuvette using a 4D-Nucleofector nuclear transfer machine (Lonza corporation) and gently bumped into the cuvette to allow the cell suspension to fully form a balanced liquid surface in the cuvette, using the program EO115 for electrotransfer. After the electrical inversion, the cuvette was carefully removed. 500uL of pre-warmed T cell medium X-VIVO 15 (Lonza, 04-418Q) was added and equilibrated in an incubator at 37℃for 5 minutes, cells were resuspended using microwell loading tips and gently blown 2-3 times. Cells were transferred to a 12-well plate with 2mL of pre-warmed medium and incubated at 37 ℃. The cell is changed for 4-6 hours after electrotransformation, which is beneficial to increasing the survival rate of the cell. The supernatant was carefully aspirated and pre-warmed fresh medium was added. At 37 ℃,5% CO 2 Culturing in incubator for 48-72 hr until detection.
2.4 DLL3 CAR-T cell electrotransport efficiency detection
After 48-72h of electrotransformation, 13 groups of T cells following electrotransformation of the PB DLL3 CAR plasmid were obtained for CAR positive rate flow analysis of T cell controls not subjected to CAR plasmid electrotransformation. Anti-human IgG (Fab) labeled with biotin 2 Antibody (jackson immunounit, 109-065-006) or human DLL3 antigen protein (ACRO, DL3-H82E 4) as CAR binding protein, and using avidin coupled PE fluorescent dye through flowCell technology detects Chimeric Antigen Receptor (CAR) expression, and the non-transduced T lymphocytes are used as negative controls, and the CAR positive rate of the T lymphocytes expressing different chimeric antigen receptors is shown in table 3, and the CAR positive rate of the T lymphocytes ranges from 10% to 50%. The experimental results show that: the CAR structures constructed by different scfvs can be used for preparing the CAR-T cells, and under the same condition, the higher the transduction efficiency is, the more favorable the subsequent application is. Fig. 2A-2N show CAR positive flow charts of detection of T lymphocytes expressing different targeted DLL3 chimeric antigen receptors by anti-human IgG (Fab) 2 antibodies, wherein fig. 2A shows T cell results of the untransduced CAR, fig. 2B shows results of the 334 DLL3 CAR-T, fig. 2C shows results of the 244DLL3 CAR-T, fig. 2D shows results of 306DLL3 CAR-T, fig. 2E shows results of 3049 DLL3 CAR-T, fig. 2F shows results of 229DLL3 CAR-T, fig. 2G shows results of 564DLL3 CAR-T, fig. 2H shows results of 254 DLL3 CAR-T, fig. 2I shows results of 108DLL3 CAR-T, fig. 2J shows results of 83DLL3 CAR-T, fig. 2K shows results of 131l 3 CAR-T, fig. 2L shows results of 136DLL3 CAR-T, fig. 2M shows results of 303l 3 CAR-T, and fig. 2M shows results of 303l 3 CAR-T. Fig. 3A-3N show CAR positive flow charts of T cells expressing different targeted DLL3 chimeric antigen receptors by DLL antigen detection, wherein fig. 3A shows T cell results for non-transduced CARs, fig. 3B shows results for 332DLL3 CAR-T, fig. 3C shows 244DLL3 CAR-T results, fig. 3D shows 306DLL3 CAR-T results, fig. 3E shows 304DLL3 CAR-T results, fig. 3F shows 229DLL3 CAR-T results, fig. 3G shows 564DLL3 CAR-T results, fig. 3H shows 654DLL3 CAR-T results, fig. 3I shows 108DLL3 CAR-T results, fig. 3J shows 83DLL3 CAR-T results, fig. 3K shows 131l 3 CAR-T results, fig. 3L shows 136DLL 3-T results, fig. 3M shows 413D 3 CAR-T results, fig. 303l 3 CAR-T shows. FIG. 4A shows an anti-human IgG (Fab) 2 Results of CAR positive rate of 13 DLL3 chimeric antigen receptor-targeted T cells detected by the antibody detection method. Figure 4B shows the CAR positive rate results for 13 DLL3 chimeric antigen receptor-targeted T cells detected by the human DLL3 antigen protein detection method.
TABLE 3 Positive Rate of T lymphocytes expressing different chimeric antigen receptors
2.5 DLL3 CAR-T cell CAR expression stability detection
Continuously culturing T cells subjected to electric transformation of 13 groups of PB DLL3 CAR plasmids and T cells not subjected to electric transformation of the CAR plasmids, simultaneously detecting the CAR positive rate on days 4, 6, 8 and 11 after electric transformation, respectively adopting biotin-labeled anti-human IgG (Fab) 2 antibodies and human DLL3 antigen proteins as CAR binding proteins, detecting Chimeric Antigen Receptor (CAR) expression by using an avidin-coupled PE fluorescent dye through a flow cytometry technology, taking non-transduced T lymphocytes as negative controls, and recording the variation of the CAR positive rate of 13 DLL3 CAR-T under different days of culture. FIG. 5A shows an anti-human IgG (Fab) 2 The change result of the CAR positive rate of 13T cells targeting the DLL3 chimeric antigen receptor in the antibody detection method under different days of culture; fig. 5B shows the results of the change in CAR positive rate of 13 DLL3 chimeric antigen receptor targeted T cells of the human DLL3 antigen protein detection method at different days of culture. The results show that the detection of the positive rate of different DLL3 CAR-T in a certain time range has basically stable CAR expression condition and no abnormal phenomena such as drop and the like.
EXAMPLE 3 in vitro toxicity test of DLL3 specific chimeric antigen receptor modified T cells
An in vitro efficacy test was established by modeling the mechanism of action (MOA) of the product. The lung cancer cell strains NCI-H460 (non-small cell lung cancer), SHP-77 (small cell lung cancer) and SHP-77-hDLL3 (small cell lung cancer) related to the DLL3 target are taken as target cells for the function verification of the DLL3CAR-T cell, wherein the SHP-77-hDLL3 cell strain is SHP-77 cell which is obtained by the over-expression of human DLL3 protein and the slow virus infection and screening, the human DLL3 antigen detection is carried out on the three lung cancer cell strains by a flow cytometry, the human DLL3 antigen of the NCI-H460 cell strain is expressed in a low mode, the human DLL3 antigen of the SHP-77 cell strain is expressed in the human DLL3 antigen, the high expression of the human DLL3 antigen of the SHP-77-hDLL3 cell strain is carried out, and the flow detection results of the human NCI-H460, SHP-77 and SHP-hDLL 3 lung cancer cell strain expression flow are shown in FIG. 6.
According to different E:T (effector cells: target cells) ratios, a co-culture system of the CAR-T cells and the targeted tumor cells is established, the biological efficacy of the CAR-T is evaluated by detecting the killing rate of the tumor cells, and a co-culture control system of the non-transduced T cells and the tumor cells is established. Fig. 7A-C show in vitro specific killing results of different DLL3CAR-T cells against lung cancer cell lines, fig. 7A shows in vitro specific killing results of 13 DLL3CAR-T cells against NCI-H460 (non-small cell lung cancer) cell lines, fig. 7B shows in vitro specific killing results of 13 DLL3CAR-T cells against SHP-77 (small cell lung cancer) cell lines, and fig. 7C shows in vitro specific killing results of 13 DLL3CAR-T cells against human DLL3 positive SHP-77-hll 3 (small cell lung cancer). T in fig. 7A-C represents T cell experimental results for the untransduced CAR. In-vitro experiment results show that under the condition of fixed number of tumor cells, except 564scfv CAR-T, when the rest 12 DLL3CAR-T are incubated with the tumor cells at an effective target ratio of 5:1, the killing efficiency of the SHP-77 cell strain expressed in the human DLL3 antigen after 24 hours is 18% -48%; for SHP-77-hDLL3 cell strain with high expression of human DLL3 antigen, the killing efficiency after 24 hours is 21% -56%; but has no obvious killing effect on the cell strain NCI-H460 with low expression of the human DLL antigen. The results demonstrate that 12 kinds of DLL3CAR-T can specifically kill tumor cells expressed by human DLL3 antigens, and the killing efficiency is positively correlated with the expression amount of the human DLL3 antigens. Table 5 shows the killing effect of 13 DLL3CAR-T on the three lung cancer cell lines.
TABLE 5 killing effect of 13 DLL3 CAR-T on the three lung cancer cell lines
Meanwhile, CAR-T biological efficacy was assessed by detecting the amount of cytokine (INF- γ) secreted into the culture supernatant, and after DLL3 CAR-T co-culture with human DLL3 highly expressed tumor cells (SHP-77-hll 3), the remaining 12 DLL3 CAR-T cell groups killed tumor cells with at least 2-fold higher levels of cytokine IFN- γ release than negative controls (non-CAR transduced T cells) except 564scfv CAR-T (see fig. 8C, table 6). Table 6 shows IFN-gamma release after co-culturing 13 DLL3 CAR-T of the present invention with three cell lines NCI-H460, SHP-77-hDLL3 without CAR transduced T cells. Fig. 8A-C show IFN- γ release results after killing 3 lung cancer cells by different DLL3 CAR-T cells, wherein fig. 8A shows IFN- γ release after co-culturing 13 DLL3 CAR-T with NCI-H460 cell lines, fig. 8B shows IFN- γ release after co-culturing 13 DLL3 CAR-T with SHP-77 cell lines, fig. 8C shows IFN- γ release after co-culturing 13 DLL3 CAR-T with SHP-77-hDLL3 cell lines, and T in fig. 8A-C represents IFN- γ release by T cells that do not transduce CARs.
TABLE 6 Co-culture supernatant cytokine IFN-gamma Release
Based on the in vitro cytotoxicity test, the 12T lymphocytes expressing different DLL3 chimeric antigen receptors can kill DLL3 positive lung cancer cells well except 564scfv CAR-T, which provides basis for in vivo drug effect study of animals.
Wherein, the specific killing detection method comprises the following steps: the lactate dehydrogenase release assay kit (LDH Release Assay Kit) (Japanese Konjac chem., CK 12) is used to detect the activity of lactate dehydrogenase released upon cytotoxicity by colorimetry based on the Diaphorase-catalyzed INT chromogenic reaction. The principle is that the disruption of cell membrane structure caused by apoptosis or necrosis can lead to the release of enzymes in the cytoplasm into the culture broth, including lactate dehydrogenase (lactate dehydrogenase, LDH) with relatively stable enzyme activity. Quantitative analysis of cytotoxicity can be achieved by detecting the activity of LDH released into the culture solution from cells ruptured in plasma membranes. LDH release is seen as an important indicator of cell membrane integrity and is widely used in cytotoxicity assays.
Wherein, cytokine detection method: the detection is carried out by using a human IFN-gamma enzyme-linked immunosorbent assay kit (R & D Systems, SIF 50), and is based on the immobilization of antigen or antibody and the enzyme labeling of antigen or antibody. The antigen or antibody bound to the surface of the solid support retains its immunological activity, and the enzyme-labeled antigen or antibody retains both its immunological activity and its enzymatic activity. In performing the detection, the test substance (antigen or antibody) in the sample binds to the immobilized antibody or antigen. The unbound material is removed by washing the plate, and enzyme-labeled antigen or antibody is added, at which time the amount of enzyme immobilized is correlated with the amount of the test substance in the sample. And (3) adding a substrate for reaction with the enzyme, developing, judging the content of the substance in the sample according to the color, and performing qualitative or quantitative analysis.
Example 4: cross-reactivity test of DLL3 specific chimeric antigen receptor modified T cells on DLL3 expressing positive cells of different genera
CAR-T product safety was assessed by immunodeficient mice, the most critical point of which was the cross-reactivity of its antibody scfv to the mouse's own target protein, and therefore the present invention performed a cross-reactivity test of DLL 3-specific chimeric antigen receptor modified T cells to DLL3 expressing positive cells of different genera.
293T cells highly expressing human DLL3 protein or murine DLL3 protein obtained by screening on 293T cells by infection with human DLL3 protein or murine DLL3 protein lentivirus purchased from Gibby organisms were designated as 293T-hDLL3 and 293T-mDLL3, respectively. The prepared DLL3CAR-T with different scfvs is taken as effector cells, 293T cells which over express human DLL3 proteins and 293T cells which over express mouse DLL3 proteins are taken as target cells, a co-culture system of the CAR-T cells and the target cells is established according to different E:T (effector cells: target cells=5:1, 2.5:1 and 1.25:1) ratios, and the specific reaction of the CAR-T to the two proteins is evaluated by detecting the killing rate of tumor cells. The in vitro results are shown in fig. 9A-9C, fig. 9A shows the in vitro killing results of 13 DLL3CAR-T cells against the 293T cell line, fig. 9B shows the in vitro killing results of 13 DLL3CAR-T cells against the human DLL3 protein over-expressed 293T cell line (293T-hll 3), and fig. 9C shows the in vitro killing results of 13 DLL3CAR-T cells against the murine DLL3 protein over-expressed 293T cell line (293T-mll 3). Tables 7-1 to 7-3 show the results of killing assays of 13 DLL3CAR-T, and non-CAR transduced T cells against 293T, 293T-hDLL3, 293T-mDLL3 cell lines. In vitro experiment results show that in the condition of fixed tumor cell number, the prepared DLL3CAR-T of different scfvs and 293T-hDLL3 tumor cells have the effective target ratio of 5: when the cell is incubated with 293T-mDLL3 tumor cells at the effective target ratio of 1.25:1, 2.5:1 and 5:1, the specific killing of DLL3CAR-T prepared by different scfvs is obviously different, which indicates that the DLL3CAR-T can attack tissues distributed on a mouse DLL3 target in a mouse body, thereby being capable of being used for evaluating the in vivo safety of the DLL3CAR-T cells.
TABLE 7-1. Killing test results of DLL3 CAR-T on 293T cell lines
TABLE 7 results of test for killing of 293T-hDLL3 cell lines by 2 DLL3 CAR-T
TABLE 7 results of test for killing of 293T-mDLL3 cell lines by 3 DLL3 CAR-T
While CAR-T specific responses were assessed by detecting the amount of cytokine (INF- γ) secreted into the culture medium supernatant, the difference in IFN- γ cytokine release expression from DLL3 CAR-T and DLL3 specific chimeric antigen receptor modified T cell co-culture supernatants of different scfvs (see fig. 10A-C, table 8) was consistent with the killing assay results (see fig. 9A-C, table 7-1 to table 7-3). Fig. 10A shows IFN- γ release results after in vitro killing of 293T cell lines by 13 DLL3 CAR-T cells, fig. 10B shows IFN- γ release results after in vitro killing of 293T cell lines (293T-hll 3) over-expressed by human DLL3 protein by 13 DLL3 CAR-T cells, and fig. 10C shows IFN- γ release results after in vitro killing of 293T cell lines (293T-mll 3) over-expressed by murine DLL3 protein by 13 DLL3 CAR-T cells. Table 8 shows the specific test results of IFN-gamma release detection after killing of 13 DLL3 CAR-T against 293T, 293T-hDLL3, 293T-mDLL3 cell lines.
The research result shows that different scfv DLL3 CAR-T cells related to the invention can generate killing and releasing cytokines for 293T cells over-expressed by human DLL3 protein, and can generate killing and releasing cytokines IFN-gamma for 293T cells over-expressed by mouse DLL3 protein, thus proving that the cell has cross reactivity to the target protein of the mouse and providing theoretical basis for evaluating safety by adopting a mouse animal model in later period.
TABLE 8 IFN-gamma Release assay results after killing of DLL3 CAR-T on 293T, 293T-hDLL3, 293T-mDLL3 cell lines
Example 5: continuous inhibition test of DLL3 specific chimeric antigen receptor modified T cells on human small cell lung cancer SHP-77 cell strain
To evaluate the long-term antitumor effect of different scfv DLL3 CAR-T cells in vitro, the present invention was designed for this example 5. The DLL3 CAR-T with different scfvs is used as effector cells, a human small cell lung cancer SHP-77 cell strain is used as target cells, a cytokine-free co-culture system is adopted according to an E:T (effector cells: target cells) ratio of 1:10, and the continuous tumor killing capacity of the DLL3 CAR-T cells is evaluated by detecting the quantity change of the CAR-T cells and tumor cells in the co-culture system.
Fig. 11A-B show the results of long-term antitumor effect of 13 DLL3 CAR-T cells on DLL 3-expressing positive cells SHP-77, wherein fig. 11A shows the results of change in the number of tumor cells of 13 DLL3 CAR-T cells on DLL 3-expressing positive cells SHP-77 long-term antitumor effect, and fig. 11B shows the results of change in the number of CAR-positive T cells of 13 DLL3 CAR-T cells on DLL 3-expressing positive cells SHP-77 long-term antitumor effect. The results of the study showed that, in effector cells: target cells were at 1:10, DLL3 CAR-T of different scfvs differ in sustained inhibition ability against human small cell lung cancer SHP-77 cell line, wherein 108, 131 and 654scfv DLL3 CAR-T were able to completely clear tumor cells.
EXAMPLE 6 in vivo efficacy test of DLL3 CAR-T animals
In this example 6, an immunodeficient mouse drug effect model of human small cell lung cancer tumor cell burden was established for evaluating the drug effect and safety of different scfv DLL3 CAR-T cells in mice.
6.1 DLL3 CAR-T cell preparation
6.1.1 T cell sorting and activation
Peripheral Blood Mononuclear Cells (PBMC) (isolated service by Shanghai Australian Co.) were provided, cells were labeled with magnetic beads by the CD3 microblades human-lyophilized Kit (available from Miltenyi Biotech), high purity CD3+ T lymphocytes were positively sorted, and the proportion of CD3 positive T cells after sorting was 95% or more. The purified T cells were then used for T lymphocyte activation and proliferation using Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher, 11132D).
6.1.2 CAR gene transduction T cells
The PB DLL3 CAR plasmids constructed from the preferred 6DLL3 antibodies (332, 304, 229, 654, 108, 131) scfv were electrotransformed. Cells for electrotransformation were first resuspended, cell pellet was blown off using a pipette tip or pipette, cell resuspension was counted, 5x10 was used 6 Cells were used for a single electrotransformation experiment. Using DPBS (GIBCO, 14190-144) 5X10 6 Diluting the cells to 5mL, centrifuging at room temperature for 10 min at 300 and x g, and removing supernatant as much as possibleCell pellet was avoided, cells were washed by resuspension with 5mL DPBS, centrifuged at 300 f x g for 10 min at room temperature, the supernatant was removed as much as possible, cell pellet was avoided, 100 μl electrotransfer buffer Entranster-E (Engreen, 98668-20) was added to resuspension cells, and the cell suspension was transferred to a 1.5mL centrifuge tube. The components in Table 9 (per electrotransport system) were added to the centrifuge tube and mixed well.
TABLE 9 electric rotating system for each group
| Component (A) | Volume (mu L) |
| PB DLL3CAR plasmid (1 ug/uL) | 5 |
| Transposase mRNA(1ug/uL) | 5 |
| |
100 |
| Total volume of | 110 |
6.1.3 DLL3CAR-T cell electrotransport efficiency detection
After 48-72h of electrotransformation, 6 groups of T cells following electrotransformation of the PB DLL3CAR plasmid and T cell controls not subjected to CAR plasmid electrotransformation were obtained for CAR positive rate flow analysis. Chimeric Antigen Receptor (CAR) expression was detected by flow cytometry using biotin-labeled human DLL3 antigen protein (ACRO, DL3-H82E 4) as CAR binding protein, using avidin-coupled PE fluorochromes as negative controls, T lymphocytes expressing 6 different chimeric antigen receptors with positive rates as shown in table 10, fig. 12A-12G showing 6 different DLL3CAR-T cell positive rate detection results for in vivo efficacy studies, with positive rates ranging between 10-40%, wherein fig. 12A shows T cell results for untransduced CARs, fig. 12B shows 332DLL 3CAR-T results, fig. 12C shows 3049 DLL3CAR-T results, fig. 12D shows 229DLL 3-T results, fig. 12E shows 654DLL 3CAR-T results, fig. 12F shows 108DLL3-T results, and fig. 12G shows 131l 3CAR-T results.
TABLE 10 Positive Rate of T lymphocytes expressing different chimeric antigen receptors
| Grouping | CAR Positive Rate |
| T cells not transduced with CAR (Mock T) | 0.12％ |
| 332DLL3 CAR-T | 24.18％ |
| 304DLL3 CAR-T | 26.50％ |
| 229DLL3 CAR-T | 26.92％ |
| 654DLL3 CAR-T | 12.77％ |
| 108DLL3 CAR-T | 33.72％ |
| 131DLL3 CAR-T | 24.63％ |
6.1.4 DLL3 CAR-T animal model test
Female NCG mice (purchased based on in vitro studies self-collecting medicine-extracting medicine-recovering) 5 x 10 percutaneous injection 6 Personal small cell lung cancer cell SHP-77, 11 th day (tumor volume at 60 mm) of inoculation of SHP-77 cells 3 Size or so), mock-T (T cell group without CAR transduction) group was 5×10 6 T cell numbers, 5X 10 for each different scfv DLL3 CAR-T cell group (332, 304, 229, 654, 108, 131scfv DLL3 CAR-T) were dosed individually 6 The CAR positive T cells were counted and the dosing volume was 200uL. The number of animals in all condition groups was 5. Tumor amount 2 times per week after administration; tumor growth curves are drawn, TGI and T/C are calculated, and all tumors are photographed at the test end point. The amplification of CAR-T cells was confirmed by qPCR method of detecting the copy number (VCN) of CAR in the peripheral blood of mice at days 2, 14 and 28 and the end point after administration of CAR-T. The results show that the CAR-T has obvious curative effect on each medicine group after 17 days of administration, the tumor inhibition rate is 25-97%, and the medicine comprises the following indexes:
(1) Weight of: compared to Mock T group (T cell group without CAR transduction), there was no significant difference in body weight among different scfv DLL3 CAR-T dosing groups, and fig. 13 shows a pharmacodynamic study of changes in body weight of mice of 6 DLL3 CAR-T cells in small cell lung cancer SHP-77 tumor-bearing NCG mice.
(2) Tumor inhibition (TGI) statistics: by 17 days after the medicine, each medicine group has obvious curative effect, the tumor inhibition rate is 25-97%, wherein 108 medicine effect is optimal, the tumor inhibition rate is 96.53%, and fig. 14 shows the medicine effect study of the change of the tumor sizes of 6 DLL3 CAR-T cells in small cell lung cancer SHP-77 tumor-bearing NCG mice.
(3) Mortality rate: by 17 days after the administration, mice in the Mock-T cell group and the different scfv DLL3 CAR-T administration groups are good in survival state, no death phenomenon of the mice occurs, and fig. 15 shows the drug effect study of the survival rate of 6 DLL3 CAR-T cells in small cell lung cancer SHP-77 tumor-bearing NCG mice.
Claims (13)
1. A Chimeric Antigen Receptor (CAR) that targets DLL3 antigen, wherein the CAR comprises an scFv of an anti-DLL 3 antibody comprising 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, wherein
The 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 93, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 58, HCDR2 shown in SEQ ID NO. 59 and HCDR3 shown in SEQ ID NO. 60;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 62, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 38, HCDR2 shown in SEQ ID NO. 64 and HCDR3 shown in SEQ ID NO. 65;
The 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 73, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 74, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 76, HCDR2 shown in SEQ ID NO. 71 and HCDR3 shown in SEQ ID NO. 77;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 19, LCDR2 shown in SEQ ID NO. 20 and LCDR3 shown in SEQ ID NO. 21, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 23, HCDR2 shown in SEQ ID NO. 24 and HCDR3 shown in SEQ ID NO. 25;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 27, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 29, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 31, HCDR2 shown in SEQ ID NO. 32 and HCDR3 shown in SEQ ID NO. 33;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 36, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 38, HCDR2 shown in SEQ ID NO. 39 and HCDR3 shown in SEQ ID NO. 40;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 42, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 29, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 44, HCDR2 shown in SEQ ID NO. 45 and HCDR3 shown in SEQ ID NO. 46;
The 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 48, LCDR2 shown in SEQ ID NO. 49 and LCDR3 shown in SEQ ID NO. 50, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 52, HCDR2 shown in SEQ ID NO. 53 and HCDR3 shown in SEQ ID NO. 54;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 28 and LCDR3 shown in SEQ ID NO. 56, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 58, HCDR2 shown in SEQ ID NO. 59 and HCDR3 shown in SEQ ID NO. 60;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 67, LCDR2 shown in SEQ ID NO. 68 and LCDR3 shown in SEQ ID NO. 69, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 44, HCDR2 shown in SEQ ID NO. 71 and HCDR3 shown in SEQ ID NO. 46;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 48, LCDR2 shown in SEQ ID NO. 49 and LCDR3 shown in SEQ ID NO. 79, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 81, HCDR2 shown in SEQ ID NO. 71 and HCDR3 shown in SEQ ID NO. 82;
the 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 84, LCDR2 shown in SEQ ID NO. 49 and LCDR3 shown in SEQ ID NO. 79, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 86, HCDR2 shown in SEQ ID NO. 71 and HCDR3 shown in SEQ ID NO. 82; or alternatively
The 3 light chain complementarity determining regions comprise LCDR1 shown in SEQ ID NO. 67, LCDR2 shown in SEQ ID NO. 68 and LCDR3 shown in SEQ ID NO. 88, and/or the 3 heavy chain complementarity determining regions comprise HCDR1 shown in SEQ ID NO. 90, HCDR2 shown in SEQ ID NO. 91 and HCDR3 shown in SEQ ID NO. 92.
2. The chimeric antigen receptor targeting DLL3 antigen according to claim 1, wherein,
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 61 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 94;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 95 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 63;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 72 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 75;
The scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 18 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 22;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 26 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 30;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 34 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 37;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 41 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 43;
The scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 47 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 51;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 55 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 57;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 66 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 70;
the scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 78 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 80;
The scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 83 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 85; or alternatively
The scFv of the anti-DLL 3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 87 and/or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 89.
3. The chimeric antigen receptor that targets DLL3 antigen of claim 1, wherein the CAR further comprises a transmembrane domain and an intracellular signaling domain;
preferably, the CAR further comprises one or more of a hinge region, a signal peptide, and a co-stimulatory signal domain;
more preferably, the transmembrane domain is a CD8 transmembrane region, the hinge region is a CD8 hinge region, the intracellular signaling domain is a cd3ζ intracellular signaling domain, the signal peptide is a CD8 a signal peptide, or the costimulatory signaling domain is a 4-1BB costimulatory signaling domain.
4. A chimeric antigen receptor targeting a DLL3 antigen according to claim 3, wherein the CAR comprises, in order from N-terminus to C-terminus, a CD8 a signal peptide, a DLL3 antibody scFv VH-linker-DLL3 antibody scFv VL, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory signal and a CD3 ζ intracellular signaling domain; optionally, the CAR does not comprise the amino acid sequence of the CD8 a signal peptide.
5. The chimeric antigen receptor targeting DLL3 antigen according to claim 3 or 4, wherein,
the CD8 a signal peptide comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 1;
the CD8 hinge region and the transmembrane region comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 2;
the 4-1BB costimulatory signal domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO. 3; or alternatively
The CD3 zeta intracellular signal domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID No. 4;
Preferably, the amino acid sequence of the CAR is shown in any one of SEQ ID NOs 5-17; more preferably, the CAR removes the amino acid sequence of the CD8 alpha signal peptide based on any of the amino acid sequences shown in SEQ ID NOs 5-17.
6. A nucleic acid encoding the chimeric antigen receptor of any one of claims 1-5 that targets DLL3 antigen.
7. An expression cassette comprising the nucleic acid of claim 6.
8. A vector comprising the nucleic acid of claim 6 or the expression cassette of claim 7.
9. A cell comprising the nucleic acid of claim 6 or the expression cassette of claim 7 or the vector of claim 8; preferably, the cell is an engineered immune cell; more preferably, the engineered immune cell is a T cell; most preferably, the T cell is a primary T cell, a gamma delta T cell or an NK T cell.
10. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-5 that targets DLL3 antigen, or the nucleic acid of claim 6, or the expression cassette of claim 7, or the vector of claim 8, or the cell of claim 9.
11. A kit comprising the chimeric antigen receptor of any one of claims 1-5 that targets DLL3 antigen, or the nucleic acid of claim 6, or the expression cassette of claim 7, or the vector of claim 8, or the cell of claim 9.
12. Use of the chimeric antigen receptor of any one of claims 1-5 that targets DLL3 antigen, or the nucleic acid of claim 6, or the expression cassette of claim 7, or the vector of claim 8, or the cell of claim 9, or the pharmaceutical composition of claim 10, or the kit of claim 11, for preventing, treating, detecting or diagnosing a disease associated with DLL 3; preferably, the DLL3 associated disease is a DLL3 high expression disease; more preferably, the disease is a DLL3 high expressing cancer or tumor; more preferably, the cancer or tumor is selected from one or more of lung cancer, melanoma, medullary thyroid cancer, glioblastoma, prostate cancer, neuroendocrine cancer; most preferably, the cancer is lung cancer, particularly small cell lung cancer.
13. A method of preparing an engineered immune cell comprising the steps of:
(1) Providing an immune cell to be modified; and
(2) Introducing the nucleic acid of claim 6, or the expression cassette of claim 7, or the vector of claim 8 into the immune cell;
preferably, the immune cells are T cells; more preferably, the T cell is a primary T cell, a gamma delta T cell or an NK T cell.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210973673X | 2022-08-15 | ||
| CN202210973673 | 2022-08-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116003628A true CN116003628A (en) | 2023-04-25 |
Family
ID=86023673
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211314320.5A Pending CN116003628A (en) | 2022-08-15 | 2022-10-25 | Fully human specific chimeric antigen receptor targeting human DLL3 antigen and application thereof |
| CN202310907882.9A Pending CN116731182A (en) | 2022-08-15 | 2023-07-24 | DLL 3-targeting antibodies, antigen binding fragments and uses thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310907882.9A Pending CN116731182A (en) | 2022-08-15 | 2023-07-24 | DLL 3-targeting antibodies, antigen binding fragments and uses thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN116003628A (en) |
| WO (1) | WO2024037288A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116813784A (en) * | 2023-05-17 | 2023-09-29 | 星奕昂(上海)生物科技有限公司 | Antibodies targeting DLL3 and uses thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116789836B (en) * | 2023-08-14 | 2024-01-05 | 浙江时迈药业有限公司 | Antibodies against DLL3 and uses thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW202011999A (en) * | 2018-04-10 | 2020-04-01 | 美商安進公司 | Chimeric receptors to dll3 and methods of use thereof |
| AU2020231308A1 (en) * | 2019-03-01 | 2021-08-19 | Allogene Therapeutics, Inc. | DLL3 targeting chimeric antigen receptors and binding agents |
| EP3997127A4 (en) * | 2019-07-11 | 2023-08-23 | Memorial Sloan Kettering Cancer Center | Dll3-targeting antibodies and uses thereof |
| CN114181311B (en) * | 2021-12-20 | 2023-06-20 | 华东师范大学 | Fully human anti-DLL 3 scFv and application thereof in CART cell therapy |
-
2022
- 2022-10-25 CN CN202211314320.5A patent/CN116003628A/en active Pending
-
2023
- 2023-07-24 WO PCT/CN2023/108807 patent/WO2024037288A1/en unknown
- 2023-07-24 CN CN202310907882.9A patent/CN116731182A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116813784A (en) * | 2023-05-17 | 2023-09-29 | 星奕昂(上海)生物科技有限公司 | Antibodies targeting DLL3 and uses thereof |
| CN116813784B (en) * | 2023-05-17 | 2024-03-01 | 星奕昂(上海)生物科技有限公司 | Antibodies targeting DLL3 and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2024037288A1 (en) | 2024-02-22 |
| CN116731182A (en) | 2023-09-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7402933B2 (en) | Genetic tags for transgenes and their use | |
| AU2020200751B2 (en) | Universal chimeric antigen receptor expressing immune cells for targeting of diverse multiple antigens and method of manufacturing the same and use of the same for treatment of cancer, infections and autoimmune disorders | |
| US11026975B2 (en) | Chimeric antigen receptor (CAR) binding to BCMA, and uses thereof | |
| US20230256018A1 (en) | T cell receptors and engineered cells expressing same | |
| US20200289561A1 (en) | Killer cell capable of efficiently and stably expressing antibody, and uses thereof | |
| US11090335B2 (en) | Chimeric antigen receptor targeting human NKG2DL and methods of preparing said receptor and pharmaceutical composition | |
| EP2884999B1 (en) | Method and compositions for cellular immunotherapy | |
| JP2023145589A (en) | Novel platforms for co-stimulation, novel car designs and other enhancements for adoptive cellular therapy | |
| CN107903324B (en) | Bispecific antibody capable of binding to human CD19 and CD3 | |
| CN108384760B (en) | Human T lymphocyte carrying CD20/CD19 bispecific chimeric antigen receptor and preparation method and application thereof | |
| CN110964121B (en) | CD19-CAR-T cell secreting IL-7 and IL-15 factors and application thereof | |
| AU2016341527A1 (en) | Methods, kits, agents and apparatuses for transduction | |
| CN116003628A (en) | Fully human specific chimeric antigen receptor targeting human DLL3 antigen and application thereof | |
| WO2017219933A1 (en) | T cell for efficiently and stably expressing antibody and application thereof | |
| WO2023109257A1 (en) | Humanized bcma antibody and bcma-car-t/bcma-car-dnt cell | |
| CN114127109A (en) | Antibody or chimeric antigen receptor targeting Claudin18.2 | |
| CN112500497A (en) | CLTX-NKG2D bispecific chimeric antigen receptor cell and preparation method and application thereof | |
| WO2021164753A1 (en) | Methods and compositions for modulating arginine levels in immune cells | |
| CN111378046B (en) | Immune effector cell conversion receptor | |
| WO2022116952A1 (en) | Antigen-binding protein targeting cd70 and use thereof | |
| WO2021139755A1 (en) | Engineered t cell, and preparation and use thereof | |
| CN114853893A (en) | Target GPC3 chimeric antigen receptor T cell and application thereof | |
| CA3229688A1 (en) | Chimeric antigen receptor and use thereof | |
| CN115397439A (en) | Quantitative control of engineered cellular activity of expression universal immunoreceptors | |
| WO2023273762A1 (en) | Spatial conformational epitope mediating efficient retention of cd3 within cells and application thereof |
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 |