CN117279651A - Transplantation cytoprotection via modified Fc receptor - Google Patents
Transplantation cytoprotection via modified Fc receptor Download PDFInfo
- Publication number
- CN117279651A CN117279651A CN202280032962.8A CN202280032962A CN117279651A CN 117279651 A CN117279651 A CN 117279651A CN 202280032962 A CN202280032962 A CN 202280032962A CN 117279651 A CN117279651 A CN 117279651A
- Authority
- CN
- China
- Prior art keywords
- cells
- cell
- modified cell
- modified
- protein
- 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
- 108010087819 Fc receptors Proteins 0.000 title claims abstract description 32
- 102000009109 Fc receptors Human genes 0.000 title claims abstract description 32
- 238000002054 transplantation Methods 0.000 title description 20
- 210000004027 cell Anatomy 0.000 claims abstract description 623
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 claims abstract description 72
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 claims abstract description 71
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 claims abstract description 71
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims abstract description 67
- 230000004540 complement-dependent cytotoxicity Effects 0.000 claims abstract description 59
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 claims abstract description 53
- 210000002889 endothelial cell Anatomy 0.000 claims abstract description 53
- 210000001685 thyroid gland Anatomy 0.000 claims abstract description 48
- 239000008280 blood Substances 0.000 claims abstract description 36
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 35
- 210000004369 blood Anatomy 0.000 claims abstract description 34
- 210000005064 dopaminergic neuron Anatomy 0.000 claims abstract description 32
- 210000004413 cardiac myocyte Anatomy 0.000 claims abstract description 23
- 210000003494 hepatocyte Anatomy 0.000 claims abstract description 22
- 239000000790 retinal pigment Substances 0.000 claims abstract description 18
- 230000005847 immunogenicity Effects 0.000 claims abstract description 16
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 14
- 210000004498 neuroglial cell Anatomy 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 170
- 238000000034 method Methods 0.000 claims description 129
- 241000282414 Homo sapiens Species 0.000 claims description 123
- 230000014509 gene expression Effects 0.000 claims description 85
- 102000004169 proteins and genes Human genes 0.000 claims description 70
- 210000000822 natural killer cell Anatomy 0.000 claims description 67
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 50
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 48
- 229920001184 polypeptide Polymers 0.000 claims description 47
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 37
- 210000004153 islets of langerhan Anatomy 0.000 claims description 36
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 claims description 34
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 claims description 33
- 206010028980 Neoplasm Diseases 0.000 claims description 30
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 28
- 201000010099 disease Diseases 0.000 claims description 27
- 238000004519 manufacturing process Methods 0.000 claims description 25
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 claims description 24
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 claims description 22
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 claims description 22
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims description 22
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 claims description 22
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 claims description 20
- 230000001939 inductive effect Effects 0.000 claims description 20
- 201000011510 cancer Diseases 0.000 claims description 19
- 230000001086 cytosolic effect Effects 0.000 claims description 18
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 18
- 230000004068 intracellular signaling Effects 0.000 claims description 16
- 241000283690 Bos taurus Species 0.000 claims description 13
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims description 13
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 12
- 241000700159 Rattus Species 0.000 claims description 12
- 230000004048 modification Effects 0.000 claims description 12
- 238000012986 modification Methods 0.000 claims description 12
- 108010076667 Caspases Proteins 0.000 claims description 11
- 102000011727 Caspases Human genes 0.000 claims description 11
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 claims description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 10
- 241000699800 Cricetinae Species 0.000 claims description 9
- 241000282326 Felis catus Species 0.000 claims description 9
- 102100021936 C-C motif chemokine 27 Human genes 0.000 claims description 8
- 241001494479 Pecora Species 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 208000019622 heart disease Diseases 0.000 claims description 8
- 241000283707 Capra Species 0.000 claims description 7
- 101000897494 Homo sapiens C-C motif chemokine 27 Proteins 0.000 claims description 7
- 208000012902 Nervous system disease Diseases 0.000 claims description 7
- 208000022873 Ocular disease Diseases 0.000 claims description 7
- 241000282898 Sus scrofa Species 0.000 claims description 7
- 241000272525 Anas platyrhynchos Species 0.000 claims description 6
- 235000002198 Annona diversifolia Nutrition 0.000 claims description 6
- 241000272814 Anser sp. Species 0.000 claims description 6
- 241001416153 Bos grunniens Species 0.000 claims description 6
- 241000282836 Camelus dromedarius Species 0.000 claims description 6
- 241000700199 Cavia porcellus Species 0.000 claims description 6
- 241000282693 Cercopithecidae Species 0.000 claims description 6
- 241000283074 Equus asinus Species 0.000 claims description 6
- 241001331845 Equus asinus x caballus Species 0.000 claims description 6
- 241000283073 Equus caballus Species 0.000 claims description 6
- 241000287828 Gallus gallus Species 0.000 claims description 6
- 101000863873 Homo sapiens Tyrosine-protein phosphatase non-receptor type substrate 1 Proteins 0.000 claims description 6
- 241000282842 Lama glama Species 0.000 claims description 6
- 208000025966 Neurological disease Diseases 0.000 claims description 6
- 208000014245 Ocular vascular disease Diseases 0.000 claims description 6
- 108010033576 Transferrin Receptors Proteins 0.000 claims description 6
- 108050003222 Transferrin receptor protein 1 Proteins 0.000 claims description 6
- 241001416177 Vicugna pacos Species 0.000 claims description 6
- 208000030172 endocrine system disease Diseases 0.000 claims description 6
- XRECTZIEBJDKEO-UHFFFAOYSA-N flucytosine Chemical group NC1=NC(=O)NC=C1F XRECTZIEBJDKEO-UHFFFAOYSA-N 0.000 claims description 6
- 229960004413 flucytosine Drugs 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 208000017701 Endocrine disease Diseases 0.000 claims description 5
- 108060003951 Immunoglobulin Proteins 0.000 claims description 5
- 102100026143 Transferrin receptor protein 2 Human genes 0.000 claims description 5
- 101710145355 Transferrin receptor protein 2 Proteins 0.000 claims description 5
- 102100029948 Tyrosine-protein phosphatase non-receptor type substrate 1 Human genes 0.000 claims description 5
- 102000018358 immunoglobulin Human genes 0.000 claims description 5
- 108010080611 Cytosine Deaminase Proteins 0.000 claims description 4
- 108020004440 Thymidine kinase Proteins 0.000 claims description 4
- 238000006471 dimerization reaction Methods 0.000 claims description 4
- 230000012202 endocytosis Effects 0.000 claims description 4
- 210000004964 innate lymphoid cell Anatomy 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 241000700584 Simplexvirus Species 0.000 claims description 3
- 230000034994 death Effects 0.000 claims description 3
- 238000010494 dissociation reaction Methods 0.000 claims description 3
- 230000005593 dissociations Effects 0.000 claims description 3
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical group O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 claims description 3
- 229960002963 ganciclovir Drugs 0.000 claims description 3
- 238000010353 genetic engineering Methods 0.000 claims description 3
- 239000000411 inducer Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 210000003890 endocrine cell Anatomy 0.000 claims description 2
- 230000019491 signal transduction Effects 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- 241000009328 Perro Species 0.000 claims 3
- 210000005229 liver cell Anatomy 0.000 claims 1
- 108010036012 Iodide peroxidase Proteins 0.000 description 56
- 102000014267 Thyroid peroxidases Human genes 0.000 description 56
- 238000003556 assay Methods 0.000 description 49
- 230000002147 killing effect Effects 0.000 description 45
- 239000002609 medium Substances 0.000 description 44
- 239000000427 antigen Substances 0.000 description 39
- 108091007433 antigens Proteins 0.000 description 39
- 102000036639 antigens Human genes 0.000 description 39
- 230000027455 binding Effects 0.000 description 34
- 210000001778 pluripotent stem cell Anatomy 0.000 description 34
- 230000000694 effects Effects 0.000 description 33
- 108010081355 beta 2-Microglobulin Proteins 0.000 description 31
- 229960000548 alemtuzumab Drugs 0.000 description 29
- 102000015736 beta 2-Microglobulin Human genes 0.000 description 29
- 239000003112 inhibitor Substances 0.000 description 29
- 108700002010 MHC class II transactivator Proteins 0.000 description 28
- 230000004069 differentiation Effects 0.000 description 27
- 210000000130 stem cell Anatomy 0.000 description 27
- 150000007523 nucleic acids Chemical group 0.000 description 22
- 210000003583 retinal pigment epithelium Anatomy 0.000 description 22
- 230000001419 dependent effect Effects 0.000 description 20
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 20
- 210000001519 tissue Anatomy 0.000 description 20
- 230000001965 increasing effect Effects 0.000 description 19
- 230000001105 regulatory effect Effects 0.000 description 19
- 241001465754 Metazoa Species 0.000 description 18
- 230000006870 function Effects 0.000 description 18
- 238000000338 in vitro Methods 0.000 description 18
- 102100024217 CAMPATH-1 antigen Human genes 0.000 description 17
- 108010065524 CD52 Antigen Proteins 0.000 description 17
- 230000001404 mediated effect Effects 0.000 description 17
- 239000013598 vector Substances 0.000 description 17
- 238000002659 cell therapy Methods 0.000 description 16
- 238000012258 culturing Methods 0.000 description 16
- 238000000684 flow cytometry Methods 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 230000001172 regenerating effect Effects 0.000 description 15
- 230000008672 reprogramming Effects 0.000 description 15
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 14
- 238000002347 injection Methods 0.000 description 14
- 239000007924 injection Substances 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- 238000005516 engineering process Methods 0.000 description 13
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 13
- 229940122531 Anaplastic lymphoma kinase inhibitor Drugs 0.000 description 12
- 108700019146 Transgenes Proteins 0.000 description 12
- 230000000735 allogeneic effect Effects 0.000 description 12
- 230000028993 immune response Effects 0.000 description 12
- 230000002163 immunogen Effects 0.000 description 12
- 102000039446 nucleic acids Human genes 0.000 description 12
- 108020004707 nucleic acids Proteins 0.000 description 12
- 210000000056 organ Anatomy 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 description 11
- 101150036449 SIRPA gene Proteins 0.000 description 11
- 206010052779 Transplant rejections Diseases 0.000 description 11
- 210000002540 macrophage Anatomy 0.000 description 11
- 230000009467 reduction Effects 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- 238000002560 therapeutic procedure Methods 0.000 description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 10
- 101001033280 Homo sapiens Cytokine receptor common subunit beta Proteins 0.000 description 10
- 102000004877 Insulin Human genes 0.000 description 10
- 108090001061 Insulin Proteins 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- 102000055647 human CSF2RB Human genes 0.000 description 10
- 229940125396 insulin Drugs 0.000 description 10
- 230000002829 reductive effect Effects 0.000 description 10
- 108091026890 Coding region Proteins 0.000 description 9
- 241000282412 Homo Species 0.000 description 9
- -1 UTF-1 Proteins 0.000 description 9
- 239000012190 activator Substances 0.000 description 9
- 230000022534 cell killing Effects 0.000 description 9
- 102000005962 receptors Human genes 0.000 description 9
- 108020003175 receptors Proteins 0.000 description 9
- 239000011435 rock Substances 0.000 description 9
- 230000004083 survival effect Effects 0.000 description 9
- 108700028369 Alleles Proteins 0.000 description 8
- 101150084532 CD47 gene Proteins 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 102000038624 GSKs Human genes 0.000 description 8
- 108091007911 GSKs Proteins 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000010459 TALEN Methods 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- 210000003719 b-lymphocyte Anatomy 0.000 description 8
- 230000001413 cellular effect Effects 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 230000000521 hyperimmunizing effect Effects 0.000 description 8
- 230000003834 intracellular effect Effects 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 230000011664 signaling Effects 0.000 description 8
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 7
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 7
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 7
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 description 7
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 description 7
- 206010057249 Phagocytosis Diseases 0.000 description 7
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 7
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 7
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 230000000295 complement effect Effects 0.000 description 7
- 239000012636 effector Substances 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 230000008782 phagocytosis Effects 0.000 description 7
- 210000004881 tumor cell Anatomy 0.000 description 7
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 6
- 101150076800 B2M gene Proteins 0.000 description 6
- 241000282472 Canis lupus familiaris Species 0.000 description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 6
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 6
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 6
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 231100000433 cytotoxic Toxicity 0.000 description 6
- 230000001472 cytotoxic effect Effects 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 6
- 230000009368 gene silencing by RNA Effects 0.000 description 6
- 210000002064 heart cell Anatomy 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 230000002688 persistence Effects 0.000 description 6
- 230000003389 potentiating effect Effects 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000007920 subcutaneous administration Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 229940034208 thyroxine Drugs 0.000 description 6
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical group CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 description 5
- 208000031229 Cardiomyopathies Diseases 0.000 description 5
- 230000004568 DNA-binding Effects 0.000 description 5
- 108010010803 Gelatin Proteins 0.000 description 5
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 5
- 101100382122 Homo sapiens CIITA gene Proteins 0.000 description 5
- 101001139134 Homo sapiens Krueppel-like factor 4 Proteins 0.000 description 5
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 5
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 5
- 102100020677 Krueppel-like factor 4 Human genes 0.000 description 5
- 101000844802 Lacticaseibacillus rhamnosus Teichoic acid D-alanyltransferase Proteins 0.000 description 5
- 241000713666 Lentivirus Species 0.000 description 5
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 238000010240 RT-PCR analysis Methods 0.000 description 5
- 241000282887 Suidae Species 0.000 description 5
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 5
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 5
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 5
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 5
- 230000004075 alteration Effects 0.000 description 5
- 229910002091 carbon monoxide Inorganic materials 0.000 description 5
- 239000006143 cell culture medium Substances 0.000 description 5
- 239000008273 gelatin Substances 0.000 description 5
- 229920000159 gelatin Polymers 0.000 description 5
- 235000019322 gelatine Nutrition 0.000 description 5
- 235000011852 gelatine desserts Nutrition 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 210000002216 heart Anatomy 0.000 description 5
- 230000006801 homologous recombination Effects 0.000 description 5
- 238000002744 homologous recombination Methods 0.000 description 5
- 210000005260 human cell Anatomy 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 108010034753 Complement Membrane Attack Complex Proteins 0.000 description 4
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 4
- 102000025850 HLA-A2 Antigen Human genes 0.000 description 4
- 108010074032 HLA-A2 Antigen Proteins 0.000 description 4
- 206010019280 Heart failures Diseases 0.000 description 4
- 108090000031 Hedgehog Proteins Proteins 0.000 description 4
- 102000003693 Hedgehog Proteins Human genes 0.000 description 4
- 101000971879 Homo sapiens Kell blood group glycoprotein Proteins 0.000 description 4
- 108010073807 IgG Receptors Proteins 0.000 description 4
- 102000009490 IgG Receptors Human genes 0.000 description 4
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 4
- 102000014429 Insulin-like growth factor Human genes 0.000 description 4
- 102100021447 Kell blood group glycoprotein Human genes 0.000 description 4
- 101000894412 Mycolicibacterium paratuberculosis (strain ATCC BAA-968 / K-10) Bacterioferritin Proteins 0.000 description 4
- 101150037203 Sox2 gene Proteins 0.000 description 4
- 230000010632 Transcription Factor Activity Effects 0.000 description 4
- 108091023040 Transcription factor Proteins 0.000 description 4
- 102000040945 Transcription factor Human genes 0.000 description 4
- 102100040076 Urea transporter 1 Human genes 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 239000005557 antagonist Substances 0.000 description 4
- 230000005875 antibody response Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 229930189065 blasticidin Natural products 0.000 description 4
- 210000000988 bone and bone Anatomy 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 210000005220 cytoplasmic tail Anatomy 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 208000032839 leukemia Diseases 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000001509 sodium citrate Substances 0.000 description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 230000009261 transgenic effect Effects 0.000 description 4
- 230000014621 translational initiation Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 208000023275 Autoimmune disease Diseases 0.000 description 3
- 238000011357 CAR T-cell therapy Methods 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 3
- 238000012286 ELISA Assay Methods 0.000 description 3
- 102000001267 GSK3 Human genes 0.000 description 3
- 108060006662 GSK3 Proteins 0.000 description 3
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 101100310645 Mus musculus Sox15 gene Proteins 0.000 description 3
- 101150086694 SLC22A3 gene Proteins 0.000 description 3
- 102000011923 Thyrotropin Human genes 0.000 description 3
- 108010061174 Thyrotropin Proteins 0.000 description 3
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 description 3
- 102000013814 Wnt Human genes 0.000 description 3
- 108050003627 Wnt Proteins 0.000 description 3
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 3
- SCJNCDSAIRBRIA-DOFZRALJSA-N arachidonyl-2'-chloroethylamide Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)NCCCl SCJNCDSAIRBRIA-DOFZRALJSA-N 0.000 description 3
- 230000005784 autoimmunity Effects 0.000 description 3
- 108091008324 binding proteins Proteins 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000005782 double-strand break Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000008030 elimination Effects 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 231100000221 frame shift mutation induction Toxicity 0.000 description 3
- 230000037433 frameshift Effects 0.000 description 3
- 238000012239 gene modification Methods 0.000 description 3
- 230000030279 gene silencing Effects 0.000 description 3
- 230000005017 genetic modification Effects 0.000 description 3
- 235000013617 genetically modified food Nutrition 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- 210000001654 germ layer Anatomy 0.000 description 3
- 210000003714 granulocyte Anatomy 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- NFVJNJQRWPQVOA-UHFFFAOYSA-N n-[2-chloro-5-(trifluoromethyl)phenyl]-2-[3-(4-ethyl-5-ethylsulfanyl-1,2,4-triazol-3-yl)piperidin-1-yl]acetamide Chemical compound CCN1C(SCC)=NN=C1C1CN(CC(=O)NC=2C(=CC=C(C=2)C(F)(F)F)Cl)CCC1 NFVJNJQRWPQVOA-UHFFFAOYSA-N 0.000 description 3
- 230000001537 neural effect Effects 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 210000002220 organoid Anatomy 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 210000001082 somatic cell Anatomy 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000010474 transient expression Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 229910052725 zinc Inorganic materials 0.000 description 3
- 239000011701 zinc Substances 0.000 description 3
- 102100023990 60S ribosomal protein L17 Human genes 0.000 description 2
- 102100033793 ALK tyrosine kinase receptor Human genes 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102100022717 Atypical chemokine receptor 1 Human genes 0.000 description 2
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 2
- 208000004736 B-Cell Leukemia Diseases 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 2
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 101100342337 Caenorhabditis elegans klf-1 gene Proteins 0.000 description 2
- 101100257359 Caenorhabditis elegans sox-2 gene Proteins 0.000 description 2
- 206010007559 Cardiac failure congestive Diseases 0.000 description 2
- 241000700198 Cavia Species 0.000 description 2
- 101150071002 Ciita gene Proteins 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 2
- 206010010144 Completed suicide Diseases 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 102000000311 Cytosine Deaminase Human genes 0.000 description 2
- 101100118093 Drosophila melanogaster eEF1alpha2 gene Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102400001368 Epidermal growth factor Human genes 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 241000283086 Equidae Species 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
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 2
- 108010075704 HLA-A Antigens Proteins 0.000 description 2
- 108010058597 HLA-DR Antigens Proteins 0.000 description 2
- 102000006354 HLA-DR Antigens Human genes 0.000 description 2
- 208000001204 Hashimoto Disease Diseases 0.000 description 2
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 2
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 2
- 101000678879 Homo sapiens Atypical chemokine receptor 1 Proteins 0.000 description 2
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 2
- 101000983523 Homo sapiens Caspase-9 Proteins 0.000 description 2
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 2
- 101000671665 Homo sapiens Urea transporter 1 Proteins 0.000 description 2
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- 206010062016 Immunosuppression Diseases 0.000 description 2
- 102000003812 Interleukin-15 Human genes 0.000 description 2
- 108090000172 Interleukin-15 Proteins 0.000 description 2
- 108010002386 Interleukin-3 Proteins 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 102100020880 Kit ligand Human genes 0.000 description 2
- 101710177504 Kit ligand Proteins 0.000 description 2
- 102000017578 LAG3 Human genes 0.000 description 2
- 101150030213 Lag3 gene Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000711408 Murine respirovirus Species 0.000 description 2
- 101100382123 Mus musculus Ciita gene Proteins 0.000 description 2
- 101100310650 Mus musculus Sox18 gene Proteins 0.000 description 2
- 101100257363 Mus musculus Sox2 gene Proteins 0.000 description 2
- 241000868066 Myadestes myadestinus Species 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 102100035591 POU domain, class 2, transcription factor 2 Human genes 0.000 description 2
- 101710084411 POU domain, class 2, transcription factor 2 Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 2
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 2
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 241000714474 Rous sarcoma virus Species 0.000 description 2
- 108091006584 SLC14A1 Proteins 0.000 description 2
- 101150099498 SOX1 gene Proteins 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 101000874347 Streptococcus agalactiae IgA FC receptor Proteins 0.000 description 2
- 206010043276 Teratoma Diseases 0.000 description 2
- 102000006601 Thymidine Kinase Human genes 0.000 description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 description 2
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 2
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 2
- 101150110932 US19 gene Proteins 0.000 description 2
- 101100043969 Xenopus laevis sox11-b gene Proteins 0.000 description 2
- 101100257370 Xenopus laevis sox3-a gene Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 101150099178 abo gene Proteins 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 206010064930 age-related macular degeneration Diseases 0.000 description 2
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001363 autoimmune Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000029918 bioluminescence Effects 0.000 description 2
- 238000005415 bioluminescence Methods 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 210000001840 diploid cell Anatomy 0.000 description 2
- 230000007783 downstream signaling Effects 0.000 description 2
- 229940116977 epidermal growth factor Drugs 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 239000006481 glucose medium Substances 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 210000005003 heart tissue Anatomy 0.000 description 2
- 230000005745 host immune response Effects 0.000 description 2
- 102000053350 human FCGR3B Human genes 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 2
- 238000012528 insulin ELISA Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000001361 intraarterial administration Methods 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 108091070501 miRNA Proteins 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 230000000921 morphogenic effect Effects 0.000 description 2
- 210000002894 multi-fate stem cell Anatomy 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 208000010125 myocardial infarction Diseases 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 230000006780 non-homologous end joining Effects 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- 150000003230 pyrimidines Chemical class 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 229930002330 retinoic acid Natural products 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 230000009131 signaling function Effects 0.000 description 2
- 230000005487 simulated microgravity Effects 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 101150089855 sox3 gene Proteins 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 229960001727 tretinoin Drugs 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 208000019553 vascular disease Diseases 0.000 description 2
- 238000011179 visual inspection Methods 0.000 description 2
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 description 1
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 1
- 101710189683 Alkaline protease 1 Proteins 0.000 description 1
- 101710154562 Alkaline proteinase Proteins 0.000 description 1
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 101710170876 Antileukoproteinase Proteins 0.000 description 1
- 101001005269 Arabidopsis thaliana Ceramide synthase 1 LOH3 Proteins 0.000 description 1
- 101001005312 Arabidopsis thaliana Ceramide synthase LOH1 Proteins 0.000 description 1
- 208000036490 Arterial inflammations Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000713842 Avian sarcoma virus Species 0.000 description 1
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 description 1
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 1
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 101150061927 BMP2 gene Proteins 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 1
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 1
- 101710112538 C-C motif chemokine 27 Proteins 0.000 description 1
- 108700012439 CA9 Proteins 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- 101100510259 Caenorhabditis elegans klf-2 gene Proteins 0.000 description 1
- 101100510263 Caenorhabditis elegans klf-3 gene Proteins 0.000 description 1
- 101100257372 Caenorhabditis elegans sox-3 gene Proteins 0.000 description 1
- 102100028801 Calsyntenin-1 Human genes 0.000 description 1
- 102100024423 Carbonic anhydrase 9 Human genes 0.000 description 1
- 206010007558 Cardiac failure chronic Diseases 0.000 description 1
- UNPLRYRWJLTVAE-UHFFFAOYSA-N Cloperastine hydrochloride Chemical compound Cl.C1=CC(Cl)=CC=C1C(C=1C=CC=CC=1)OCCN1CCCCC1 UNPLRYRWJLTVAE-UHFFFAOYSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 206010056370 Congestive cardiomyopathy Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000008265 DNA repair mechanism Effects 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 241000539679 Defiwi virus Species 0.000 description 1
- 102100030074 Dickkopf-related protein 1 Human genes 0.000 description 1
- 201000010046 Dilated cardiomyopathy Diseases 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 240000002989 Euphorbia neriifolia Species 0.000 description 1
- 101150021185 FGF gene Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 230000010558 Gene Alterations Effects 0.000 description 1
- 208000008069 Geographic Atrophy Diseases 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102100028976 HLA class I histocompatibility antigen, B alpha chain Human genes 0.000 description 1
- 102100028971 HLA class I histocompatibility antigen, C alpha chain Human genes 0.000 description 1
- 102100031546 HLA class II histocompatibility antigen, DO beta chain Human genes 0.000 description 1
- 108010058607 HLA-B Antigens Proteins 0.000 description 1
- 108010052199 HLA-C Antigens Proteins 0.000 description 1
- 108010010378 HLA-DP Antigens Proteins 0.000 description 1
- 102000015789 HLA-DP Antigens Human genes 0.000 description 1
- 108010062347 HLA-DQ Antigens Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 108010027412 Histocompatibility Antigens Class II Proteins 0.000 description 1
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 1
- 241001272567 Hominoidea Species 0.000 description 1
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 1
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000864646 Homo sapiens Dickkopf-related protein 1 Proteins 0.000 description 1
- 101000866281 Homo sapiens HLA class II histocompatibility antigen, DO beta chain Proteins 0.000 description 1
- 101001103039 Homo sapiens Inactive tyrosine-protein kinase transmembrane receptor ROR1 Proteins 0.000 description 1
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 1
- 101000998120 Homo sapiens Interleukin-3 receptor subunit alpha Proteins 0.000 description 1
- 101000623901 Homo sapiens Mucin-16 Proteins 0.000 description 1
- 101001051490 Homo sapiens Neural cell adhesion molecule L1 Proteins 0.000 description 1
- 101001103036 Homo sapiens Nuclear receptor ROR-alpha Proteins 0.000 description 1
- 101001094700 Homo sapiens POU domain, class 5, transcription factor 1 Proteins 0.000 description 1
- 101000685824 Homo sapiens Probable RNA polymerase II nuclear localization protein SLC7A6OS Proteins 0.000 description 1
- 101000781955 Homo sapiens Proto-oncogene Wnt-1 Proteins 0.000 description 1
- 101000666640 Homo sapiens Rho-related GTP-binding protein RhoJ Proteins 0.000 description 1
- 101000633784 Homo sapiens SLAM family member 7 Proteins 0.000 description 1
- 101000851696 Homo sapiens Steroid hormone receptor ERR2 Proteins 0.000 description 1
- 101000666775 Homo sapiens T-box transcription factor TBX3 Proteins 0.000 description 1
- 101000799461 Homo sapiens Thrombopoietin Proteins 0.000 description 1
- 101000694103 Homo sapiens Thyroid peroxidase Proteins 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 1
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 1
- 102100039615 Inactive tyrosine-protein kinase transmembrane receptor ROR1 Human genes 0.000 description 1
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 1
- 102000000646 Interleukin-3 Human genes 0.000 description 1
- 102100033493 Interleukin-3 receptor subunit alpha Human genes 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 206010048858 Ischaemic cardiomyopathy Diseases 0.000 description 1
- 101150072501 Klf2 gene Proteins 0.000 description 1
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 description 1
- 108010017123 Kruppel-Like Transcription Factors Proteins 0.000 description 1
- 102000004434 Kruppel-Like Transcription Factors Human genes 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010050513 Metastatic renal cell carcinoma Diseases 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 241000713333 Mouse mammary tumor virus Species 0.000 description 1
- 102100023123 Mucin-16 Human genes 0.000 description 1
- 101100165560 Mus musculus Bmp7 gene Proteins 0.000 description 1
- 101100510267 Mus musculus Klf4 gene Proteins 0.000 description 1
- 101000969137 Mus musculus Metallothionein-1 Proteins 0.000 description 1
- 101001094698 Mus musculus POU domain, class 5, transcription factor 1 Proteins 0.000 description 1
- 101100310657 Mus musculus Sox1 gene Proteins 0.000 description 1
- 101100257376 Mus musculus Sox3 gene Proteins 0.000 description 1
- 101100043050 Mus musculus Sox4 gene Proteins 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 108091057508 Myc family Proteins 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 108700026495 N-Myc Proto-Oncogene Proteins 0.000 description 1
- 102100030124 N-myc proto-oncogene protein Human genes 0.000 description 1
- 102100024964 Neural cell adhesion molecule L1 Human genes 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 102100035593 POU domain, class 2, transcription factor 1 Human genes 0.000 description 1
- 101710084414 POU domain, class 2, transcription factor 1 Proteins 0.000 description 1
- 102100026466 POU domain, class 2, transcription factor 3 Human genes 0.000 description 1
- 101710084413 POU domain, class 2, transcription factor 3 Proteins 0.000 description 1
- 102100026459 POU domain, class 3, transcription factor 2 Human genes 0.000 description 1
- 101710133394 POU domain, class 3, transcription factor 2 Proteins 0.000 description 1
- 102100026456 POU domain, class 3, transcription factor 3 Human genes 0.000 description 1
- 101710133393 POU domain, class 3, transcription factor 3 Proteins 0.000 description 1
- 102100026450 POU domain, class 3, transcription factor 4 Human genes 0.000 description 1
- 101710133389 POU domain, class 3, transcription factor 4 Proteins 0.000 description 1
- 101150054854 POU1F1 gene Proteins 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 208000005764 Peripheral Arterial Disease Diseases 0.000 description 1
- 208000030831 Peripheral arterial occlusive disease Diseases 0.000 description 1
- BJGNCJDXODQBOB-SSDOTTSWSA-N Photinus luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-SSDOTTSWSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241001505332 Polyomavirus sp. Species 0.000 description 1
- 102100023136 Probable RNA polymerase II nuclear localization protein SLC7A6OS Human genes 0.000 description 1
- 101710168705 Protamine-1 Proteins 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 101710151715 Protein 7 Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102100029143 RNA 3'-terminal phosphate cyclase Human genes 0.000 description 1
- 206010070308 Refractory cancer Diseases 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 206010038748 Restrictive cardiomyopathy Diseases 0.000 description 1
- 206010038848 Retinal detachment Diseases 0.000 description 1
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 102100038337 Rho-related GTP-binding protein RhoJ Human genes 0.000 description 1
- 101150011212 SOX13 gene Proteins 0.000 description 1
- 101150073471 SOX14 gene Proteins 0.000 description 1
- 238000006085 Schmidt reaction Methods 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 101150117830 Sox5 gene Proteins 0.000 description 1
- 102100040435 Sperm protamine P1 Human genes 0.000 description 1
- 101000668858 Spinacia oleracea 30S ribosomal protein S1, chloroplastic Proteins 0.000 description 1
- 208000027073 Stargardt disease Diseases 0.000 description 1
- 102100036831 Steroid hormone receptor ERR2 Human genes 0.000 description 1
- 206010061372 Streptococcal infection Diseases 0.000 description 1
- 101000898746 Streptomyces clavuligerus Clavaminate synthase 1 Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 102100038409 T-box transcription factor TBX3 Human genes 0.000 description 1
- 108010006877 Tacrolimus Binding Protein 1A Proteins 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102100022435 Transcription factor SOX-13 Human genes 0.000 description 1
- 102000007238 Transferrin Receptors Human genes 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 206010060872 Transplant failure Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 229940124674 VEGF-R inhibitor Drugs 0.000 description 1
- 208000024248 Vascular System injury Diseases 0.000 description 1
- 208000012339 Vascular injury Diseases 0.000 description 1
- 208000009982 Ventricular Dysfunction Diseases 0.000 description 1
- 235000018936 Vitellaria paradoxa Nutrition 0.000 description 1
- 208000000208 Wet Macular Degeneration Diseases 0.000 description 1
- 102000052547 Wnt-1 Human genes 0.000 description 1
- 101100043968 Xenopus laevis sox11-a gene Proteins 0.000 description 1
- 101100366235 Xenopus laevis sox13 gene Proteins 0.000 description 1
- 101100043971 Xenopus laevis sox18-a gene Proteins 0.000 description 1
- 101100043972 Xenopus laevis sox18-b gene Proteins 0.000 description 1
- 101100257371 Xenopus laevis sox3-b gene Proteins 0.000 description 1
- 101100096235 Xenopus laevis sox9-a gene Proteins 0.000 description 1
- 101100096236 Xenopus laevis sox9-b gene Proteins 0.000 description 1
- 101100043970 Xenopus tropicalis sox17a gene Proteins 0.000 description 1
- 101710185494 Zinc finger protein Proteins 0.000 description 1
- 102100023597 Zinc finger protein 816 Human genes 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 108010023082 activin A Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 102000005840 alpha-Galactosidase Human genes 0.000 description 1
- 108010030291 alpha-Galactosidase Proteins 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 210000003663 amniotic stem cell Anatomy 0.000 description 1
- 239000002269 analeptic agent Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000003208 anti-thyroid effect Effects 0.000 description 1
- 230000009227 antibody-mediated cytotoxicity Effects 0.000 description 1
- 229940043671 antithyroid preparations Drugs 0.000 description 1
- 210000002403 aortic endothelial cell Anatomy 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 208000010928 autoimmune thyroid disease Diseases 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 210000001109 blastomere Anatomy 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 210000001043 capillary endothelial cell Anatomy 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000036978 cell physiology Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000447 dimerizing effect Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 210000000604 fetal stem cell Anatomy 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000003198 gene knock in Methods 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000007773 growth pattern Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 102000044459 human CD47 Human genes 0.000 description 1
- 102000053400 human TPO Human genes 0.000 description 1
- 206010020871 hypertrophic cardiomyopathy Diseases 0.000 description 1
- 208000003532 hypothyroidism Diseases 0.000 description 1
- 230000002989 hypothyroidism Effects 0.000 description 1
- 230000008004 immune attack Effects 0.000 description 1
- 102000027596 immune receptors Human genes 0.000 description 1
- 108091008915 immune receptors Proteins 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000002650 immunosuppressive therapy Methods 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006882 induction of apoptosis Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000006662 intracellular pathway Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 208000023589 ischemic disease Diseases 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- ZGSXEXBYLJIOGF-BOPNQXPFSA-N iwr-1 Chemical group C=1C=CC2=CC=CN=C2C=1NC(=O)C(C=C1)=CC=C1N1C(=O)[C@@H]2C(C=C3)CC3[C@@H]2C1=O ZGSXEXBYLJIOGF-BOPNQXPFSA-N 0.000 description 1
- 101150105127 klf1 gene Proteins 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 201000002818 limb ischemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000012153 long-term therapy Methods 0.000 description 1
- 231100000355 lymphocytotoxic Toxicity 0.000 description 1
- 230000001391 lymphocytotoxic effect Effects 0.000 description 1
- 210000003738 lymphoid progenitor cell Anatomy 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052618 mica group Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000003643 myeloid progenitor cell Anatomy 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- AEMBWNDIEFEPTH-UHFFFAOYSA-N n-tert-butyl-n-ethylnitrous amide Chemical compound CCN(N=O)C(C)(C)C AEMBWNDIEFEPTH-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000010309 neoplastic transformation Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 102000045246 noggin Human genes 0.000 description 1
- 108700007229 noggin Proteins 0.000 description 1
- 230000037434 nonsense mutation Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000002747 omentum Anatomy 0.000 description 1
- 210000004789 organ system Anatomy 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000009984 peri-natal effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229950008679 protamine sulfate Drugs 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 108091006082 receptor inhibitors Proteins 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 208000016691 refractory malignant neoplasm Diseases 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 208000037803 restenosis Diseases 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 230000004264 retinal detachment Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 101150095164 rex1 gene Proteins 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 229910052703 rhodium Inorganic materials 0.000 description 1
- 102200015453 rs121912293 Human genes 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000010845 search algorithm Methods 0.000 description 1
- 208000027653 severe early-childhood-onset retinal dystrophy Diseases 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 210000001988 somatic stem cell Anatomy 0.000 description 1
- 101150077014 sox10 gene Proteins 0.000 description 1
- 101150055666 sox6 gene Proteins 0.000 description 1
- 101150108218 sox7 gene Proteins 0.000 description 1
- 101150045711 sox8 gene Proteins 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000011301 standard therapy Methods 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 125000002328 sterol group Chemical group 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 239000005495 thyroid hormone Substances 0.000 description 1
- 229940036555 thyroid hormone Drugs 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000011277 treatment modality Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 230000006815 ventricular dysfunction Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention provides for the first time cells expressing truncated or modified Fc receptor proteins (e.g., CD16t, CD32t, or CD64 t) to evade Antibody Dependent Cellular Cytotoxicity (ADCC) or Complement Dependent Cytotoxicity (CDC). The cells may be pluripotent cells expressing truncated or modified Fc receptors, including low immunogenicity pluripotent cells (HIP) or ABO blood group O rhesus factor negative HIP cells (HIPO-). The invention encompasses cells derived from pluripotent cells and primary cells. The cells may also be differentiated cells including Chimeric Antigen Receptor (CAR) cells, T cells, natural Killer (NK) cells, endothelial cells, dopaminergic neurons, glial cells, islet beta cells, thyroid cells, fibroblasts, hepatocytes, cardiomyocytes, or retinal pigment endothelial cells.
Description
I. Cross-reference to related applications
The present application claims priority from U.S. c. ≡119 (e) to U.S. provisional application No. 63/168,225 filed on 3/30 of 2021 and U.S. provisional application No. 63/305,587 filed on 1 of 2022, and is incorporated herein by reference in its entirety.
Technical Field
The present invention relates to regenerative or tumor cell therapies. In some embodiments, regenerative cell therapy comprises transplanting cells or cell lines into a patient in need thereof. In some embodiments, the cell line comprises pluripotent cells that express cell surface Fc receptors that have been truncated to remove intracellular signaling domains. In other embodiments, the cell surface Fc receptor is a truncated CD16, CD32, or CD64 protein (CD 16t, CD32t, or CD64t, respectively). In other embodiments, they are hypoimmunogenic, have O-blood group or are Rh factor negative. In other embodiments, the regenerative or tumor cell therapy product exhibits prolonged survival in allogeneic recipients. In some embodiments, regenerative cell therapy is used to treat injured organs and tissues, and immune tumor cell products are used to treat cancer. In some embodiments, regenerative cell therapies of the invention utilize islet cells, thyroid cells, hepatocytes, chimeric Antigen Receptor (CAR) cells, endothelial cells, dopaminergic neurons, glial cells, cardiomyocytes, or retinal pigment endothelial cells for treating a disease or repairing damaged tissue. In other embodiments, the immune tumor cell products of the invention utilize T cells, natural Killer (NK) cells, or other immune cells such as Innate Lymphocytes (ILC).
II technical background
Regenerative cell therapy is an important potential therapy for regenerating damaged organs and tissues. The possibility of regenerating tissue by transplanting readily available cell lines into a patient is understandably attractive due to the low availability of organs for transplantation and the concomitant long waiting time. Regenerative cell therapy has shown promising initial results in animal models (e.g., post-myocardial infarction) for repair of damaged tissue after transplantation. However, the propensity of the immune system of the transplant recipient to reject allografts greatly reduces the potential efficacy of the treatment and reduces the possible positive effects surrounding such treatment.
Although Chimeric Antigen Receptor (CAR) T cell therapies have made significant progress in the treatment of refractory cancer patients, strategies must be formulated to benefit a large number of solid tumor individuals. Before this type of therapy becomes a widely accepted standard therapy for different cancers, some obstacles need to be overcome. Among these disorders, poor persistence of infused cells is a key challenge in successful cancer treatment. It is well known that poor persistence of infused CAR T cells is inversely related to persistent clinical remission in cancer patients, and that limited CAR T cell persistence is related to immunogenicity of engineered T cell products (Korde, n. et al A phase II trial of pan-KIR2Dblockade with IPH2101in smoldering multiple myela. Haemallogic 99, e81-83 (2014), schmidts, a. & Maus, m.v. making CAR T Cells a Solid Option for Solid tunes. Front Immunol 9,2593 (2018)). In some cases of autologous CAR T therapy, it has been determined that the specific immune response is directed against the CAR itself and eliminates the cell therapeutic agent before its effect is fully achieved (Hege, k.m. et al, safety, tumor trafficking and immunogenicity of Chimeric Antigen Receptor (CAR) -T cells specific for TAG-72in colorectal cancer.J Immunother Cancer 5,22 (2017), lamers, c.h. et al, treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target specificity.mol thor 21,904-912 (2013)). CD19 expression is maintained in B lineage cells that have undergone neoplastic transformation, and thus CD19 can be used to diagnose B cell leukemia and as a target for CAR T cell therapy. Modern leukemia treatment includes cytotoxic pretreatment followed by administration of anti-CD 19CAR T cells that target all B cells, including leukemia and benign populations. Thus, host versus graft immune responses in patients treated for B-cell leukemia are greatly reduced and show long-term inhibition of antibody production. Thus, CAR T cells are better protected from immune rejection when treating leukemia patients. In solid organ cancer patients, the immune response against CAR T products is enhanced (Hege, k.m. et al, safety, tumor trafficking and immunogenicity of Chimeric Antigen Receptor (CAR) -T cells specific for TAG-72in colorectal cancer.J Immunother Cancer 5,22 (2017)) and the efficacy of this therapy is minimized. Poor persistence can hinder effector function of infused cells and prevent success of long-term therapy.
Autologous induced pluripotent stem cells (ipscs) theoretically constitute an unlimited source of cells for organ repair strategies based on patient-specific cells. However, their generation presents technical and manufacturing challenges and is a lengthy process conceptually preventing any acute treatment modality. From a manufacturing perspective, allogeneic iPSC-based therapies or embryonic stem cell-based therapies are easier and allow for the production of well-screened, standardized, high quality cell products. However, such cell products are subject to rejection due to their allogeneic origin. With reduced or eliminated cellular antigenicity, a generally acceptable cellular product can be produced. Because pluripotent stem cells can differentiate into any cell type of the three germ layers, the potential uses of stem cell therapies are broad. Differentiation may be performed ex vivo or in vivo by transplanting progenitor cells that continue to differentiate and mature in the organ environment at the implantation site. Ex vivo differentiation allows researchers or clinicians to closely monitor the process and ensure that the appropriate cell population is generated prior to transplantation.
However, in most cases, undifferentiated pluripotent stem cells are avoided for clinical transplantation therapies due to their propensity to form teratomas. In contrast, such therapies tend to use differentiated cells (e.g., stem cell derived cardiomyocytes transplanted into the myocardium of heart failure patients). Clinical use of such pluripotent cells or tissues would benefit from a "safety feature" that controls the growth and survival of the cells after cell transplantation.
The art seeks to be able to generate stem cells for regenerating or replacing cells that are diseased or defective. Pluripotent Stem Cells (PSCs) can be used because they proliferate and differentiate into many possible cell types. The PSC family consists of several members that are produced by different technologies and have different immunogenic characteristics. Patient compatibility with engineered cells or tissues derived from PSCs determines the risk of immune rejection and the need for immunosuppression.
Embryonic Stem Cells (ESCs) isolated from the inner cell mass of the blastocyst exhibit histocompatibility antigens that do not match the recipient. This immune barrier cannot be addressed by a pool of ESC typed for Human Leukocyte Antigens (HLA) because even HLA-matched PSC grafts would be rejected due to mismatch of non-HLA molecules as minor antigens. The same is true of allogeneic induced pluripotent stem cells (ipscs).
Low immunogenicity multipotent (hypoimmunogenic pluripotent, HIP) cells and cell products have gene knockouts or transgenes to protect them from cellular components of the immune system including T cells, NK cells and macrophages. They may also be ABO blood group type O and Rh negative (HIPO-).
Immune rejection is a major obstacle to the success of cell therapy, and efforts are currently underway to develop universal allogeneic ready cells that evade cell rejection (Yoshihara, e. Et al, nature 586,606-611 (2020); wang, b. Et al, nat Biomed Eng 5,429-440 (2021); cause, t. Et al, proc Natl Acad Sci U S A, (2021)). However, such genetically engineered, low-immunogenicity cells are still susceptible to antibody killing against non-HLA epitopes, cell type specific autoantigens, or xenogeneics (Klee, g.g. arch Pathol Lab Med 124,921-923 (2000)) or synthetic constructs (Choe, j.h. et al, sci trans l Med 13 (2021)) in engineered cells and virus products. Such cells are produced by the transduction process (polymers, C.H. et al, blood 117,72-82 (2011); jensen, M.C. et al, biol Blood Marrow Transplant, 1245-1256 (2010)). Cytotoxic antibodies may be pre-existing or therapy-induced (Wagner, d.l. et al Nat Rev Clin Oncol, 379-393 (2021)) and compromise the persistence and efficacy of cell therapies.
Two of the most relevant killing mechanisms involved in antibodies are antibody-dependent cellular cytotoxicity (ADCC) by Natural Killer (NK) cells, macrophages, B cells or granulocytes and complement-dependent cytotoxicity (CDC) by activating the complement cascade. All of these killing mechanisms utilize antibodies that bind to target cells and activate effector immune cells or complement (fig. 1). IgG antibodies may be potent mediators of both ADCC and CDC. IgG antibodies have two variable Fab regions that bind to a specific epitope. The crystallizable Fc region protrudes and is used to bind NK cells, B cells, macrophages, granulocytes or complement.
Receptors that recognize the Fc portion of IgG fall into four distinct classes: fcγri (CD 64), fcγrii (CD 32), fcγriii (CD 16) and fcγriv. Fcγri exhibits high affinity for antibody constant regions and has restricted isotype specificity, while fcγrii and fcγriii have low affinity for the Fc region of IgG but have a broader isotype binding pattern, and fcγriv is a recently identified receptor with moderate affinity and restricted subclass specificity. Physiologically, fcyri plays a role during early immune reactions, while fcyrii and RIII recognize IgG in aggregate form surrounding multivalent antigens during late immune reactions.
If the antibody binds to unprotected cells through its Fab region, the Fc can be bound by NK cells (predominantly through its CD16 receptor), macrophages (predominantly through CD64, CD16 or CD 32), B cells (predominantly through CD 32) or granulocytes (predominantly through CD32, CD16 or CD 64) and mediate ADCC. Complement can also bind to Fc and activate its cascade, forming a Membrane Attack Complex (MAC) for CDC killing.
III summary of the invention
The present invention provides cells expressing cell surface Fc receptors that have been truncated or modified to remove an intracellular signaling function or intracellular signaling domain. Cells may be transplanted into a patient in need thereof. In some embodiments, regenerative or tumor cell therapies of the invention utilize islet cells, thyroid cells, chimeric Antigen Receptor (CAR) cells, T cells, NK cells, ILC, hepatocytes, endothelial cells, dopaminergic neurons, glial cells, cardiomyocytes, or retinal pigment endothelial cells for the treatment of cancer, disease, or repair of damaged tissue.
The present invention provides cells expressing truncated CD16, CD32 or CD64 (fig. 2) that sequester the Fc portion of local antibodies and thus inhibit ADCC and CDC. (compare FIGS. 1 and 3). The cells may be primary cells, differentiated cells, pluripotent cells including low immunogenicity pluripotent cells (HIP), ABO blood group O rhesus factor negative HIP cells (HIPO-) Induced Pluripotent Stem Cells (iPSCs), iPSCs that are O-, embryonic Stem Cells (ESCs), or ESCs that are O-, any of which further comprises truncated CD64 expression. Primary cells are isolated directly from the tissue.
In other embodiments, the cells may be islet cells, thyroid cells, chimeric Antigen Receptor (CAR) cells, T cells, NK cells, ILCs, endothelial cells, hepatocytes, dopaminergic neurons, glial cells, cardiomyocytes, or retinal pigment endothelial cells for treating a disease or repairing damaged tissue.
CD64 is only constitutively present on macrophages and monocytes and is not normally expressed on tissue cells. It is commonly referred to as Fc-gamma receptor 1 (Fcgamm) and binds with high affinity to the IgG Fc region. CD64 over-expression on target cells sequesters IgG Fc and binds it to target cells. In cells that do not have intracellular pathways for cellular activation by CD64, there may be no functional impact on such cells. For all cells that allow the cytoplasmic tail of CD64 to induce intracellular activation pathways, fc binding affects the cells and may disrupt their physiology. This can be avoided by truncating or modifying the intracellular signaling domain of the Fc receptor (e.g., CD 64). Fc receptors without intracellular domains do not trigger cell activation but are still able to sequester IgG Fc. The same is true for CD64, CD32 and CD 16. Even though the free Fab region will bind to adjacent target cells, occupancy of Fc will prevent any ADCC or CDC.
Thus, the invention provides a modified cell, wherein the modified cell expression comprises a truncated or modified Fc receptor protein, wherein the Fc receptor protein expression results in the modified cell being less sensitive to Antibody Dependent Cellular Cytotoxicity (ADCC) or Complement Dependent Cytotoxicity (CDC), wherein the truncation or modification reduces or eliminates intracellular signaling. In some aspects of the invention, the Fc receptor protein is selected from the group consisting of truncated CD16 (CD 16 t), truncated CD32 (CD 32 t), and truncated CD64 (CD 64 t). In other aspects, the cell is a primary cell, a differentiated cell, or a pluripotent cell.
In some aspects of the invention, the Fc receptor protein is selected from the group consisting of SEQ ID NO:16, a CD64t protein having at least 90% sequence identity to SEQ ID NO:14 and a CD16t protein having at least 90% sequence identity to SEQ ID NO:15, a CD32t protein having at least 90% sequence identity. In a preferred aspect, the CD64t protein has the amino acid sequence of SEQ ID NO: 16.
In some aspects of the invention, the modified cells are derived from human low-immunogenicity pluripotent (HIP) cells, human low-immunogenicity pluripotent ABO blood group O rhesus factor negative (HIPO-) cells, human Induced Pluripotent Stem Cells (iPSC), or human Embryonic Stem Cells (ESCs).
In other aspects, the modified cell is from a species selected from the group consisting of human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig.
The invention provides a modified cell as disclosed herein further comprising a suicide gene that is activated by a trigger that causes death of the modified cell. In some aspects, the suicide gene is the herpes simplex virus thymidine kinase gene (HSV-tk) and the trigger is ganciclovir. In a preferred aspect, the HSV-tk gene encodes a sequence identical to SEQ ID NO:4, a protein having at least 90% sequence identity. In another preferred aspect, the HSV-tk gene encodes a polypeptide comprising SEQ ID NO:4, and a protein of the sequence of 4. In another aspect, the suicide gene is the E.coli cytosine deaminase gene (EC-CD) and the trigger is 5-fluorocytosine (5-FC). In a preferred aspect, the EC-CD gene encodes a sequence that hybridizes to SEQ ID NO:5, a protein having at least 90% sequence identity. In another preferred aspect, the EC-CD gene encodes a polypeptide comprising SEQ ID NO:5, and a protein of the sequence of 5. In another aspect, the suicide gene encodes an inducible caspase protein and the trigger is a dimerization Chemical Inducer (CID). In a preferred aspect, the suicide gene encodes a polypeptide that hybridizes with SEQ ID NO:6, an inducible caspase protein having at least 90% sequence identity. In another preferred aspect, the suicide gene encodes a polypeptide comprising SEQ ID NO:6 sequence of 6 is of the order of (a) and (b) caspase proteins. In another aspect, CID is AP1903.
The invention provides a modified cell disclosed herein selected from the group consisting of Chimeric Antigen Receptor (CAR) cells, T cells, NK cells, ILC, endothelial cells, dopaminergic neurons, islet cells, islet beta cells, thyroid cells, fibroblasts, hepatocytes, cardiomyocytes, and retinal pigment endothelial cells. In some aspects, the cell is a CAR-T or CAR-NK cell.
The present invention provides a method comprising transplanting a cell disclosed herein into a subject, wherein the subject is a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, guinea pig. In some aspects of the method, the modified cell is selected from the group consisting of a Chimeric Antigen Receptor (CAR) cell, a T cell, an NK cell, an ILC, an endothelial cell, a dopaminergic neuron, an islet cell, a cardiomyocyte, and a retinal pigment endothelial cell.
The invention provides methods of treating a disease comprising administering to a subject a cell disclosed herein or cells derived therefrom. In some aspects of the method, the cell or derived cell is selected from the group consisting of a Chimeric Antigen Receptor (CAR) cell, a T cell, an NK cell, an ILC, an endothelial cell, a dopaminergic neuron, an islet cell, an islet beta cell, a thyroid cell, a fibroblast cell, a hepatocyte, a cardiomyocyte, and a retinal pigment endothelial cell. In other aspects, the disease is selected from the group consisting of type I diabetes, heart disease, neurological disease, endocrine disease, cancer, ocular disease, and vascular disease.
The invention provides methods for producing a modified cell disclosed herein comprising expressing a CD16t, CD32t or CD64t protein in a parent non-modified form of the cell. In some aspects, the modified cell has a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, or guinea pig origin. In other aspects, the modified cells are derived from HIP cells, HIPO cells, iPSC cells, or ESC cells.
In some aspects of the method, CD16t, CD32t or CD64t expression is caused by introducing at least one copy of the human CD16t, CD32t or CD64t gene under the control of a promoter into the parental form of the modified cell. In a preferred aspect, the promoter is a constitutive promoter.
The invention provides a pharmaceutical composition for treating a disease comprising a modified cell disclosed herein or a cell derived therefrom and a pharmaceutically acceptable carrier. In some aspects, the cell is selected from the group consisting of a Chimeric Antigen Receptor (CAR) cell, a T cell, an NK cell, an ILC, an endothelial cell, a dopaminergic neuron, a glial cell, an islet beta cell, a thyroid cell, another endocrine cell, a fibroblast, a hepatocyte, a cardiomyocyte, and a retinal pigment endothelial cell. In other aspects, the disease is selected from the group consisting of type I diabetes, heart disease, neurological disease, endocrine disease, cancer, ocular disease, and vascular disease.
The present invention provides a medicament for treating a disease comprising a cell as described herein or a cell derived from a modified cell as described herein. In some aspects, the cell or derived cell is selected from the group consisting of Chimeric Antigen Receptor (CAR) cells, T cells, NK cells, ILC, endothelial cells, dopaminergic neurons, glial cells, islet beta cells, thyroid cells, fibroblasts, hepatocytes, cardiomyocytes, and retinal pigment endothelial cells. In other aspects, the disease is selected from the group consisting of type I diabetes, heart disease, neurological disease, endocrine disease, cancer, ocular disease, and vascular disease.
The present invention provides a modified cell comprising a CD16t, CD32t or CD64t protein, wherein protein expression results in the modified cell being less sensitive to Antibody Dependent Cellular Cytotoxicity (ADCC) or Complement Dependent Cytotoxicity (CDC). In some aspects, the modified cells comprise enhanced levels of CD16t, CD32t, or CD64t protein produced by genetic engineering. In other aspects, the cell is selected from the group consisting of Chimeric Antigen Receptor (CAR) cells, T cells, NK cells, ILC, endothelial cells, dopaminergic neurons, islet cells, islet beta cells, thyroid cells, fibroblasts, hepatocytes, cardiomyocytes, and retinal pigment endothelial cells.
The invention provides a modified cell as disclosed herein, wherein the cell comprises an Fc receptor chimera comprising a cytoplasmic domain that does not mediate an Fc receptor signaling pathway, and wherein the cytoplasmic domain facilitates endocytosis of an antibody that binds to the extracellular domain of the Fc receptor chimera by its Fc. In some aspects, the cytoplasmic domain is from a transferrin receptor. In other aspects, the transferrin receptor is TfR1 or TfR2. In a preferred aspect, the Fc receptor chimera comprises a CD16, CD32 or CD64 cell surface domain and a TfR1 or TfR2 cytoplasmic domain.
The invention provides a modified cell as disclosed herein, wherein the cell expresses a recombinant sirpa-conjugated protein. In other aspects, the sirpa-engaging protein comprises an immunoglobulin superfamily domain, an antibody Fab domain, or a single chain variable fragment (scFV). In other aspects, the sirpa-binding protein binds sirpa with an affinity as measured by its dissociation constant (Kd), wherein Kd is about 10 -7 To 10 -13 M。
In some aspects, the modified cells disclosed herein comprise a B2M-/-phenotype, CIITA-/-phenotype, CD64t, and SIRP alpha-binding molecules. In other aspects, the sirpa-engaging protein is CD47. In other aspects, the cell is an engineered NK cell.
Brief description of the drawings
FIG. 1 is a schematic diagram of ADCC and CDC. When an antibody of the IgG type finds an antigen on the surface of a target islet cell, it activates NK cells or complement to kill the target cell by antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
FIG. 2 is a schematic representation of one aspect of the invention that utilizes an engineered Fc receptor that sequesters antibodies without altering target cell physiology. Truncated or modified CD16, CD32 or CD64 Coding Sequences (CDs) lack functional intracellular signaling domains. Protein domains according to Uniprot are shown. Cytoplasmic domains for truncation or modification are shown.
Figure 3 shows that truncated CD64 (CD 64 t) captures free IgG Fc without inducing any signaling in target cells.
Fig. 4A and 4B. FIG. 4A shows that wild-type (wt) human iEC expresses full-length CD64. Fig. 4B shows that these iecs captured alemtuzumab Fc in a concentration-dependent manner.
Fig. 5A and 5B. Wild-type (wt) human iEC expressing CD64t similarly captured alemtuzumab Fc in a concentration-dependent manner. FIG. 5A shows CD64t expression on wt iEC. Fig. 5B shows that iEC captures alemtuzumab Fc in a concentration-dependent manner.
FIGS. 6A and 6B show flow cytometry histograms of CD64 expression on human HIPiEC (FIG. 6A). Assessment of free IgG1F Using alemtuzumab c Alemtuzumab is an anti-CD 52 antibody that has no specific binding site on HIP iEC. No IgG1F was present throughout the concentration range tested c Binding (fig. 6B).
FIGS. 7A and 7B show flow cytometry histograms of CD64 expression on human HIPiECs transduced to express CD64 transgenes (HIPiECs) CD64 Fig. 7A). Assessment of free IgG1F Using alemtuzumab c Alemtuzumab is a binding in HIPiECs CD64 An anti-CD 52 antibody without specific binding sites. There was concentration-dependent binding of IgG1 (fig. 7B). Due to absence of F ab Binding epitopes, so this binding must pass F c And CD 64.
FIGS. 8A and 8B show flow cytometry histograms of CD64t expression on human HIPiECs transduced to express CD64t transgenes (HIPiECs) CD64t Fig. 8A). There was concentration-dependent binding of IgG1 (fig. 8B). Due to absence of F ab Binding epitopes, so this binding must pass F c And CD64 t.
Figure 9 shows HIP iEC transduced to express CD52, which is the target epitope of the human anti-CD 52IgG1 antibody alemtuzumab. HIPiEC CD52 Challenge with different alemtuzumab concentrations in the impedance NK cell ADCC assay and were killed in a concentration-dependent manner (mean ± SD, three independent replicates per group and time point).
FIG. 10 shows the use of different alemts in a resistance CDC assayHIPiEC with monoclonal antibody concentration challenge CD52 。HIP iEC CD52 Are killed in a concentration-dependent manner (mean ± SD, three independent replicates per group and time point).
Figure 11 shows HIP iEC transduced to express CD52, which is the target epitope of the human anti-CD 52IgG1 antibody alemtuzumab, as well as CD64 t. HIPiEC CD52,CD64t Challenge with different alemtuzumab concentrations in the impedance NK cell ADCC assay and were completely resistant to killing (mean ± SD, three independent replicates per group and time point).
FIG. 12 shows HIPiEC challenged with different alemtuzumab concentrations in a impedance CDC assay CD52,CD64t 。HIP iEC CD52,CD64t Killing was completely resisted (mean ± SD, three independent replicates per group and time point).
FIGS. 13A and 13B show human thyroid epiCs TPO (A) And epiCs TPO,CD64t (B) Representative flow cytometry histograms of upper Thyroid Peroxidase (TPO) and CD64t expression.
FIGS. 14A and 14B show free IgG1F c (alemtuzumab, which lacks F on thyroepics ab Binding site) in human thyroid epiCs TPO (FIG. 14A) and epi Cs TPO,CD64t (FIG. 14B) representative flow cytometry histograms of the binding on. Only epi Cs TPO,CD64t Capable of binding IgG1.
FIGS. 15A and 15B show the epiCs measured in the Elisa assay TPO (FIG. 15A) and epi Cs TPO,CD64t Thyroxine production (FIG. 15B). There was no difference in thyroxine production (mean.+ -. SD, three independent replicates per group and time point) with or without 1 μg/ml of anti-CD 52IgG1 antibody.
FIG. 16 human thyroid epithelial cells (epiCs) expressing Thyroid Peroxidase (TPO) in vitro undergo antibody-mediated rejection (AMR) by NK cell ADCC at increasing concentrations of anti-TPO antibodies (upper panel). In addition, human thyroep expressing CD64t was protected from ADCC at all antibody concentrations (bottom row).
FIG. 17 shows human thyroid epochs in an impedance NK cell ADCC assay with varying concentrations of anti-TPOIgG 1 antibody TPO . Thyroid epi cs TPO Are killed in a concentration-dependent manner (mean ± SD, three independent replicates per group and time point).
FIG. 18 shows human thyroepics in a resistance CDC assay using varying concentrations of anti-TPOIgG 1 antibody TPO . Thyroid epi cs TPO Are killed in a concentration-dependent manner (mean ± SD, three independent replicates per group and time point).
FIG. 19 shows human thyroid epochs in an impedance NK cell ADCC assay with varying concentrations of anti-TPOIgG 1 antibody TPO,CD64t . Thyroid epi cs TPO,CD64t Are fully protected from killing (mean ± SD, three independent replicates per group and time point).
FIG. 20 shows human thyroepics in a resistance CDC assay using varying concentrations of anti-TPOIgG 1 antibody TPO . Thyroid epi cs TPO,CD64t Are fully protected from killing (mean ± SD, three independent replicates per group and time point).
FIGS. 21A, 21B and 21C human thyroepics were assayed in impedance CDC TPO And epiCs TPO,CD64t Incubated with serum from bridgehead patients 1 (fig. 21A), 2 (fig. 21B) and 3 (fig. 21C). The anti-TPO antibody titers of these patients were 21-fold (FIG. 21A), 26.3-fold (FIG. 21B) and 29.6-fold (FIG. 21C) of the upper limit of normal (0.03U/ml). Thyroid epi cs TPO Are rapidly killed in these assays, but the epi-cs TPO,CD64t Survival was not affected at all (mean ± SD, three independent replicates per group and time point).
Fig. 22A, 22B, and 22C: fig. 22A shows experimental setup for an in vivo antibody killing assay. Will total 5 x 10 4 Personal thyroid epiCs TPO Or epi cs TPO,CD64t And 10 (V) 6 Subcutaneous injection of NK cells into immunodeficient NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ). Both groups received 3 subcutaneous doses of 1mg of anti-TPO on days 0, 1 and 2. Tracking thyroid epi cs TPO (FIG. 22B) and epi Cs TPO,CD64t (FIG. 22C) BLI signal showing thyroepics TPO The transplants disappeared rapidly, whereas all the epiCs TPO,CD64t Are all alive (is plottedAll individual animals are shown, and each group shows a BLI image of a representative animal).
FIGS. 23A and 23B show human beta cells (FIG. 23A) or CD64t expressing beta cells CD64t (FIG. 23B) representative flow cytometry histograms of CD64t expression on. Beta cells CD64t Exhibits strong CD64t expression.
FIGS. 24A and 24B show free IgG1F c (alemtuzumab) in beta cells (FIG. 24A) and beta cells CD64t (FIG. 24B) combined flow cytometry histograms over a sample. Beta cells only CD64t Shows significant IgG1F c Binding was in a concentration-dependent manner.
FIGS. 25A and 25B show human beta cells (FIG. 25A) and beta cells in ELISA assays CD64t Glucose sensing and insulin production (fig. 25B). The assay was performed at low (2 mM) and high (20 mM) glucose. Beta cells and beta cells CD64t Significantly more insulin is produced under high glucose conditions. Glucose sensing and insulin production were not affected by the presence or absence of 1 μg/ml anti-CD 52IgG1 antibody (mean ± SD, three independent replicates per group and time point).
FIG. 26 human islet cells (HLA-A 2 positive) underwent in vitro antibody-mediated killing (top row) when challenged with anti-HLA-A 2IgG antibodies and NK effector cells. Islet cells transduced to express CD64t are protected from anti-HLA-A 2 antibody-mediated killing at all antibody concentrations (bottom row).
FIG. 27 shows human beta cells challenged with varying concentrations of anti-HLA-A 2IgG1 antibodies in a resistance CDC assay. Beta cells expressing HLA-A2 were killed in a concentration-dependent manner (mean ± SD, three independent replicates per group and time point).
FIG. 28 shows human beta cells challenged with different concentrations of anti-HLA-A 2IgG1 antibodies in a resistance CDC assay CD64t . Beta cells expressing HLA-A2 CD64t CDC killing was completely resisted (mean ± SD, three independent replicates per group and time point).
Fig. 29A, 29B, 29C, and 29D: fig. 29A shows the experimental setup of an in vivo β cell survival experiment. Will total 5 x 10 4 Personal beta cells or beta cells CD64t And 10 (V) 6 Individual NK cells were subcutaneously injected into NSG mice. Both groups received 3 subcutaneous 1mg doses of anti-HLA-A 2IgG1 on days 0, 1 and 2. Tracking beta cells (FIG. 29B) and beta cells CD64t (fig. 29C) BLI signal. All beta cell grafts were rejected within only 2 days. All beta cells CD64t None of the grafts was affected and survived the study period. As a control, 5X 10 4 Personal beta cells were injected subcutaneously into NSG mice without NK cells and without antibodies (fig. 29D). Beta cells challenged with anti-HLA-A 2 and NK cells CD64t The survival kinetics of the grafts were not different from those of beta cell grafts without antibodies and NK cells. This indicates that beta cells CD64t The graft is fully protected in vivo from antibody-mediated killing. All individual animals were mapped and a BLI image of one representative animal was displayed for each group.
FIG. 30 shows human CAR-T (A) and CAR-T CD64t (B) Representative flow cytometry histograms of anti-CD 19scFv and CD64t expression. anti-CD 19scFv antibodies specifically recognized anti-CD 19 CARs (mean ± SD, three independent replicates per group and time point).
FIGS. 31A and 31B show free IgG1F c (anti-TPOIgG 1) and CAR-T cells (FIG. 31A) and CAR-T CD64t Representative flow cytometry histograms of cell (fig. 31B) binding. CAR-T only CD64t IgG1 can be bound in a concentration-dependent manner (mean ± SD, three independent replicates per group and time point).
FIG. 32 shows T cells, CAR-T cells and CAR-T CD64t CD19 of (C) + Kinetics of NALM target cell killing. The killing rate is expressed as the number of hours required for the cell index to drop from 1 to 0.5. Different T cell to NALM ratios (mean ± SD, three independent replicates per group and time point) are shown.
FIG. 33 shows CAR-T in the presence and absence of 1 μg/ml anti-CD 52 CD64t CD19 of (C) + Kinetics of NALM target cell killing. The killing rate is expressed as the number of hours required for the cell index to drop from 1 to 0.5. Different CAR-T cell to NALM ratios (mean ± SD, three independent replicates per group and time point) are shown.
Figure 34 shows human CAR-T cells (mean ± SD, three independent replicates per group and time point) in an impedance NK cell ADCC assay using 1 μg/ml antibodies against HLA (anti-HLA-a 2), non-HLA (anti-CD 52, anti-CD 3), rhesus blood group antigen D (anti-Rh (D)) and CAR (anti-CD 19 scFv). In the case of all antibodies, CAR-T cells were killed very rapidly.
Figure 35 shows human CAR-T cells (mean ± SD, three independent replicates per group and time point) in an impedance CDC assay using 1 μg/ml antibodies against HLA (anti-HLA-a 2), non-HLA (anti-CD 52, anti-CD 3), rhesus blood group antigen D (anti-Rh (D)) and CAR (anti-CD 19 scFv). Also, in the case of all antibodies, CAR-T cells were killed very rapidly.
FIG. 36 shows human CAR-T in an impedance NK cell ADCC assay using 1 μg/ml antibodies against HLA (anti-HLA-A 2), non-HLA (anti-CD 52, anti-CD 3), rhesus blood group antigen D (anti-Rh (D)) and against CAR (anti-CD 19 scFv) CD64t Cells (mean ± SD, three independent replicates per group and time point). CAR-T CD64t The cells were completely resistant to ADCC killing.
FIG. 37 shows human CAR-T in an impedance CDC assay using 1 μg/ml antibodies to HLA (anti-HLA-A 2), non-HLA (anti-CD 52, anti-CD 3), rhesus blood group antigen D (anti-Rh (D)) and to CAR (anti-CD 19 scFv) CD64t Cells (mean ± SD, three independent replicates per group and time point). CAR-T CD64t Cells were completely resistant to CDC killing.
FIGS. 38A and 38B show human NK cells (FIG. 38A) and NK cells CD64t Representative flow cytometry histograms of CD64t expression on cells (fig. 38B). Only NK CD64t Cells express CD64t.
FIGS. 39A and 39B show free IgG1F c (anti-TPOIgG 1) and NK cells (FIG. 39A) and NK CD64t Representative flow cytometry histograms of cell (fig. 39B) binding. Only NK CD64t Cells are able to bind IgG1 in a concentration-dependent manner.
Figure 40 shows human NK cells challenged with different concentrations of anti-CD 52IgG1 antibody (alemtuzumab) in an impedance NK cell ADCC assay. NK cells expressing CD52 were killed in a concentration-dependent manner (mean ± SD, three independent replicates per group and time point).
Fig. 41 shows human NK cells challenged with different concentrations of anti-CD 52 antibody in a resistance CDC assay. NK cells expressing CD52 were killed in a concentration-dependent manner (mean ± SD, three independent replicates per group and time point).
FIG. 42 shows human NK challenged with different concentrations of anti-CD 52IgG1 antibody (alemtuzumab) in an impedance NK cell ADCC assay CD64t And (3) cells. NK expressing CD52 CD64t Cells were completely protected from ADCC killing (mean ± SD, three independent replicates per group and time point).
FIG. 43 shows human NK challenged with different concentrations of anti-CD 52IgG1 antibody (alemtuzumab) in a impedance CDC assay CD64t And (3) cells. NK expressing CD52 CD64t Cells were completely protected from CDC killing (mean ± SD, three independent replicates per group and time point).
V. detailed description of the invention
A. Introduction to the invention
The present invention provides for the first time cells comprising truncated CD16, CD32 or CD64 expression to evade Antibody Dependent Cellular Cytotoxicity (ADCC) or Complement Dependent Cytotoxicity (CDC) while eliminating intracellular signaling. The cells may be primary cells or pluripotent cells, including low immunogenicity pluripotent cells (HIP) or ABO blood group O rhesus factor negative HIP cells (HIPO-), which further comprise enhanced CD64 expression. The cell may also be an islet cell, thyroid cell, chimeric Antigen Receptor (CAR) cell, T cell, NK cell, ILC, endothelial cell, dopaminergic neuron, cardiomyocyte, or retinal pigment endothelial cell for treating a disease or repairing damaged tissue.
Antibody-mediated rejection (AMR) after solid organ transplantation was first discovered in 1997 as a unique clinical pathology entity. After decades, the concept of cell rejection has been widely accepted and standardized nomenclature and diagnostic criteria have been developed for kidney, heart, lung, liver and pancreas transplantation. One indicator of AMR is the presence of graft specific antibodies and graft damage.
When an antibody binds to an HLA or non-HLA antigen on a cell through its Fab region, fc is bound by NK cells (primarily through its CD16 receptor) and mediates antibody-mediated cytotoxicity (ADCC) or AMR in the context of transplantation. Complement can also bind to Fc and activate its cascade and form a Membrane Attack Complex (MAC) for CDC killing (fig. 1).
There are three layers of antigens to which antibodies can be directed. Also of primary relevance in the context of transplantation is Human Leukocyte Antigen (HLA), the most polymorphic, histocompatibility complex critical for T cell activation. Patients with antibodies to their grafts always showed poor graft survival in various organ systems.
The second is a highly polymorphic non-HLA antigen, such as MHC class I related sequence a (MICA), which is a cell surface glycoprotein (glyco-protein) encoded within the human HLA gene. It does not bind to β2-microglobulin nor does it present a peptide. anti-MICA antibodies are associated with accelerated transplant failure in solid organ transplants. MICA polymorphism and expression on islet cells are associated with type 1 diabetes (T1 DM).
Third, antibodies raised after solid organ transplantation against cell type specific surface antigens mediate AMR.
These AMR reactions occur in the transplant recipients despite the use of potent systemic immunosuppression. In some autoimmune diseases, autoantibodies cause or enhance destruction of target cells and persist as part of the disease. Patients with autoimmune thyroiditis or type 1 diabetes (T1 DM) have a high prevalence of anti-thyroid epithelial or anti-beta antibodies, respectively, and they may last for years.
Patients undergoing cell transplantation in immunocompetent patients using long-term benign regeneration methods may eventually experience some form of antibody-mediated immune attack, depending on the source of the cells and the disease being treated. Thyroid cells and islet cells express all three layers of antigens and are most vulnerable to all forms of AMR. Thus, these cell types exemplify the antibody evasion techniques disclosed herein. Other cell types are contemplated, including allogeneic human embryonic stem cell-derived cardiac progenitor cells and mesenchymal stromal cell derivatives.
The present invention provides a gene editing strategy that is effective in protecting transplanted cells, such as islet cells and thyroid cells, from AMR. It establishes protection against antibodies against HLA, non-HLA and cell type specific antigens. In some embodiments, it is combined with additional low immunity editing to silence cell rejection.
The present invention provides antibody resistance constructed based on HIP concepts and utilizing non-immunogenic components. Systemic use of microbial IgG degrading enzymes has been successfully used to eliminate total IgG and HLA antibodies in highly sensitized patients prior to their kidney transplantation (Jordan, s.c. et al, N Engl J med377,442-453 (2017)). Recently, this endopeptidase has been shown to cleave IgG binding to target cells (Peraro, L. Et al, mol Ther (2021)). However, pre-existing antibodies to these bacterial enzymes are widely available in healthy humans (Akesson, P. Et al, J. Effect Dis189,797-804 (2004)). They proliferate during streptococcal infection (Lei, B.et al, nat Med 7,1298-1305 (2001); okamoto, S.et al, vaccine 23,4852-4859 (2005)), and may themselves increase unwanted immunogenicity. Human CD64 and its truncated form CD64t are not immunogenic, have high affinity for IgG (Bruhns, p. Et al, blood 113,3716-3725 (2009)), and are very effective against antibody killing in several translation-related cell types.
The present invention applies this technology to regenerative cell therapy, particularly for diseases with potential autoimmune components, where antibodies exist that would destroy the transplanted cells. Regenerative cell therapy would be destroyed like natural cells if autoimmunity were not circumvented (Hollenberg, a.n. et al, mol Cell Endocrinol 445,35-41 (2017)).
The present invention shows that Thyroid Peroxidase (TPO) and CD64 (epi Cs) TPO,CD64t ) Is protected from clinically relevant anti-TPO killing. Islet cells derived from current stem cells in clinical trials conducted in patients with type I diabetes are currently produced from embryonic stem cells (Melton, d.the promise of stemcell-derived islet replacement therapyia 64,1030-1036 (2021)). They are transplanted to where HLA does not match. Although encapsulation strategies are used to protect islet cells from the host immune system, antibodies to microencapsulated transplanted cells have been observed. (Desai, T).&Shea, l.d., nat Rev Drug Discov, 16,338-350 (2017); duviier-Kali et al, am J Transplant 4,1991-2000 (2004)). Forced overexpression of CD64t on human beta cells is sufficient to protect them from anti-HLA ADCC and CDC.
Most of the success of CAR-T cell therapy was achieved using B-cell tumors, where patients received treatment with lymphocytotoxic drugs, and CAR-T cells were directly targeted to the source of antibody production (Brudno, J.N.&Kochenderfer, j.n., nat Rev Clin Oncol, 31-46 (2018)). However, antibody induction was still observed regularly (Till, B.G. et al, blood112,2261-2271 (2008)). Antibody responses against CAR-T cells have been shown to prevent CAR-T cells from expanding upon reinfusion (Peraro, l. Et al Incorporation of bacterial immunoevasins to protect cell therapies from host antibody-mediated immune injection. Mol ter (2021)) and hinder persistence. Expression of CD64T on CAR-T cells enables them to resist anti-CAR-T cell antibodies without affecting their specific killing capacity. Since early immune clearance is more common in non-B cell cancers, CAR-T CD64t May be more effective for these indications. F (F) c The quarantine mechanism reliably establishes protection against antibodies in a variety of cell types and further advances the concept of immune evasion for allogeneic regeneration and immune tumor cell therapy. (see WO2021076427, the entire contents of which are incorporated herein by reference).
CD64 is a high affinity human IgG receptor fcyri capable of capturing free serum IgG. To achieve resistance against AMR, the Fc binding capacity of CD64 was exploited. However, to avoid engineering cells to be altered by unwanted intracellular signaling, the present invention provides a truncated form of CD64 (CD 64 t) on cells that are incapable of inducing intracellular signaling. The intracellular tail is cleaved off prior to cloning the CD64 Coding Sequence (CDs) into lentiviruses for expression in pluripotent cells. CD64t captured free IgG Fc without inducing downstream signaling (fig. 3).
One particular indication of this technology is the cellular treatment of autoimmune diseases. Antibodies against the autologous loci cause beta cell death in type 1 diabetes and thyroid cell death in autoimmune thyroiditis. The antibodies can bind to HLA class II molecules. In addition, HLA independent antibodies are also described. If the cells used for regenerative therapy have the same epitope, they will also be bound and killed by these antibodies. The CD64 isolation of the present invention prevents ADCC and CDC.
In some embodiments of the invention, low immunogenic pluripotent ("HIP") cells are modified to express CD16t, CD32t, or CD64t (HIP/CD 16t, HIP/CD32t, or HIP/CD64t cells). HIP cells avoid host immune responses due to several genetic manipulations outlined herein. The cells lack the primary immune antigen that elicits an immune response and are engineered to avoid phagocytosis and NK cell killing. In some embodiments, the activity of both alleles of the B2M gene in pluripotent stem cells (ipscs) is induced by elimination; eliminating the activity of both alleles of the CIITA gene in ipscs; and increasing expression of CD47 in ipscs to prepare HIP cells. HIP cells are described in detail in WO2018132783 (incorporated herein by reference in its entirety). In other embodiments, HIP cells express SIRPalpha engager molecules as disclosed in PCT/US2021/062008, which is incorporated herein by reference in its entirety.
In other embodiments of the invention, low-immunogenicity pluripotent O blood group Rh- ("HIPO-") cells are modified to express CD16t, CD32t, or CD64t (HIPO-/CD 16t cells, HIPO-/CD32t, or HIPO-/CD64t cells). HIPO-cells avoid host immune responses due to several genetic or enzymatic manipulations outlined herein. The cells lack the major blood group and immune antigens that elicit immune responses and are engineered to avoid rejection, phagocytosis, or killing. This allows the derivation of "off-the-shelf" cell products for the production of specific tissues and organs. The benefits of being able to use human allogeneic HIPO-cells and derivatives thereof in human patients provide significant benefits, including the ability to avoid long-term adjuvant immunosuppressive therapies and drugs that are common in allografts. They also provide significant cost savings because cell therapies can be used without requiring separate treatments for each patient. HIPO-/CD16, HIPO-/CD32 or HIPO-/CD64 cells can be used as a general cell source for the production of universally acceptable derivatives. HIPO-cells are described in detail in U.S. provisional application Ser. Nos. 62/846,399 and 62.855,499, each of which is incorporated by reference herein in its entirety.
B. Definition of the definition
The term "pluripotent cell" refers to a cell that is capable of self-renewal and proliferation while remaining in an undifferentiated state, and that is capable of being induced to differentiate into a specialized cell type under appropriate conditions. As used herein, the term "pluripotent cells" encompasses embryonic stem cells and other types of stem cells, including fetal, amniotic, or somatic stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Other exemplary stem cell lines include those obtainable by the national institutes of health human embryonic stem cell registry and HUES deposit of the HUES institute of medicine, HUES (as described in Cowan, c.a. et al, new England j.med.350:13 (2004), incorporated herein by reference in its entirety).
As used herein, a "pluripotent stem cell" has the ability to differentiate into three germ layers: the potential of any of the endodermal layers (e.g., gastric mucosa, gastrointestinal tract, lung, etc.), mesodermal layers (e.g., muscle, bone, blood, genitourinary tissue, etc.), or ectodermal layers (e.g., epidermal tissue and nervous system tissue). As used herein, the term "pluripotent stem cell" also encompasses "induced pluripotent stem cell," or "iPSC," a pluripotent stem cell type derived from non-pluripotent cells. Examples of parent cells include somatic cells that have been reprogrammed by various means to induce a pluripotent undifferentiated phenotype. Such "iPS" or "iPSC" cells may be produced by inducing the expression of certain regulatory genes or by exogenous application of certain proteins. Methods of inducing iPS cells are known in the art and will be described further below. (see, e.g., zhou et al, stem Cells 27 (11): 2667-74 (2009); huangfu et al, nature Biotechnol.26 (7): 795 (2008); woltjen et al, nature 458 (7239): 766-770 (2009); and Zhou et al, cell Stem Cells 8:381-384 (2009); each of which is incorporated herein by reference in its entirety). The production of induced pluripotent stem cells (ipscs) is summarized below. As used herein, "hiPSC" is a human induced pluripotent stem cell and "miPSC" is a murine induced pluripotent stem cell.
"pluripotent stem cell characteristics" refers to cellular characteristics that distinguish pluripotent stem cells from other cells. The ability to produce offspring capable of differentiating under appropriate conditions into cell types that collectively display characteristics associated with cell lineages from all three germ layers (endoderm, mesoderm and ectoderm) is a pluripotent stem cell characteristic. The expression or non-expression of certain combinations of molecular markers is also a feature of pluripotent stem cells. For example, human pluripotent stem cells express at least several, and in some embodiments, all markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, sox2, E-cadherin, UTF-1, oct4, rex1 and Nanog. The cell morphology associated with pluripotent stem cells is also a characteristic of pluripotent stem cells. As described herein, the cells need not be reprogrammed to endodermal progenitor cells and/or hepatocytes by pluripotency.
As used herein, "multipotent" or "multipotent cells" refers to cell types capable of producing a limited number of other specific cell types. For example, induced pluripotent cells are capable of forming endodermal cells. In addition, pluripotent blood stem cells can differentiate themselves into several types of blood cells, including lymphocytes, monocytes, neutrophils, and the like.
As used herein, the term "oligokinetic" refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells are capable of forming cells of lymphoid or myeloid lineages, respectively.
As used herein, the term "unipotent" (unitotent) refers to the ability of a cell to form a single cell type. For example, spermatogonial stem cells can only form sperm.
As used herein, the term "totipotent" refers to the ability of a cell to form a whole organism. For example, in mammals, only fertilized eggs and first cleavage stage blastomeres are totipotent.
As used herein, "non-pluripotent cells" refers to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are not limited to, cells from tissue selected from the group consisting of bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells. The starter cells used to produce induced pluripotent cells, endodermal progenitor cells, and hepatocytes may be non-pluripotent cells.
Differentiated cells include, but are not limited to, pluripotent cells, oligopotent cells, monopotent cells, progenitor cells, and terminally differentiated cells. In particular embodiments, cells that are less potent (less potent) than more potent (more potent) cells are considered "differentiated".
A "somatic cell" is a cell of an organism that forms the organism. Somatic cells include cells that constitute organs, skin, blood, bone, and connective tissue in an organism, but do not include germ cells.
The cells may be derived from, for example, a human or non-human mammal. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cows, and non-human primates. In some embodiments, the cell is from an adult or non-human mammal. In some embodiments, the cells are from a human neonate, adult, or non-human mammal.
As used herein, the term "subject" or "patient" refers to any animal, such as a domestic animal, zoo animal, or human. The "subject" or "patient" may be a mammal, such as a dog, cat, bird, livestock, or human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) suffering from diseases or conditions associated with liver, heart, lung, kidney, pancreas, brain, neural tissue, blood, bone marrow, and the like.
Mammalian cells may be from human or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cows, and non-human primates (e.g., chimpanzees, macaques, and apes).
"Low immunogenicity pluripotent" cells or "HIP" cells herein refer to pluripotent cells that retain their pluripotent properties but which, when transferred into an allogeneic host, cause reduced immune rejection. In a preferred embodiment, HIP cells do not elicit an immune response. Thus, "low immunogenicity" refers to an immune response that is significantly reduced or eliminated as compared to the immune response of the parental (i.e., "wt") cells prior to the immunization project outlined herein. In many cases, HIP cells are immunosilent, but still retain multipotent. The determination of HIP features is summarized below.
By "HIP/CD16t", "HIP/CD32t" or "HIP/CD64t" cells herein is meant HIP cells having truncated CD16, CD32 or CD64 proteins, respectively, on the cell surface. Truncations remove intracellular signaling domains. In alternative embodiments, the signaling domain is not truncated, but is modified to eliminate signaling function. Such modifications may be amino acid substitutions or internal deletions.
"Low-immunogenic pluripotent cells O-", "Low-immunogenic pluripotent ORh-" cells or "HIPO-" cells are meant herein to refer to HIPO-cells that are also ABO blood group O and rhesus factor Rh-. HIPO-cells may be produced by O-cells, enzymatically modified to O-, or genetically engineered to O-.
As used herein, "HIPO-/CD16t", "HIPO-/CD32t" or "HIPO-/CD64t" cells refer to HIPO-cells having truncated CD16, CD32 or CD64 proteins, respectively, on the cell surface. Truncations remove intracellular signaling domains. In an alternative embodiment, the signaling domain is not truncated, but is mutated to eliminate signaling function.
An "HLA" or "human leukocyte antigen" complex is a complex of genes encoding Major Histocompatibility Complex (MHC) proteins in humans. These cell surface proteins constituting the HLA complex are responsible for modulating the immune response to the antigen. In humans, there are two classes of MHC, class I and class II ("HLA-I" and "HLA-II"). HLA-I includes three proteins, HLA-A, HLA-B and HLA-C, which present peptides from the cell interior, and antigen presented by the HLA-I complex attracts killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins associate with beta-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from outside the cell to T lymphocytes. This stimulates cd4+ cells (also known as T helper cells). It will be appreciated that the use of "MHC" or "HLA" is not meant to be limiting, as this depends on whether the gene is from a Human (HLA) or from a Murine (MHC). Thus, these terms are used interchangeably herein when referring to mammalian cells.
By "gene knockout" herein is meant that inactivation of a particular gene in the host cell in which it resides results in the production of no protein of interest or results in an inactivated form. As will be appreciated by those skilled in the art and described further below, this can be accomplished in a number of different ways, including removal of a nucleic acid sequence from a gene, or disruption of the sequence with other sequences, altering the reading frame, or altering regulatory components of the nucleic acid. For example, all or part of the coding region of the target gene may be removed or replaced with a "nonsensical" sequence, all or part of a regulatory sequence such as a promoter may be removed or replaced, a translation initiation sequence may be removed or replaced, and so forth.
"Gene knock-in" as used herein refers to the process of adding genetic functions to a host cell. This results in an elevated level of encoded protein. As will be appreciated by those skilled in the art, this may be accomplished in several ways, including adding one or more additional copies of the gene to the host cell, or altering regulatory components of the endogenous gene, thereby increasing expression of the protein. This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer or modifying other gene expression sequences.
"beta-2 microglobulin" or "beta 2M" or "B2M" protein refers to a human beta 2M protein having the amino acid and nucleic acid sequences shown below; the accession number of the human gene is NC_000015.10:4471487-44718159.
"CD47 protein" protein refers to a human CD47 protein having the amino acid and nucleic acid sequences shown below; the accession number of the human gene is NC_000016.10:10866208-10941562.CD47 is a ligand for sirpa ("sirpa-binding protein" molecule). CD47 is a "self-labeling" protein that can be widely overexpressed in a variety of tumor types. It is becoming a novel effective macrophage immune checkpoint for cancer immunotherapy. CD47 in tumor cells signals "do not eat me", which inhibits phagocytosis by macrophages.
"CIITA protein" protein refers to a human CIITA protein having the amino acid and nucleic acid sequences shown below; the accession number of the human gene is NC_000003.12:108043094-108094200.
"wild type" in the context of a cell refers to a cell found in nature. However, in the case of pluripotent stem cells, as used herein, it is also meant that ipscs may contain nucleic acid changes that result in pluripotency, but are not subjected to the gene editing program of the present invention to achieve low immunogenicity.
"syngeneic" refers herein to the genetic similarity or identity of a cell graft in which there is immune compatibility (e.g., no immune response is generated) with a host organism.
"allogeneic" as used herein refers to the genetic difference of a cell graft in which an immune response is generated from a host organism.
"B2M-/-" herein means that diploid cells have inactivated B2M genes on both chromosomes. This may be accomplished in a variety of ways, as described herein.
"CIITA-/-" herein means that a diploid cell has inactivated CIITA genes on both chromosomes. This may be accomplished in a variety of ways, as described herein.
"CD47tg" (representing a "transgene") or "CD47+") herein means that the host cell expresses CD47, in some cases by having at least one additional copy of the CD47 gene.
"Oct polypeptide" refers to any naturally occurring member of the octamer family of transcription factors, or variants thereof that retain transcription factor activity that is similar (in the range of at least 50%, 80% or 90% activity) as compared to the most closely related naturally occurring family members, or polypeptides comprising at least the DNA binding domain of a naturally occurring family member, and may further comprise a transcriptional activation domain. Exemplary Oct polypeptides include Oct-1, oct-2, oct-3/4, oct-6, oct-7, oct-8, oct-9, and Oct-11.Oct3/4 (referred to herein as "Oct 4") comprises a POU domain (a 150 amino acid sequence conserved in Pit-1, oct-2 and uric-86). (see Ryan, A.K. & Rosenfeld, M.G., genes Dev.11:1207-1225 (1997), which is incorporated herein by reference in its entirety). In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity over its entire sequence compared to a naturally occurring Oct polypeptide family member, such as compared to those listed above or in Genbank accession No. NP-002692.2 (human Oct 4) or NP-038661.1 (mouse Oct 4). The Oct polypeptide (e.g., oct3/4 or Oct 4) may be from a human, mouse, rat, cow, pig, or other animal. Typically, the same class of proteins will be used with the cell type being manipulated. The Oct polypeptide may be a multipotent factor that can help induce pluripotency in non-pluripotent cells.
"Klf polypeptide" refers to any naturally occurring member of the kruppel-like factor (Klf) family, a zinc finger protein comprising an amino acid sequence similar to that of the drosophila embryo pattern modulator kruppel, or a variant of a naturally occurring member that retains similar (in a range of at least 50%, 80% or 90% activity) transcription factor activity compared to the most closely related naturally occurring family member, or a polypeptide comprising at least the DNA binding domain of the naturally occurring family member, and may further comprise a transcriptional activation domain. (see, dang, D.T., pevsner, J. & Yang, V.W., cell biol.32:1103-1121 (2000), which is incorporated herein by reference in its entirety). Exemplary Klf family members include Klf1, klf2, klf3, klf-4, klf5, klf6, klf7, klf8, klf9, klf10, klf11, klf12, klf13, klf14, klf15, klf16, and Klf17. Klf2 and Klf-4 were found to be factors capable of producing iPS cells in mice, as were the relevant genes Klf1 and Klf5, albeit with reduced efficiency. (see Nakagawa et al, nature Biotechnology 26:101-106 (2007), which is incorporated herein by reference in its entirety). In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity over its entire sequence compared to a naturally occurring Klf polypeptide family member, e.g., compared to those listed above or listed in Genbank accession No. CAX16088 (mouse Klf 4) or CAX14962 (human Klf 4). The Klf polypeptides (e.g., klf1, klf4, and Klf 5) may be from humans, mice, rats, cows, pigs, or other animals. Typically, the same class of proteins will be used with the cell type being manipulated. The Klf polypeptide may be a pluripotency factor. Expression of the Klf4 gene or polypeptide can help induce pluripotency in the starter cell or starter cell population.
"Myc polypeptide" refers to any naturally occurring member of the Myc family. (see, e.g., adhikary, S. & eiers, m., nat. Rev. Mol. Cell biol.6:635-645 (2005), which is incorporated herein by reference in its entirety). It also includes variants that retain similar transcription factor activity (i.e., within a range of at least 50%, 80%, or 90% activity) when compared to the most closely related naturally occurring family members. It also includes polypeptides comprising at least the DNA-binding domain of a naturally occurring family member, and may also comprise a transcriptional activation domain. Exemplary Myc polypeptides include, for example, c-Myc, N-Myc, and L-Myc. In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity over its entire sequence compared to a naturally occurring Myc polypeptide family member, such as compared to those listed above or such as listed in Genbank accession number CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc) may be from humans, mice, rats, cows, pigs, or other animals. Typically, the same class of proteins will be used with the cell type being manipulated. The Myc polypeptide may be a pluripotency factor.
"Sox polypeptide" refers to any naturally occurring member of the SRY-related HMG-box (Sox) transcription factor characterized by the presence of a high-mobility group (HMG) domain or variant thereof that retains similar transcription factor activity (i.e., within a range of at least 50%, 80% or 90% activity) when compared to the most closely related naturally occurring family members. It also includes polypeptides comprising at least the DNA-binding domain of a naturally occurring family member, and may also comprise a transcriptional activation domain. (see, e.g., dang, D.T. et al, int.J. biochem.cell biol.32:1103-1121 (2000), which is incorporated herein by reference in its entirety). Exemplary Sox polypeptides include, for example, sox1, sox-2, sox3, sox4, sox5, sox6, sox7, sox8, sox9, sox10, sox11, sox12, sox13, sox14, sox15, sox17, sox18, sox-21, and Sox30. The efficiency of Sox1 to generate iPS cells has been shown to be similar to Sox2, and the genes Sox3, sox15 and Sox18 have also been shown to generate iPS cells, although the efficiency is slightly lower than Sox2. (see Nakagawa, et al, nature Biotechnology 26:101-106 (2007), which is incorporated herein by reference in its entirety). In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity over its entire sequence as compared to a naturally occurring Sox polypeptide family member, such as those listed above or such as those listed in Genbank accession No. CAA83435 (human Sox 2). Sox polypeptides (e.g., sox1, sox2, sox3, sox15, or Sox 18) may be from humans, mice, rats, cows, pigs, or other animals. Typically, the same class of proteins will be used with the cell type being manipulated. The Sox polypeptide may be a pluripotency factor. As described herein, SOX2 proteins have particular utility in the production of ipscs.
By "differentiated low-immunogenicity pluripotent cells" or "differentiated HIP cells" or "dHIP cells" herein is meant iPS cells that have been engineered to have low immunogenicity (e.g., by knockout of B2M and CIITA and knockout of CD 47), and then differentiated into cell types that are ultimately transplanted into a subject. Thus, for example, HIP cells can differentiate into hepatocytes ("dHIP hepatocytes"), β -like pancreatic cells or islet organoids ("dHIP β cells"), endothelial cells ("dHIP endothelial cells"), and the like. Parallel definitions apply to "differentiated HIP/CD64" and differentiated HIPO-/CD64 cells.
In the context of two or more nucleic acid or polypeptide sequences, the term "percent identity" refers to two or more sequences or subsequences that have a specified percentage of identical nucleotide or amino acid residues, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the "percentage of identity" may be present over the region of the sequences being compared, for example over the functional domain, or over the full length of the two sequences being compared. For sequence comparison, typically one sequence is used as a reference sequence for comparison with the test sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.
The optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, adv. Appl. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), by the similarity search algorithm of Pearson & Lipman, proc. Nat' l. Acad. Sci. USA 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA, genetics Computer Group,575Science Dr. Madison, wis., in the Wis.) or by visual inspection (see generally Ausubel et al, see below).
One example of an algorithm suitable for determining the percent sequence identity and percent sequence similarity is the BLAST algorithm, which is described in Altschul et al, J.mol. Biol.215:403-410 (1990). Software for performing BLAST analysis is publicly available through the national center for biotechnology information (www.ncbi.nlm.nih.gov /).
"inhibitors", "activators" and "modulators" affect the function or expression of biologically relevant molecules. The term "modulator" includes inhibitors and activators. They can be identified by measuring the expression or activity of the target molecule in vitro and in vivo.
An "inhibitor" is an agent that, for example, inhibits expression or binding to a target molecule or protein. They may partially or fully block stimulation or have protease inhibitor activity. They may reduce, prevent or delay activation, including inactivation, desensitization or downregulation of the activity of the target protein. The modulator may be an antagonist of the target molecule or protein.
An "activator" is an agent that, for example, induces or activates the function or expression of a target molecule or protein. They can bind to, stimulate, increase, open, activate or promote the activity of a target molecule. The activator may be an agonist of the target molecule or protein.
A "homolog" is a biologically active molecule that is similar to a reference molecule at the nucleotide sequence, peptide sequence, functional or structural level. A homologue may comprise a sequence derivative sharing a certain percentage identity with the reference sequence. Thus, in one embodiment, homologous or derived sequences share at least 70% sequence identity. In a specific embodiment, homologous or derived sequences share at least 80% or 85% sequence identity. In a specific embodiment, homologous or derived sequences share at least 90% sequence identity. In a specific embodiment, homologous or derived sequences share at least 95% sequence identity. In a more specific embodiment, homologous or derived sequences share at least 50%, 55%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Homologous or derivative nucleic acid sequences can also be defined by their ability to remain bound to a reference nucleic acid sequence under high stringency hybridization conditions. A homolog having structural or functional similarity to the reference molecule may be a chemical derivative of the reference molecule. Methods for detecting, generating and screening structural and functional homologs and derivatives are known in the art.
"hybridization" generally depends on the ability of denatured DNA to re-anneal when the complementary strand is present in an environment below its melting temperature. The higher the degree of homology desired between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Thus, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures make the reaction conditions less stringent. For additional details and explanation of hybridization reaction stringency, see Ausubel et al, current Protocols in Molecular Biology, wiley Interscience Publishers (1995), which is incorporated herein by reference in its entirety.
The "stringency" of hybridization reactions can be readily determined by one of ordinary skill in the art and is typically calculated empirically based on probe length, wash temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures.
As defined herein, "stringent conditions" or "highly stringent conditions" may be determined by the following conditions: (1) Washing with low ionic strength and high temperature, e.g., 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium dodecyl sulfate at 50 ℃; (2) Denaturing agents, such as formamide, such as 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer, ph6.5 with 750mM sodium chloride, 75mM sodium citrate, 42 ℃ are used during hybridization; or (3) hybridization in a solution of 50% formamide, 5 XSSC (0.75M NaCl,0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. Mu.l/m 1), 0.1% SDS and 10% dextran sulfate at 42℃overnight, washing in 0.2XSSC (sodium chloride/sodium citrate) at 42℃for 10 minutes followed by 10 minutes high stringency washing consisting of 0.1 XSSC containing EDTA at 55 ℃.
Every maximum numerical limitation given throughout this specification is intended to include every smaller numerical limitation, as if such smaller numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every larger numerical limitation, as if such larger numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
As used herein, the term "modification" refers to a change that physically distinguishes a modified molecule from a parent molecule. In one embodiment, amino acid changes in a CD16, CD32, CD64, CD47, HSVtk, EC-CD, or iCasp9 variant polypeptide prepared according to the methods described herein distinguish it from a corresponding parent gene or cell that is not modified according to the methods described herein, such as a wild-type protein, a naturally occurring mutant protein, or another engineered protein that does not include modification of such variant polypeptide. In another embodiment, the variant polypeptide comprises one or more modifications that distinguish the variant polypeptide from the function of the unmodified polypeptide. For example, amino acid changes in a variant polypeptide affect its receptor binding profile (receptor binding profile). In other embodiments, the variant polypeptide comprises a substitution, deletion, or insertion modification, or a combination thereof. In another embodiment, the variant polypeptide comprises one or more modifications that increase its affinity for the receptor as compared to the affinity of the unmodified polypeptide.
In one embodiment, the variant polypeptide comprises one or more substitutions, insertions or deletions relative to the corresponding native or parent sequence. In certain embodiments, the variant polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-40, 41 to 50, or 51 or more modifications.
"episomal vector" herein refers to a genetic vector that can exist in the cytoplasm of a cell and autonomously replicate; for example, it is not integrated into the genomic DNA of the host cell. Many episomal vectors are known in the art and are described below.
"knockout" in the context of a gene refers to a functional protein product that does not produce the gene by the host cell containing the knockout. As outlined herein, a knockout can be caused by a variety of ways: removal of all or part of the coding sequence, introduction of frameshift mutations such that no functional protein (truncated or nonsense sequence) is produced, removal or alteration of regulatory components (e.g., promoters) such that the gene is not transcribed, prevention of translation by binding to mRNA, etc. Typically, the knockout is effected at the genomic DNA level such that the progeny of the cell also permanently carry the knockout.
By "knock-in" in the context of a gene is meant that the host cell containing the knock-in has more active functional protein in the cell. As outlined herein, the knock-in may be performed in a variety of ways, typically by introducing at least one copy of the transgene (tg) encoding the protein into the cell, although this may also be performed by replacing regulatory components, e.g. by adding a constitutive promoter to the endogenous gene. In general, knock-in techniques result in the integration of additional copies of the transgene into the host cell.
VI cells of the invention
The present invention provides compositions and methods for producing pluripotent cells expressing CD16t, CD32t or CD64 t. In some aspects of the invention, the cells will be derived from, or derived from, induced Pluripotent Stem Cells (IPSCs), O-induced pluripotent stem cells (iPSCO-), embryonic Stem Cells (ESCs), O-embryonic stem cells (ESCO-), low immunogenic pluripotent (HIP) cells, low immunogenic pluripotent O- (HIPO-) cells, or cells derived or differentiated therefrom. In other aspects, the cell is a primary cell, including a gene-edited primary cell. The cells of the invention may also be islet cells, thyroid cells, chimeric Antigen Receptor (CAR) cells, endothelial cells, dopaminergic neurons, cardiomyocytes, or retinal pigment endothelial cells for use in treating a disease or repairing damaged tissue.
A. Methods for gene alteration
The invention includes methods of modifying a nucleic acid sequence under intracellular or cell-free conditions to produce a cell having expression of CD16t, CD32t, or CD64 t. Exemplary techniques include homologous recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly interspaced short palindromic repeats)/Cas 9, and other site-specific nuclease techniques. These techniques enable double-stranded DNA to be broken at desired sites. These controlled double strand breaks promote homologous recombination at specific locus sites. This process focuses on targeting a specific sequence of a nucleic acid molecule, such as a chromosome, with an endonuclease that recognizes and binds to the sequence and induces a double strand break of the nucleic acid molecule. Double strand breaks can be repaired by error-prone non-homologous end joining (NHEJ) or Homologous Recombination (HR).
As will be appreciated by those skilled in the art, many different techniques may be used to engineer the pluripotent cells of the invention, as well as to engineer ipscs to be low immunogenic, as outlined herein.
In general, these techniques may be used alone or in combination. For example, these techniques can be used to generate cells expressing Fc receptors, wherein the receptor is truncated to remove some or all of the cytoplasmic signaling domain. Alternatively, the domain may be modified by deletion, substitution, or nonsense mutation.
Upon Fc binding, the cytoplasmic tail of CD64 interacts with src related families of tyrosine kinases (e.g., fyn and Lyn) and Syk family kinases. They phosphorylate a cytoplasmic amino acid motif called the immune receptor tyrosine activation motif (ITAM) on the related FcR gamma chain (encoded by its own gene FCER 1G). This mediates activation of immune cells.
Thus, the present invention provides modification of cytoplasmic tails that interact with src-related family and Syk family kinases of tyrosine kinases to prevent activation of FcR gamma chain ITAM and thus downstream signaling.
CD64 cytoplasmic tail is from SEQ ID NO:7 of the following 60 amino acids:
RKELKRKKKWDLEISLDSGHEKKVISSSLQEDRHLEEELKC QEQKEEQLQEGVHRKEPQGAT
in some embodiments, the cytoplasmic domain of the Fc receptor is exchanged for another cytoplasmic domain having a different physiology. For example, cytoplasmic domains of transferrin receptors (e.g., tfR1 or TfR 2) promote sustained endocytosis, resulting in high receptor turnover rates. Thus, in certain embodiments, the Fc receptor is a chimera between the extracellular domain of the Fc receptor and the cytoplasmic domain of the transferrin receptor. This will promote endocytosis and disintegration of IgG antibodies that initially bind to Fc receptor domains on the cell surface. Thus, in a preferred embodiment, the cells of the invention express a chimera comprising a CD16, CD32 or CD64 cell surface domain and a TfR1 or TfR2 cytoplasmic domain.
Furthermore, these techniques can be used for HIP cell production. CRISPR can be used to reduce expression of active B2M and/or CIITA proteins in engineered cells, using viral techniques (e.g., lentivirus) to knock in CD47 or other sirpa-junction protein expression. Furthermore, although one embodiment knocks out B2M sequentially with a CRISPR step followed by a CIITA with a CRISPR step and a final step with lentiviral knockin of CD47 or other sirpa-binding protein expression, these genes can be manipulated in different sequences using different techniques, as will be appreciated by those skilled in the art.
As discussed more fully below, transient expression of the reprogramming genes is typically performed to generate/induce pluripotent stem cells.
CRISPR technology
In one embodiment, cells are manipulated using clustered regularly interspaced short palindromic repeats)/Cas ("CRISPR") techniques known in the art. CRISPR can be used to produce starting ipscs or to produce HIP cells from ipscs. There are a number of CRISPR-based techniques, see, e.g., doudna and Charpentier, science doi 10.1126/science.1258096, which are incorporated herein by reference. CRISPR technology and kits are commercially available.
TALEN technology
In some embodiments, HIP cells of the present invention are prepared using a transcription activator-like effector nuclease (TALEN) method. TALENs are restriction enzymes that bind to nucleases and can be engineered to bind to and cleave virtually any desired DNA sequence. TALEN kits are commercially available.
c. Zinc finger technology
In one embodiment, the cell is manipulated using zinc finger nuclease technology. Zinc finger nucleases are artificial restriction enzymes produced by fusing a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target a specific desired DNA sequence, which enables the zinc finger nuclease to target unique sequences in a complex genome. By utilizing endogenous DNA repair mechanisms, these agents can be used to precisely alter the genome of higher organisms, similar to CRISPR and TALENs.
d. Virus-based techniques
There are a variety of viral techniques that can be used to generate HIP cells of the present invention (and for initial generation of iPCS), including but not limited to the use of retroviral vectors, lentiviral vectors, adenoviral vectors, and Sendai virus vectors. Additional vectors for generating ipscs are described below.
e. Down-regulating genes using interfering RNAs
In other embodiments, genes encoding proteins used in HLA molecules are down-regulated by RNAi technology. RNA interference (RNAi) is a process in which RNA molecules typically inhibit gene expression by causing degradation of specific mRNA molecules. Two types of RNA molecules, micrornas (mirnas) and small interfering RNAs (sirnas), are central to RNA interference. They bind to the target mRNA molecule, enhancing or reducing its activity. RNAi helps cells resist parasitic nucleic acids, such as nucleic acids from viruses and transposons. RNAi also affects development.
sdRNA molecules are a class of asymmetric siRNA comprising a 19-21 base guide (antisense) strand. They contain 5 'phosphates, 2' ome or 2'f modified pyrimidines and phosphorothioates at 6 3' positions. They also contain a sense strand containing a 3' conjugated sterol moiety, 2 phosphates at the 3' position and a 2' ome modified pyrimidine. Both chains contain 2' ome purines, wherein the unmodified purines have a continuous extension of no more than 3.sdRNA is disclosed in U.S. Pat. No. 8,796,443, which is incorporated herein by reference in its entirety.
For all of these techniques, well-known recombinant techniques are used to generate the recombinant nucleic acids outlined herein. In certain embodiments, a recombinant nucleic acid (encoding a desired polypeptide, e.g., CD47, or encoding a disruption sequence) may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences are generally suitable for the host cell and subject to be treated. For a variety of host cells, many types of suitable expression vectors and suitable regulatory sequences are known in the art. In general, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader sequences or signal sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are also contemplated. The promoter may be a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter. The expression construct may be present on an episome (such as a plasmid) in the cell, or the expression construct may be inserted into a chromosome. In particular embodiments, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Certain embodiments include expression vectors comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequences as used herein include promoters, enhancers and other expression control elements. In certain embodiments, the expression vector is designed for the selection of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the copy number of the vector, the ability to control that copy number, or the expression of any other protein encoded by the vector (such as an antibiotic marker).
Examples of suitable mammalian promoters include, for example, promoters from the following genes: hamster ubiquitin/S27 a promoter (WO 97/15664), simian vesicular virus 40 (SV 40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, long terminal repeat of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), moloney murine leukemia virus long terminal repeat and human Cytomegalovirus (CMV) early promoter. Examples of other heterologous mammalian promoters are actin, immunoglobulin or heat shock promoters.
In further embodiments, the promoter for the mammalian host cell may be obtained from the genome of viruses such as polyomavirus, fowlpox virus (UK 2,211,504 published 7.5 in 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis b virus, and simian virus 40 (SV 40). In further embodiments, heterologous mammalian promoters are used. Examples include actin promoters, immunoglobulin promoters and heat shock promoters. The early and late promoters of SV40 are conveniently obtained as SV 40-restricted fragments which also contain the SV40 viral origin of replication. Fiers et al, nature 273:113-120 (1978). The immediate early promoter of human cytomegalovirus is conveniently obtained in the form of HindIII E restriction fragments. Greenaway, P.J. et al, gene 18:355-360 (1982). The foregoing references are incorporated by reference in their entirety.
B. Production of pluripotent cells
The present invention provides methods for producing non-immunogenic pluripotent cells from pluripotent cells. Thus, the first step is to provide pluripotent stem cells.
The generation of mouse and human pluripotent stem cells (commonly referred to as iPSCs; either the miPSCs of murine cells or the hiPSCs of human cells) is well known in the art. As will be appreciated by those skilled in the art, there are a number of different methods of generating iPCS. Initial induction from mouse embryo or adult fibroblasts using viral introduction of four transcription factors Oct3/4, sox2, c-Myc and Klf 4; see Takahashi and Yamanaka Cell 126:663-676 (2006), which are incorporated herein by reference in their entirety, particularly for the techniques outlined therein. Since then, a number of methods have been developed; for reviews see Seki et al, world J.stem Cells 7 (1): 116-125 (2015), and Lakshmathy and Vermuri, editions, methods in Molecular Biology: pluripotent Stem Cells, methods and Protocols, springer2013, which are expressly incorporated herein by reference in their entirety, particularly for methods for producing hiPSCs (see, e.g., chapter 3 of the latter reference).
In general, ipscs are produced by transient expression of one or more "reprogramming factors" (typically introduced using episomal vectors) in a host cell. Under these conditions, a small number of cells were induced to iPSC (typically, this step is less efficient because no selection marker is used). Once cells are "reprogrammed" and rendered pluripotent, they lose one or more episomal vectors and utilize endogenous genes to produce factors. This loss of one or more episomal vectors results in cells known as "zero footprint" cells. This is desirable because the fewer the genetic modification (particularly in the genome of the host cell) the better. Therefore, it is preferred that the resulting hipscs have no permanent genetic modification.
As also understood by those skilled in the art, the number of reprogramming factors that may be used or that have been used may vary. In general, when fewer reprogramming factors are used, the efficiency of the cell's conversion to the pluripotent state decreases, as does the "pluripotency", e.g., fewer reprogramming factors may result in cells that are not fully pluripotent, but only differentiate into fewer cell types.
In some embodiments, a single reprogramming factor OCT4 is used. In other embodiments, two reprogramming factors OCT4 and KLF4 are used. In other embodiments, three reprogramming factors OCT4, KLF4, and SOX2 are used. In other embodiments, four reprogramming factors OCT4, KLF4, SOX2, and c-Myc are used. In other embodiments, 5, 6, or 7 reprogramming factors selected from the group consisting of sokmnt, SOX2, OCT4 (POU 5F 1), KLF4, MYC, NANOG, LIN, 28, and SV40L T antigens may be used.
Typically, these reprogramming factor genes are provided on episomal vectors (such as those known in the art and commercially available). For example, thermo fisher/Invitrogen sells sendai virus reprogramming kits for zero footprint generation of hipscs, see catalog No. a34546.ThermoFisher also sells EBNA based systems, see catalog number A14703.
In addition, there are many hiPSC lines commercially available; see, for exampleEpisonal hiPSC line K18945, a zero footprint, virus-free integrated human iPSC cell line (see also Burridge et al 2011, supra).
Typically, ipscs are produced from non-pluripotent cells such as cd34+ umbilical cord blood cells, fibroblasts, etc. by transient expression of reprogramming factors as described herein, as known in the art.
For example, using only Oct3/4, sox2, and Klf4, while omitting C-Myc, also produced a successful iPSC, despite reduced reprogramming efficiency.
In general, iPSC is characterized by the expression of certain factors including KLF4, nanog, OCT4, SOX2, ESRRB, TBX3, c-Myc and TCL 1. For the purposes of the present invention, the novel or increased expression of these factors may be by induction or regulation of endogenous loci or expression from transgenes.
For example, diecke et al, sci Rep.2015, jan.28 may be used; 5:8081 (doi: 10.1038/srep 08081) (incorporated herein by reference in its entirety, particularly for methods and reagents for generating a miPSC) to generate a mouse iPSC. See also, e.g., burridge et al, PLoS One,2011 (4): 18293, which is incorporated herein by reference in its entirety, particularly for the methods outlined herein.
In some cases, the pluripotency of the cells is measured or confirmed as outlined herein, for example, by analysis of reprogramming factors or by conducting a differentiation reaction as outlined herein and in the examples.
C. Production of low immunogenic pluripotent (HIP) cells
The generation of HIP cells from pluripotent cells is performed with as few as three genetic alterations that result in minimal disruption of cellular activity, but confer immune silencing on the cells.
As discussed herein, one embodiment utilizes the reduction or elimination of protein activity of MHC I and II (HLA I and II when the cell is a human). This can be achieved by altering the gene encoding its component. In one embodiment, a coding region or regulatory sequence of a CRISPR disruption gene is used. In another embodiment, the use of interfering RNA techniques reduces gene translation. The third is the alteration of genes that regulate susceptibility to phagocytosis by macrophages, such as CD47 or sirpa-engageenans, which are typically genes "knockins" using viral technology.
In some cases, hiPSC cells containing Cas9 constructs that enable efficient editing of cell lines may be used when CRISPR is used for genetic modification; see, e.g., human-attached Cas9iPSC cell line a33124 from Life Technologies.
HLA-I reduction
HIP cells of the present invention include a decrease in MHC I function (HLA I when the cells are derived from human cells).
As will be appreciated by those of skill in the art, the reduction of function may be achieved in a variety of ways, including removal of a nucleic acid sequence from a gene, disruption of the sequence with other sequences, or alteration of regulatory components of the nucleic acid. For example, all or part of the coding region of the target gene may be removed or replaced with a "nonsense" sequence, a frameshift mutation may be generated, all or part of a regulatory sequence such as a promoter may be removed or replaced, a translation initiation sequence may be removed or replaced, and the like.
As will be appreciated by those skilled in the art, successful reduction of MHC I function (HLA I when the cell is derived from a human cell) in pluripotent cells can be measured using techniques known in the art and described below; for example, FACS techniques using labeled antibodies that bind to HLA complexes; for example, commercially available HLA-a, B, C antibodies that bind to the alpha chain of human major histocompatibility class I HLA antigen are used.
a.B2M Change
In one embodiment, the reduction of HLA-I activity is achieved by disrupting the expression of the beta-2 microglobulin gene in pluripotent stem cells, the human sequences of which are disclosed herein. Such changes are generally referred to herein as gene "knockouts," which are made on both alleles of a host cell in HIP cells of the present invention. In general, the technique of performing both interrupts is the same.
Particularly useful embodiments use CRISPR techniques to disrupt genes. In some cases, CRISPR techniques are used to introduce small deletions/insertions into the coding region of a gene such that no functional protein is produced, typically as a result of a frame shift mutation that results in the production of a stop codon such that a truncated non-functional protein is produced.
Thus, a useful technique is to use CRISPR sequences designed to target the coding sequence of the B2M gene in mice or the B2M gene in humans. After gene editing, transfected iPSC cultures were dissociated into single cells. Single cells were expanded to full-size colonies and CRISPR editing was tested by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones were selected in which both alleles were deleted. Such clones did not express B2M, as shown by PCR, nor HLA-I, as shown by FACS analysis (see, e.g., examples 1 and 6).
Assays for testing whether the B2M gene has been inactivated are known and described herein. In one embodiment, the assay is a western blot of cell lysates probed with antibodies to B2M proteins. In another embodiment, the reverse transcriptase polymerase chain reaction (rt-PCR) confirms the presence of an inactivation change.
In addition, cells can be tested to confirm that HLA I complexes are not expressed on the cell surface. As discussed above, this can be determined by FACS analysis using antibodies to one or more HLA cell surface components.
Notably, others have not performed well when attempting to silence the B2M gene on both alleles. See, e.g., gornaguse et al, nature Biotech. Doi/10.1038/nbt.3860).
HLA-II reduction
In addition to the reduction of HLA I, HIP cells of the invention also lack MHC II function (HLA II when the cells are derived from human cells).
As will be appreciated by those of skill in the art, the reduction in function may be achieved in a variety of ways, including removal of a nucleic acid sequence from a gene, addition of a nucleic acid sequence to a gene, disruption of the reading frame, disruption of the sequence with other sequences, or alteration of regulatory components of the nucleic acid. In one embodiment, all or part of the coding region of the gene of interest may be removed or replaced with a "nonsense" sequence. In another embodiment, regulatory sequences such as promoters may be removed or replaced, translation initiation sequences may be removed or replaced, and the like.
Successful reduction of MHC II function (HLA II when the cells are derived from human cells) in pluripotent cells or derivatives thereof can be measured using techniques known in the art, such as western blotting using antibodies to proteins, FACS techniques, rt-PCR techniques, and the like.
CIITA Change
In one embodiment, the reduction in HLA-II activity is achieved by disrupting expression of the CIITA gene in the pluripotent stem cells, the human sequence of the CIITA gene being as set forth herein. Such alterations are generally referred to herein as gene "knockouts". In the HIP cells of the present invention, this is done on both alleles of the host cell.
Assays for testing whether the CIITA gene has been inactivated are known and described herein. In one embodiment, the assay is a western blot of cell lysates probed with antibodies to CIITA proteins. In another embodiment, the reverse transcriptase polymerase chain reaction (rt-PCR) confirms the presence of an inactivation change.
In addition, cells can be tested to confirm that HLA II complexes are not expressed on the cell surface. Again, this determination is made according to methods known in the art. Exemplary assays include western blot or FACS analysis using commercial antibodies that bind to human HLA class II HLA-DR, DP and most DQ antigens as described below.
One particularly useful embodiment uses CRISPR technology to disrupt CIITA genes. CRISPR is designed to target the coding sequence of the Ciita gene of mice or the Ciita gene of humans, which is an essential transcription factor for all MHC II molecules. After gene editing, transfected iPSC cultures were dissociated into single cells. They were amplified to full-size colonies and tested for successful CRISPR editing by screening for the presence of abnormal sequences from the CRISPR cleavage site. Clones with deletions did not express CIITA (as determined by PCR) and MHC II/HLA-II (as determined by FACS analysis).
3. Phagocytosis is reduced
In addition to the reduction of HLA I and II (or MHC I and II), B2M and CIITA knockouts are typically used, and the HIP cells of the invention have reduced susceptibility to macrophage phagocytosis and NK cell killing. Due to one or more CD47 transgenes, the resulting HIP cells "escape" immune macrophages and the innate pathway.
Increased CD47
In some embodiments, the decrease in macrophage phagocytosis and NK cell killing susceptibility results from an increase in CD47 on the surface of the HIP cells. As will be appreciated by those skilled in the art, this is accomplished in several ways using "knock-in" or transgenic techniques. In some cases, the increase in CD47 expression is caused by one or more CD47 transgenes.
Thus, in some embodiments, one or more copies of the CD47 gene are added to the HIP cells under the control of an inducible or constitutive promoter (the latter being preferred). In some embodiments, the CD47 gene may be integrated into the genome of the host cell under the control of a suitable promoter known in the art using lentiviral constructs, as described herein or known in the art.
HIP cell lines were generated from B2M-/-CIITA-/-iPSC. Cells containing lentiviral vectors expressing CD47 were selected using blasticidin markers. The CD47 gene sequence was synthesized and the DNA was cloned into plasmid lentivirus pLenti6/V5 (Thermo Fisher Scientific, waltham, mass.) with blasticidin resistance.
In some embodiments, expression of the CD47 gene may be increased by altering the regulatory sequences of the endogenous CD47 gene (e.g., by replacing the endogenous promoter with a constitutive promoter or a different inducible promoter). This can be typically accomplished using known techniques such as CRISPR.
Once altered, the presence of adequate CD47 expression can be determined using known techniques, such as those described in the examples, such as western blotting, ELISA assays, or FACS assays using anti-CD 47 antibodies. In general, "sufficiency" in this specification refers to an increase in expression of CD47 on the surface of HIP cells that silences NK cell killing. Once MHC I is removed, the native expression levels on cells are too low to protect them from NK cell lysis.
The participation of SIRP can be accomplished by engineering molecules or replacing molecules. In some aspects of the invention, the binding molecule is a protein. In other aspects, the protein is a fusion protein. In other aspects, the fusion protein comprises a CD47 extracellular domain (ECD). In other aspects of the invention, the SIRP-alpha mating cell comprises an immunoglobulin superfamily domain. In other aspects of the invention, the binding molecule comprises an antibody Fab or single chain variable fragment (scFV) that binds sirpa. In other aspects, the Fab or scFV binds to sirpa with an affinity as measured by its dissociation constant (Kd), wherein Kd is about 10 -7 To 10 -13 M. See PCT/US2021/062008, incorporated herein by reference in its entirety.
In some aspects of the invention, the binding molecule comprises one or more antibody Complementarity Determining Regions (CDRs) that bind sirpa.
4. Suicide gene
In some embodiments, the invention provides low immunogenicity multipotent cells comprising a "suicide gene" or "suicide switch". They are integrated as "safety switches" that can cause the death of low-immunogenic pluripotent cells if they grow and divide in an undesired manner. The "suicide gene" ablation method includes suicide genes in gene transfer vectors, which suicide genes encode proteins that cause cell killing only when activated by a specific compound. Suicide genes may encode enzymes that selectively convert non-toxic compounds to highly toxic metabolites. The result is a specific elimination of cells expressing the enzyme. In some embodiments, the suicide gene is a herpes virus thymidine kinase (HSV-tk) gene and the initiating agent is ganciclovir. In other embodiments, the suicide gene is the E.coli cytosine deaminase (EC-CD) gene, the initiator is 5-fluorocytosine (5-FC) (Baress et al mol. Therapeutic.20 (10): 1932-1943 (2012), xu et al, cell Res.8:73-8 (1998), both of which are incorporated herein by reference in their entirety).
In other embodiments, the suicide gene is an inducible caspase protein. The inducible caspase protein comprises at least a portion of a caspase protein capable of inducing apoptosis. In one embodiment, the sequence set forth in SEQ ID NO:6 illustrates a portion of a caspase protein. In a preferred embodiment, the inducible caspase protein is iCasp9. Comprising the sequence of the human FK506 binding protein FKBP12 with the F36V mutation linked by a series of amino acids to the gene encoding human caspase 9. FKBP12-F36V binds with high affinity to the small molecule dimerization agent AP1903. Thus, the suicide function of iCasp9 in the present invention is triggered by the application of a dimerizing Chemical Inducer (CID). In some embodiments, CID is small molecule drug AP1903. Dimerization leads to rapid induction of apoptosis. (see WO2011146862; stasi et al, N.Engl. J. Med 365;18 (2011); tey et al, biol. Blood Marrow transfer.13:913-924 (2007), each of which is incorporated herein by reference in its entirety).
CD16t, CD32t or CD64t expression
The cells of the invention have reduced susceptibility to ADCC and CDC due to CD16t, CD32t or CD64t expression. The resulting cells sequester antibodies due to increased expression. In one embodiment, the cell comprises one or more CD16t, CD32t or CD64t transgenes.
In some embodiments, the decrease in ADCC or CDC susceptibility is caused by CD16t, CD32t, or CD64t on the cell surface. As will be appreciated by those skilled in the art, this is accomplished in several ways using "knock-in" or transgenic techniques. In some cases, CD16t, CD32t, or CD64t expression is caused by one or more transgenes.
Thus, in some embodiments, one or more copies of the CD16t, CD32t or CD64t gene are added to cells under the control of an inducible or constitutive promoter (preferably the latter). In some embodiments, lentiviral constructs are used as described herein or as known in the art. The gene may be integrated into the genome of the host cell under the control of a suitable promoter known in the art.
Cells containing lentiviral vectors expressing CD16t, CD32t or CD64t were selected using blasticidin markers. The gene sequences were synthesized and the DNA cloned into, for example, a plasmid lentivirus pLenti6/V5 (Thermo Fisher Scientific, waltham, mass.) having blasticidin resistance.
In some embodiments, gene expression may be increased by altering the regulatory sequences of the endogenous CD16, CD32, or CD64 genes in combination with truncations or mutations described herein. This can be achieved, for example, by replacing the endogenous promoter with a constitutive promoter or with a different inducible promoter. This can be typically accomplished using known techniques such as CRISPR.
Once altered, the presence of sufficient expression can be determined using known techniques, such as those described in the examples, such as Western blotting, ELISA assays, or FACS assays using anti-CD 16, CD32, or CD64 antibodies. In general, "sufficiency" in the present specification refers to the isolation of antibodies and the inhibition of an increase in cell surface expression of ADCC or CDC.
HIP phenotype and determination of pluripotency maintenance
Once HIP cells are produced, their retention of low immunogenicity and/or pluripotency can be determined as generally described herein and in the examples.
For example, low immunogenicity is determined using a variety of techniques. One exemplary technique involves transplanting into an allogeneic host and monitoring HIP cell growth (e.g., teratomas) that evade the host's immune system. HIP derivatives were transduced to express luciferase and then can be tracked using bioluminescence imaging. Similarly, the host animal was tested for T cell and/or B cell responses to HIP cells to confirm that HIP cells did not elicit an immune response in the host animal. T cell function was assessed by Elispot, elisa, FACS, PCR or mass flow Cytometry (CYTOF). FACS or luminex was used to assess B cell responses or antibody responses. Additionally, or alternatively, the ability of the cells to avoid an innate immune response (e.g., NK cell killing) can be determined. NK cytolytic activity is assessed in vitro or in vivo using techniques known in the art.
Similarly, retention of pluripotency is tested in a variety of ways. In one embodiment, pluripotency is determined by expression of certain pluripotency-specific factors, as generally described herein. Additionally or alternatively, HIP cells differentiate into one or more cell types as an indicator of pluripotency.
Production of HIPO-CD16t, CD32t or CD64t expressing cells
In some aspects of the invention, the HIP cells produced above will already be HIPO-cells, as the process will begin with pluripotent cells with blood of type O.
Other aspects of the invention include enzymatic conversion of the a and B antigens. In a preferred aspect, an enzyme is used to convert the B antigen to the O antigen. In a more preferred aspect, the enzyme is an alpha-galactosidase. This enzyme eliminates the terminal galactose residues of the B antigen. Other aspects of the invention include enzymatic conversion of the a antigen to the O antigen. In a preferred aspect, alpha-N-acetylgalactosamine enzymes are used to convert the A antigen to the O antigen. Enzymatic conversions are discussed, for example, in the following documents: olsson et al Transfusion Clinique et Biologique 11:33-39 (2004); U.S. Pat. nos. 4,427,777, 5,606,042, 5,633,130, 5,731,426, 6,184,017, 4,609,627 and 5,606,042; and international publication No. WO9923210, each of which is incorporated herein by reference in its entirety.
Other embodiments of the invention include genetically engineering cells by knocking out exo 7 of the ABO gene or silencing the SLC14A1 (JK) gene. Other embodiments of the invention include knockout of the C and E antigens in the Rh blood group system (Rh), the K antigen in the Kell blood group system (KEL), the Fya and FY3 antigens in the Duffy blood group system (FY), the Jkb antigen in the Kidd blood group system (JK), or the U and S antigens in the MNS blood group system. Any knockout method known in the art or described herein, such as CRISPR, talens or homologous recombination, may be used.
E. Preferred embodiments of the invention
The HIP, HIPO-, iPSC, iPSCO-, ESC-or ESCO-cells expressing CD16t, CD32t or CD64t or derivatives thereof of the present invention are useful in the treatment of, for example, type 1 diabetes, heart disease, neurological diseases, cancer, blindness, vascular diseases and other diseases responsive to regenerative medicine therapies. In particular, the present invention contemplates the use of cells to differentiate into any cell type. Thus, provided herein are cells that express CD16t, CD32t, or CD64t and exhibit multipotency, but do not result in host ADCC or CDC response when transplanted as multipotential cells or their differentiation products into an allogeneic host, such as a human patient. In addition, intracellular signaling functions of CD16, CD32 or CD64 are reduced or eliminated by truncations or mutations.
In one aspect, the cells of the invention comprise a nucleic acid encoding a Chimeric Antigen Receptor (CAR) having CD16t, CD32t, or CD64t expression. The CAR may comprise an extracellular domain, a transmembrane domain, and an intracellular signaling domain.
In some embodiments, the extracellular CAR domain binds to an antigen selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CS1, CD171, BCMA, MUC16, ROR1, and WT 1. In certain embodiments, the extracellular domain comprises a single chain variable fragment (scFv). In some embodiments, the CAR transmembrane domain comprises cd3ζ, CD4, CD8 a, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, and BTLA. In certain embodiments, the CAR intracellular signaling domain comprises cd3ζ, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, and BTLA.
In certain embodiments, the CAR comprises an anti-CD 19scFv domain, a CD28 transmembrane domain, and a CD3 zeta signaling intracellular domain. In some embodiments, the CAR comprises an anti-CD 19scFv domain, a CD28 transmembrane domain, a 4-1BB signaling intracellular domain, and a CD3 zeta signaling intracellular domain.
In another aspect of the invention, an isolated CAR-T cell expressing CD16T, CD32T or CD64T is produced by differentiating any of the cells described herein in vitro. In some embodiments, the cell is a cytotoxic low immune CAR-T cell.
In various embodiments, in vitro differentiation comprises culturing cells carrying the CAR construct in a medium comprising one or more growth factors or cytokines selected from bFGF, EPO, flt L, IGF, IL-3, IL-6, IL-15, GM-CSF, SCF, and VEGF. In some embodiments, the culture medium further comprises one or more selected from BMP activators, GSK3 inhibitors, ROCK inhibitors, tgfp receptor/ALK inhibitors, and NOTCH activators.
In a particular embodiment, the isolated CAR-T cells of the invention produced by in vitro differentiation are used as a treatment for cancer.
In another aspect of the invention, there is provided a method of treating a cancer patient by administering a composition comprising a therapeutically effective amount of any of the isolated CAR-T cells described herein. In some embodiments, the composition further comprises a therapeutically effective carrier.
In some embodiments, the administering step includes intravenous administration, subcutaneous administration, intranode administration, intratumoral administration, intrathecal administration, intrapleural administration, and intraperitoneal administration. In some cases, administering further includes bolus injection or continuous infusion.
In some embodiments, the cancer is a blood cancer selected from leukemia, lymphoma, and myeloma. In various embodiments, the cancer is a solid tumor cancer or a liquid tumor cancer.
In another aspect, the invention provides a method of making any of the isolated CAR-T/CD16T, CD32T, or CD64T cells described herein. The method comprises differentiating any of the cells of the invention in vitro, wherein in vitro differentiation comprises culturing them in a medium comprising one or more growth factors or cytokines selected from bFGF, EPO, flt L, IGF, IL-2, IL-3, IL-6, IL-7, IL-15, GM-CSF, SCF, and VEGF. In some embodiments, the culture medium further comprises one or more selected from BMP activators, GSK3 inhibitors, ROCK inhibitors, tgfp receptor/ALK inhibitors, and NOTCH activators.
In some embodiments, in vitro differentiation comprises culturing HIPO-cells on feeder cells. In other embodiments, in vitro differentiation comprises culturing under simulated microgravity. In some cases, the culture is performed under simulated microgravity for at least 72 hours.
In some aspects, provided herein are isolated, engineered low immune heart cells (low immunogenic heart cells) differentiated from cells expressing CD16t, CD32t, or CD64 t.
In some aspects, provided herein are methods of treating a patient suffering from a heart condition or heart disease. The method comprises administering a composition comprising a therapeutically effective amount of any population of isolated, engineered low immune cardiac cells derived from the cells of the invention described herein. In some embodiments, the composition further comprises a therapeutically effective carrier.
In some embodiments, administering comprises implanting cardiac tissue of the patient, intravenous injection, intra-arterial injection, intra-coronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, endocardial injection, epicardial injection, or infusion.
In some embodiments, the heart condition or disease is selected from pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, perinatal cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial ischemia reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
In some aspects, provided herein are methods of generating a low immune cardiac cell population from a population of HIPO-/CD16t, CD32t, or CD64t cells by in vitro differentiation, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been deleted, while expression of CD47 or another sirpa-engaging protein in HIPO-cells is increased. The method comprises the following steps: (a) Culturing a population of HIPO-cells in a medium comprising a GSK inhibitor; (b) Culturing a population of HIPO-cells in a medium comprising a WNT antagonist to produce a population of precordial cells; and (c) culturing the pre-cardiac cell population in a medium comprising insulin to produce a low immune cardiac cell population.
In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of GSK inhibitor ranges from about 2 μm to about 10 μm. In some embodiments, the WNT antagonist is IWR1, a derivative or variant thereof. In some cases, the concentration of WNT antagonist ranges from about 2 μm to about 10 μm.
In some aspects, provided herein are isolated, engineered endothelial cells that overexpress CD16, CD32, or CD64 that differentiate from HIPO-cells. In other aspects, the isolated engineered endothelial cells of the invention are selected from the group consisting of capillary endothelial cells, vascular endothelial cells, aortic endothelial cells, brain endothelial cells, and kidney endothelial cells.
In some aspects, provided herein are methods of treating a patient suffering from a vascular condition or disease. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of the isolated engineered endothelial cell population of the invention.
The method comprises administering a composition comprising a therapeutically effective amount of any of the isolated, engineered endothelial cell populations described herein that overexpress CD16, CD32, or CD 64. In some embodiments, the composition further comprises a therapeutically effective carrier or excipient. In some embodiments, administering comprises implanting cardiac tissue of the patient, intravenous injection, intra-arterial injection, intra-coronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, endocardial injection, epicardial injection, or infusion.
In some embodiments, the vascular condition or disease is selected from the group consisting of vascular injury, cardiovascular disease, vascular disease, ischemic disease, myocardial infarction, congestive heart failure, hypertension, ischemic tissue injury, limb ischemia, stroke, neuropathy, and cerebrovascular disease.
In some aspects, provided herein are methods of generating a population of low immune endothelial cells from a population of cells expressing CD16t, CD32t, or CD64t by in vitro differentiation, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been deleted and CD47 expression in HIPO-cells is increased. The method comprises the following steps: (a) Culturing a population of HIPO-cells in a first medium comprising a GSK inhibitor; (b) Culturing the population of HIPO-cells in a second medium comprising VEGF and bFGF to produce a population of pre-endothelial cells; and (c) culturing the population of pre-endothelial cells in a third medium comprising a ROCK inhibitor and an ALK inhibitor to produce a population of low immune endothelial cells.
In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of GSK inhibitor ranges from about 1 μm to about 10 μm. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the concentration of ROCK inhibitor ranges from about 1 μm to about 20 μm. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 0.5 μm to about 10 μm.
In some embodiments, the first medium comprises from 2 μm to about 10 μm CHIR-99021. In some embodiments, the second medium comprises 50ng/ml VEGF and 10ng/ml bFGF. In other embodiments, the second medium further comprises Y-27632 and SB-431542. In various embodiments, the third medium comprises 10. Mu. M Y-27632 and 1. Mu.M SB-431542. In certain embodiments, the third medium further comprises VEGF and bFGF. In certain cases, the first medium and/or the second medium is free of insulin.
In some aspects, provided herein are isolated, engineered low immune Dopaminergic Neurons (DNs) differentiated from cells expressing CD16t, CD32t, or CD64 t. In some embodiments, endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated and CD47 or another sirpa-engaging protein expression has been increased, the neurons being O and Rh-blood groups.
In some embodiments, the isolated dopaminergic neurons are selected from the group consisting of neuronal stem cells, neuronal progenitor cells, immature dopaminergic neurons, and mature dopaminergic neurons.
In some aspects, provided herein are methods of treating a patient suffering from a neurodegenerative disease or condition. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of any of the isolated low immune dopaminergic neuron populations. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the isolated population of low immune dopaminergic neurons is on a biodegradable scaffold. Administration may include transplantation or injection. In some embodiments, the neurodegenerative disease or condition is selected from parkinson's disease, huntington's disease, and multiple sclerosis.
In some aspects, provided herein are methods of producing a population of dopaminergic neurons expressing CD16t, CD32t, or CD64t by in vitro differentiation. In some embodiments, endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated and CD47 or another sirpa-engaging protein expression has been increased, the blood group being O-type and Rh-type. In some embodiments, the method comprises (a) culturing a population of cells in a first medium comprising one or more factors selected from the group consisting of sonic hedgehog (SHH), BDNF, EGF, bFGF, FGF, WNT1, retinoic acid, gsk3β inhibitor, ALK inhibitor, and ROCK inhibitor, to produce a population of immature dopaminergic neurons; and (b) culturing the population of immature dopaminergic neurons in a second medium, different from the first medium, to produce a population of dopaminergic neurons.
In some embodiments, the gskβ inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of gskβ inhibitor ranges from about 2 μm to about 10 μm. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 1 μm to about 10 μm. In some embodiments, the first medium and/or the second medium is free of animal serum.
In some embodiments, the method further comprises separating the hyperimmune dopaminergic neuron population from the non-dopaminergic neurons. In some embodiments, the method further comprises cryopreserving the isolated population of hyperimmune dopaminergic neurons.
In some aspects, provided herein are isolated engineered hyperimmune islet cells differentiated from cells expressing CD16t, CD32t, or CD64 t. In some embodiments, endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated and CD47 or another sirpa-engaging protein expression has been increased, the blood group being O-type and Rh-type.
In some embodiments, the isolated hyperimmune islet cells are selected from the group consisting of islet progenitor cells, immature islet cells, and mature islet cells.
In some aspects, provided herein are methods of treating a diabetic patient. The method comprises administering a composition comprising a therapeutically effective amount of a population of isolated islet cells described herein that express any one of CD16t, CD32t, or CD64 t. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of isolated hyperimmune islet cells is on a biodegradable scaffold. In some cases, administering includes transplanting or injecting.
In some aspects, provided herein are methods of generating a hyperimmune islet cell population from a cell population expressing CD16t, CD32t, or CD64t by in vitro differentiation. In some embodiments, in HIPO-cells, endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been deleted and CD47 or another SIRPalpha-adaptor protein expression has been increased, with blood groups of O-type and Rh-type. The method comprises the following steps: (a) Culturing a population of cells expressing CD16t, CD32t, or CD64t in a first culture medium comprising one or more factors selected from the group consisting of insulin-like growth factor (IGF), transforming Growth Factor (TGF), fibroblast growth factor (EGF), epidermal Growth Factor (EGF), hepatocyte Growth Factor (HGF), sonic hedgehog (SHH), and Vascular Endothelial Growth Factor (VEGF), transforming growth factor- β (tgfp) superfamily, bone morphogenic protein-2 (BMP 2), bone morphogenic protein-7 (BMP 7), GSK3 p inhibitor, ALK inhibitor, BMP type 1 receptor inhibitor, and retinoic acid to produce an immature islet cell population; and (b) culturing the population of immature islet cells in a second medium, different from the first medium, to produce a population of hyperimmune islet cells.
In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of GSK inhibitor ranges from about 2 μm to about 10 μm. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 1 μm to about 10 μm. In some embodiments, the first medium and/or the second medium is free of animal serum.
In some embodiments, the method further comprises isolating the population of islet cells expressing CD16t, CD32t, or CD64t from the non-islet cells. In some embodiments, the method further comprises cryopreserving the isolated hyperimmune islet cell population.
In some aspects, provided herein are isolated, engineered low immune Retinal Pigment Epithelium (RPE) cells differentiated from cells expressing CD16t, CD32t, or CD64t, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, CD47 or another sirpa-engageenans expression has been increased, blood types of O-type and Rh-type.
In some embodiments, the isolated low immune RPE cells are selected from RPE progenitor cells, immature RPE cells, mature RPE cells, and functional RPE cells.
In some aspects, provided herein are methods of treating a patient having an ocular condition. The method comprises administering a composition comprising a therapeutically effective amount of any of the isolated populations of RPE cells expressing CD16t, CD32t or CD64t described herein. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the isolated population of low immune RPE cells is on a biodegradable scaffold. In some embodiments, administering comprises transplanting or injecting the patient's retina. In some embodiments, the ocular condition is selected from wet macular degeneration, dry macular degeneration, juvenile macular degeneration, leber's congenital opacity, retinitis pigmentosa, and retinal detachment.
In some aspects, provided herein are methods of producing a population of Retinal Pigment Epithelium (RPE) cells expressing CD16t, CD32t, or CD64t from a population of cells by in vitro differentiation. In some embodiments, endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been deleted and CD47 or another sirpa-engaging protein expression has been increased in HIPO-cells. The method comprises the following steps: (a) Culturing a population of HIPO-cells in a first medium comprising any one factor selected from the group consisting of activin A, bFGF, BMP/7, DKK1, IGF1, noggin, BMP inhibitor, ALK inhibitor, ROCK inhibitor, and VEGFR inhibitor to produce a population of pre-RPE cells; and (b) culturing the population of pre-RPE cells in a second medium different from the first medium to produce a population of low immune RPE cells.
In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 2 μm to about 10 μm. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the concentration of ROCK inhibitor ranges from about 1 μm to about 10 μm.
In some embodiments, the first medium and/or the second medium lacks animal serum.
In some embodiments, the method further comprises separating the low immune RPE cell population from the non-RPE cells. In some embodiments, the method further comprises cryopreserving the isolated low immune RPE cell population.
In one aspect, a human Pluripotent Stem Cell (PSC) is resistant to ADCC or CDC by CD16t, CD32t, or CD64t expression. In some embodiments, they are low immune pluripotent stem cells (hipscs). They are rendered hypoimmunogenic by a) disruption of the B2M gene (e.g., B2M-/-) at each allele, B) disruption of the CIITA gene (e.g., CIITA-/-) at each allele, and c) by overexpression of the CD47 gene (CD47+, e.g., by introducing one or more additional copies of the CD47 gene or an activating genomic gene). This makes the hiPSC population B2M-/-CIITA-/-CD47tg. In a preferred aspect, the cells are non-immunogenic. In another embodiment, HIP cells are rendered non-immunogenic B2M-/-CIITA-/-CD47tg as described above and are further modified by the inclusion of an inducible suicide gene that is induced to kill the cells in vivo when desired. In other aspects, HIPO cells that overexpress CD16, CD32, or CD64 are produced when HIP cells are rendered Rh-type blood by knocking out exon 7 of the ABO gene or silencing the SLC14A1 (JK) gene, and by knocking out the C and E antigens of the Rh blood group system (RH), the K antigen in the Kell blood group system (KEL), the Fya and Fy3 antigens in the Duffy blood group system (FY), the Jkb antigen in the Kidd blood group system (JK), or the U and S antigens in the MNS blood group system.
Maintenance of HIPO-/CD16t, CD32t or CD64t cells
Once produced, HIPO-CD16t, CD32t or CD64t cells can be maintained in an undifferentiated state as known to maintain the state of iPSC. For example, HIP cells are cultured on matrigel using a medium that prevents differentiation and maintains pluripotency.
Differentiation of HIPO-/CD16t, CD32t or CD64t cells
The present invention provides HIPO-/CD16t, CD32t or CD64t cells differentiated into different cell types for subsequent transplantation into a subject. As will be appreciated by those skilled in the art, the method of differentiation depends on the desired cell type using known techniques. Cells are differentiated in suspension and then placed in a gel matrix form (such as a matrix gel, gelatin, or fibrin/thrombin form) to promote cell survival. Differentiation is typically determined by assessing the presence of cell-specific markers, as is known in the art.
In some embodiments, HIPO-/CD16t, CD32t, or CD64t cells are differentiated into hepatocytes to address the loss of hepatocyte function or cirrhosis. There are a number of techniques available for differentiating HIPO-cells into hepatocytes; differentiation is also measured from functions such as ammonia metabolism, storage and uptake of LDL, uptake and release of ICG and storage of glycogen, as known in the art.
In some embodiments, HIPO-/CD16T, CD32T, or CD64T cells differentiate into β -like cells or islet organoids for transplantation to treat type I diabetes (T1 DM). Cell systems are a promising approach to address T1DM, see, e.g., ellis et al, doi/10.1038/nrgastro.2017.93, incorporated herein by reference. In addition, pagliuca et al report successful differentiation from hiPSC to beta cells (see doi/10.106/j. Cell.2014.09.040, which is incorporated herein by reference in its entirety, particularly for the methods and reagents outlined therein for large-scale production of functional human beta cells from human pluripotent stem cells). Furthermore, vegas et al show that human beta cells are produced from human pluripotent stem cells and then encapsulated to avoid immune rejection by the host; (doi: 10.1038/nm.4030), incorporated herein by reference in its entirety, particularly for the methods and reagents outlined therein for large-scale production of functional human beta cells from human pluripotent stem cells.
Differentiation is typically determined by assessing the presence of beta cell-related or specific markers, including but not limited to insulin, as is known in the art. Differentiation can also be measured functionally, for example glucose metabolism, see generally Muraro et al, doi: 10.1016/j.cells.2016.09.002, which is incorporated herein by reference in its entirety, particularly for the biomarkers outlined therein.
Once dhpo-/CD 16t, CD32t or CD64t beta cells are produced, they can be transplanted (as a cell suspension or within the gel matrix described herein) into the portal vein/liver, omentum, gastrointestinal mucosa, bone marrow, muscle or subcutaneous sac (subcutaneous pouches).
In some embodiments, the HIPO-/CD16t, CD32t, or CD64t cells are differentiated into Retinal Pigment Epithelium (RPE) to treat vision-threatening ocular diseases. Human pluripotent stem cells have been differentiated into RPE cells using the techniques outlined in Kamao et al Stem Cell Reports 2014:2:205-18 (incorporated herein by reference in its entirety, particularly for the methods and reagents for differentiation techniques and reagents outlined therein); see also Mandai et al, doi 10.1056/NEJMoa1608368, also incorporated in its entirety for techniques for producing RPE cell sheets and transplanting into patients.
Differentiation can be determined, as known in the art, typically by assessing the presence of RPE-related and/or specific markers or by functional measurements. See, e.g., kamao et al, doi:10.1016/j.stemcr.2013.12.007, which is incorporated herein by reference in its entirety, particularly for the markers outlined in the first paragraph of the results section.
In some embodiments, the HIPO-/CD16t, CD32t, or CD64t cells are differentiated into cardiomyocytes to treat cardiovascular disease. Techniques for differentiating hipscs into cardiomyocytes are known in the art and are discussed in the examples. Differentiation can be determined, as known in the art, typically by assessing the presence of cardiomyocyte-related or specific markers or by functional measurements; see, for example, loh et al, doi 10.1016/j.cell.2016.06.001, incorporated herein by reference in its entirety, particularly for methods of differentiating stem cells, including cardiomyocytes.
In some embodiments, HIPO-/CD16t, CD32t, or CD64t cells are differentiated into Endothelial Colony Forming Cells (ECFCs) to form new blood vessels to treat peripheral arterial disease. Techniques for differentiating endothelial cells are known. See, e.g., prasain et al, doi:10.1038/nbt.3048, which is incorporated by reference in its entirety, particularly for methods and reagents for the production of endothelial cells from human pluripotent stem cells, and transplantation techniques. Differentiation can be determined, as known in the art, typically by assessing the presence of endothelial cell-related or specific markers or by functional measurements.
In some embodiments, HIPO-/CD16t, CD32t, or CD64t cells are differentiated into thyroid progenitor cells and thyroid follicular organoids, which secrete thyroid hormones to treat autoimmune thyroiditis. Techniques for differentiating thyroid cells are known in the art. See, e.g., kurmann et al, doi:10.106/j.stem.2015.09.004, which is expressly incorporated herein by reference in its entirety, particularly for methods and reagents for generating thyroid cells from human pluripotent stem cells, as well as transplantation techniques. Differentiation can be determined, as known in the art, typically by assessing the presence of thyroid cell associated or specific markers or by functional measurements.
H. Transplantation of differentiated HIPO-/CD16t, CD32t or CD64t cells
As will be appreciated by those skilled in the art, differentiated HIPO-/CD16t, CD32t or CD64t derivatives are transplanted using techniques known in the art, depending on the cell type and the end use of the cells. In general, the cells of the invention are transplanted by intravenous route or by injection at a specific site in a patient. When transplanted to a specific location, cells may be suspended in a gel matrix to prevent dispersion when they are immobilized.
The following examples are presented in order to provide a more complete understanding of the invention described herein. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any way.
Examples VII. Examples
HIP and HIPO-cells are produced as disclosed in WO2018/132783, PCT/US19/42123, PCT/US19/42117 and provisional application Nos. 62/698,973, 62/698,978, 62/698,981, 62/698,984, 62/846,399 and 62/855,499. Protection against NK cell and ADCC killing when CD64 is overexpressed on pluripotent cells is disclosed in PCT/US 20/55120. Each of the foregoing documents is incorporated by reference herein in its entirety.
Example 1: CD64t expression sequestered antibodies by Fc domains
Fig. 3 is a schematic showing that CD64t captures free IgG Fc without inducing any signaling in target cells. Human wild-type (wt) iPSC-derived endothelial cells (iEC) were transduced to express CD64 or CD64t. One hundred thousand iECs were plated in six-well plates coated with gelatin and at 37℃with 5% CO 2 Incubate overnight. The next day, the medium was changed and 20 μl of transgenic lentiviral particles carrying truncated CD64 under the control of the re-engineered EF1a promoter (1 x10 8 IFU/ml) or 200. Mu.l of lentiviral particles carrying full length CD64 (1X 10) 7 IFU/ml) (custom product, genTarget, san Diego, calif.) was added to 1.5ml of medium. After 36 hours, 1ml of cell culture medium was added. After a further 24 hours of time period, the process was continued,complete medium exchange was performed. Two days later CD64 or CD64t expression was measured by flow cytometry (fig. 4A and 5A). Flow cytometry analysis of CD64 or CD64t was performed using PE-labeled mouse anti-human CD64 antibodies (BD Biosciences, sanJose, CA, catalog No. 558592). Antibody Fc binding capacity was measured using the humanized IgG1 anti-CD 52 antibody alemtuzumab (ichorobio, wantage OX12 9ff, uk). The antibody did not recognize an epitope on iPSC, but only bound in the presence of CD64 or CD64t (fig. 4A and 5A, respectively). Fc binding was quantified using QDot 655 labeled goat anti-human IgG (h+l) F (ab') 2 secondary antibody (catalog No. Q-11221MP,Thermo Fisher Scientific,Carlsbad,CA). Flow cytometry showed an increase in alemtuzumab Fc binding with increasing concentrations of CD64 and CD64t antibodies (fig. 4B and 5B, respectively).
Example 2: HIPiEC expressing CD64t are protected from ADCC and CDC
HIPiEC did not express CD64 (FIG. 6A). Since they also do not express any CD52, they do not pass through their F ab Or F c Binds to anti-CD 52IgG1 (alemtuzumab) (fig. 6B). HIPiEC were transduced to express CD64 (FIG. 7A). Such HIPiECs CD64 Can pass through F thereof c IgG1 was bound in a concentration-dependent manner (fig. 7B).
To avoid intracellular signaling, HIP iEC was transduced to express intracellular truncated CD64 (CD 64 t) (fig. 8A). Such HIPiECs CD64t Can pass through F thereof c IgG1 was bound in a concentration-dependent manner (fig. 8B).
HIPiEC and HIPiECs then CD64t Is transduced to additionally express CD52, which is a target of the highly cytotoxic IgG1 antibody alemtuzumab. Human primary NK cells were purchased from Stemcell Technologies (70036, vancouver, canada) and cultured in RPMI-1640 supplemented with 10% FCS hi and 1% pen/strep before performing the assay. In the CDC assay, target cells are incubated with fresh ABO-compatible human serum.
Real-time killing assays were performed on xcelligent SP platform and MP platform (ACEA Biosciences, san Diego, ca.). A special 96-well E plate (ACEA Biosciences) coated with collagen (Sigma-Aldrich) was used. Will total 4 x 10 4 Individual iEC, epiC, beta cells orCAR-T cells were plated in 100 μl of medium. After the cell index reached 0.7, 4X 10 4 Individual NK cells were added to the ADCC assay or 50 μl of blood group compatible human serum was added to the CDC assay. Patient serum samples were treated with DTT prior to use to eliminate blood group IgM antibodies.
For the Nalm6 kill assay, 4×10 plates were plated 4 Nalm6 cells were used and 4X 10 cells were used 4 The individual CAR-T cells act as effector cells. The following antibodies were used and added after mixing with NK cells in 50 μl medium or in the serum indicated: humanized anti-CD 52IgG1 (alemtuzumab, ichorbrio, catalog No. ICH 4002), humanized anti-HLA-A 2IgG1 (clone 3PF12,Absolute Antibody, catalog No. AB 00947-10.0), humanized anti-Rh (D) IgG1 (clone F5, creative Biolabs, catalog No. FAMAB-0089 WJ), humanized anti-TPOIgG 1 (clone B8, creative Biolabs, catalog No. FAMAB-0014 JF), humanized anti-CD 3IgG1 (Creative Biolabs, custom product), humanized anti-CD 19scFv (FMC 63) IgG1 (clone 136.20.1,Creative Biolabs, catalog No. HPAB-0440-YJ-m/h). Different concentrations of 0.0001. Mu.g/ml to 1. Mu.g/ml were used. As a negative control, cells were treated with 2% Triton X-100 medium (data not shown). Data were normalized and analyzed using RTCA software (ACEA).
HIPiECs in ADCC and CDC killing assays CD52 Is killed by NK cells (fig. 9) or complement (fig. 10), respectively, in a concentration-dependent manner. Discovery of HIPiECs CD52,CD64t Is completely protected against ADCC (fig. 11) and CDC (fig. 12).
Example 3: engineering human thyroid epithelial cells to evade antibody-mediated autoimmune killing
Hashimoto thyroiditis is a prototype disease in which cytotoxic autoantibodies cause thyroid tissue destruction. anti-TPO antibodies (predominantly IgG1 subclass) are present in high concentrations in 90% of patients (Rapoport, B. & McLachlan, S.M., gyroid Autoimmunity. JClin Invest 108,1253-1259 (2001); doullay, F. Et al, autoimmunity 9,237-244 (1991)). They mediate ADCC (Bogner, U.S. Pat. No. 4, J Clin Endocrinol Metab 59,734,734-738 (1984); stathatos, N. & Daniels, G.H., autoimmune thyroid disease. Curr Opin Rheumatol 24,70-75 (2012); stassi, G. & De Maria, R.Autoimmuta thyrate disease: new models of cell death in auto immuta. Nat Rev Immunol 2,195-204 (2002)) and CDC (Rebuffat, S. et al J Clin Endocrinol Metab 93,929,929-934 (2008); chiovat, L. Et al J Clin Endocrinol Metab, 1700-1705 (1993)). This impairs thyroid function (Pearce, E.N., et al, N Engl J Med 348,2646-2655 (2003)). The present invention provides thyroid epithelial cells (epi cs) resistant to TPO antibodies for use in surviving the innate immune response and reconstructing organ function when transplanted into hypothyroidism patients.
Immortalized human thyroid epithelial cells (epi cs, instreenex, braunschweig, germany, cat. No. INS-CI-1017) and primary human islet cells (Takara, mountain View, CA, cat. # Y10106) were used. Because the epiCs do not exhibit physiological responses to Thyroid Stimulating Hormone (TSH) in vitro, their Thyroid Peroxidase (TPO) levels are well below physiological expression levels. Thus, lentiviral transduction methods were used to artificially increase their TPO expression. One hundred thousand thyroid epiCs were plated onto gelatin coated six well plates and incubated at 37℃with 5% CO 2 Incubate overnight. The next day, the medium was changed and 200. Mu.l of lentiviral particles carrying the human TPO re-engineered EF1a promoter transgene (1X 10 7 IFU/ml) (GenTarget) was added to 1.5ml of medium. After 36 hours, 1ml of cell culture medium was added.
These epiCs TPO Shows good TPO expression but no CD64 (FIG. 13A). Then, the epiCs TPO Transduced to also express CD64t and produce epi Cs TPO,CD64t (FIG. 13B). Although thyroid epi cs TPO Does not bind any human IgG1, even at high concentrations (FIG. 14A), but the epi Cs TPO,CD64t Effective binding to free human IgG1F c (FIG. 14B).
Thyroxine production was measured by ELISA. The 96-well plate was coated with gelatin and 3×10 wells per well 4 Personal thyroid epiCs TPO Or epi cs TPO,CD64t Inoculated in 100 mu l H H medium and 5% CO at 37 ℃ 2 And incubated for 24 hours. The next day, H7H medium was changed and 1mU/mL of native bovine thyroid stimulating agent was supplementedHormone protein (TSH, catalog number TSH-1315B,Creative Biomart,Shirley,NY). Three wells of each epiC group were also supplemented with 1. Mu.g/mL of anti-CD 52IgG1 (alemtuzumab, clone Campath-1H, biorad). After 72 hours, supernatants were collected and thyroxine levels were assessed using a thyroxine (T4) competitive ELISA kit (catalogue No. EIAT4C, invitrogen) according to the manufacturer's instructions. The results are expressed as the change in Optical Density (OD) between groups with and without alemtuzumab. Thyroid epiCs, whether or not IgG1 antibodies are present TPO And epiCs TPO,CD64t Thyroxine was produced (figures 15A and B).
The epiCs (TPO) with and without CD64t expression were plated on the xcelligent platform for in vitro impedance determination (fig. 16). They adhere to plastic dishes and grow in multiple layers. Thus, the cellular index calculated by the xcelligent machine does not reach a steady state level of endothelial cell known formation even of a monolayer. As these cells grow in multi-layered clusters, the cell index increases. In this assay, increasing concentrations of rabbit anti-TPO antibody (Abcam, cambridge, MA, catalog #ab 203057) were used to induce antibody-mediated killing. 40,000 human NK cells were added as effector cells. Because rabbit Ig Fc does bind to human CD16, epi cs (TPO) are killed in a concentration-dependent manner. However, engineered epi cs (TPO, CD64 t) effectively evade antibody-mediated killing at all antibody concentrations used in this assay.
Although rabbit IgG Fc binds human CD64 and CD64t, we next used humanized anti-TPOIgG 1 antibodies. In NK cell ADCC assay (FIG. 17) and CDC assay (FIG. 18), this human IgG1 antibody effectively killed thyroepics TPO . However, thyroid epi cs TPO,CD64t Is fully protected from ADCC (fig. 19) and CDC (fig. 20).
Serum samples from anti-TPO antibody titers of 21, 26.3 and 29.6 fold with hashimoto thyroiditis and with normal upper limit levels (0.03U/ml) rapidly killed thyroepics in CDC assay TPO (FIGS. 21A-C). Thyroid epi cs TPO,CD64t Is fully protected from killing in patient serum and is therefore able to withstand clinically relevant autoimmunityConditions.
NSG (NOD.Cg-Prkdc) at 6-12 weeks of age scid Il2rg tm1Wjl /SzJ, 005557) mice were purchased from Jackson Laboratories (Sacramento, CA). The number of animals used in the examples is presented in each figure. NSG recipients subcutaneously inject 5 x 10 on days 0, 1 and 2 4 Individual epiCs TPO Or epi cs TPO,CD64t 100 ten thousand human NK cells and 1mg dose of anti-TPO (FIG. 22A). All of the epiCs TPO The graft disappeared rapidly (FIG. 22B), whereas all the epiCs had TPO,CD64t Graft survival (fig. 22C).
Example 4: engineering human beta cells to evade HLA antibody mediated killing
Type 1 diabetes (T1 DM) is another autoimmune disease, which is mediated by T cells with an antibody response. Our objective was to test whether CD64t expression could render them resistant to HLA antibody killing. Human iPSC-derived islet beta cells were purchased from TaKaRa (ChiPSC 22, catalog No. Y10106) and cultured in Cellartis hiPS Beta cell culture media kit (TaKaRa, catalog No. Y10108). Cells were seeded into 12-well plates according to the manufacturer's protocol. Some cells were transduced with CD64t lentiviral particles (GenTarget). Human iPSC-derived islet cells (beta cells) did not express CD64 (fig. 23A). Human iPSC-derived beta cells were then transduced to express CD64t (fig. 23B). Although beta cells were unable to bind IgG1 (fig. 24A), beta cells CD64t Capable of capturing and binding free IgG F c (FIG. 24B).
Insulin production was measured by ELISA. The 24-well plate was coated with gelatin and each well was 5×10 4 Individual iPSC-derived beta cells and beta cells CD64t Cells (catalog number Y10108, takara Bio, san Jose, calif.) were seeded in 500. Mu.l Celloartis hiPS beta cell culture medium and incubated at 37℃with 5% CO 2 Incubate for 24 hours. The next day, the Celloartis hiPS beta cell culture medium was changed to RPMI 1640 without glucose (catalog number 11879-020, gibco) for 2 hours. After 2 hours, the medium was changed to RPMI without glucose and supplemented with 2mM glucose (catalog No. G7528, sigma Aldrich). Three wells of each betacell group were also supplemented with 1 μg/mL of anti-CD 52IgG1 (alemtuzumab, clone Campath-1H, catalog No. MCA6101, biorad). 20After minutes, the supernatant was collected and the medium was replaced with RPMI without glucose and supplemented with 20mM glucose. Again 1 μg/mL alemtuzumab was added to 3 wells per group. After 20 minutes, the supernatant was collected and the level of human insulin was determined using a human insulin ELISA kit (catalog number KAQ1251, invitrogen) according to the manufacturer's instructions. The results are expressed as the change in Optical Density (OD) between groups with and without alemtuzumab. Beta cells (FIG. 25A) and beta cells CD64t (FIG. 25B) shows complete glucose sensing and insulin production. These data show Optical Density (OD) measurements in insulin ELISA. Beta cells and beta cells in low glucose medium (2 mM) compared to high glucose medium (20 mM) CD64t All showed lower insulin production. In the presence of 1 μg/ml of anti-CD 52 (alemtuzumab) antibody, insulin production was unchanged.
Islet cells (islet beta cells) with and without CD64t expression were then plated on the xcelligent platform (fig. 26). They adhere to plastic dishes but also grow in clusters and the cell index increases over time. Despite the aggregation growth pattern, the assay is still very sensitive in detecting target cell killing. Islet cells display HLA type A2. Thus, we used humanized anti-human HLA-A2 (Absolute Antibody, boston, MA, catalog # Ab 00947-10.0) with human IgG1Fc to induce antibody-mediated killing. Human NK cells (40,000) were used as effector cells. Corresponding to an increase in anti-HLA-A 2 concentration, islet cells are killed more and more rapidly. In contrast, islet beta cell (CD 64 t) cells are protected from anti-HLA-A 2 antibody mediated killing.
In the CDC assay, the HLA-A2 expressing beta cells were killed faster and faster as the concentration of anti-HLA-A 2IgG1 antibodies increased (FIG. 27). However, beta cells CD64t HLA antibody-mediated killing was completely resisted in CDC assays (fig. 28).
We then subcutaneously injected 5 x 10 into NSG mice 4 Personal beta cells or beta cells CD64t And one million human NK cells (fig. 29A). Three 1mg doses of anti-HLA-A 2 were subcutaneously injected on days 0, 1 and 2. All injected cells were Luc + . For imaging, D-luciferin firefly potassium salt (375 mg/kg; biosynth AG, stard, switzerland) was dissolved in PBS (pH 7.4, gibco) and 250. Mu.l was injected into the peritoneum of anesthetized mice. Animals were imaged in AMI HT (spectroscopic instrument imaging). Region of interest (ROI) bioluminescence is measured as the maximum number of photons per square centimeter per second per steradian (p/s/cm) 2 Per sr) as a unit. The maximum signal from the ROI was measured using the Aura Image software (Spectral Instruments). All beta cell grafts disappeared within 2 days (fig. 29B), while all beta cells CD64t The graft was resistant to antibody-mediated killing (fig. 29C) and similar to the survival of β cells without antibody challenge (fig. 29D).
Example 5: engineering human CAR-T cells to evade HLA-, non-HLA-and CAR-directed antibody killing
Clinical CAR-T cell therapy induces an antibody response that is even more pronounced in solid tumor patients. Thus, antibody protection is engineered into CAR-T cells. Human T cells are transduced to express the CD19scFv-4-1BB-CD3 zeta construct with or without additional CD64T expression. Human anti-CD 19CAR-T cells were generated from human PBMC using transgenic lentiviral particles carrying the CD19scFv-4-1BB-CD3 zeta construct (ProMab, catalog number PM-CAR 1002-V). PBMC were stimulated overnight with IL-2 and incubated with protamine sulfate and 1. Mu.g/ml IL-2 (Peprotech) per well 10 5 The density of individual cells was seeded in 96-well U-shaped bottom plates. Lentiviruses were added to wells at a MOI of 20. Some wells were additionally transduced with CD64t lentiviral particles at MOI (GenTarget) of 20. Spinfection was performed at 1800rpm for 30 minutes at 25 ℃. Thereafter, the cells were put back in wet 5% CO 2 The incubator was left overnight. After 2 days the medium was changed and the cells were plated at 10 per ml 6 The density of the individuals was inoculated in T cell medium (OpTmizer, thermo Fisher Scientific). CD3/CD28 beads (Thermo Fisher Scientific) are used for T cell expansion. For CAR on BD Aria Fusion + And CAR + /C64t + The population sorts the cells and is used for further assays.
CAR-T cells showed significant expression of CAR receptor (anti-CD 19 scFv), but did not express CD64T (fig. 30A). Then CAR-T cell channelsTransduction to express CD64T and such CAR-T CD64t CAR was still expressed and CD64t was additionally expressed (fig. 30B).
Although the CAR-T cells did not bind any free IgG1 (using anti-TPO antibodies, fig. 31A), the CAR-T cells did not bind any free IgG1 CD64t Capable of capturing and binding free IgG1F c (FIG. 31B).
CAR-T, CAR-T was evaluated CD64t And the killing efficacy of conventional T cells against CD19 positive NALM target cells (fig. 32). Although control T cells did not show killing, CAR-T and CAR-T CD64t Is equally effective in killing wounded at various effector to target cell ratios. Furthermore, CAR-T CD64t Is free from F c The effect of the presence of bound antibodies (fig. 33).
ADCC (figure 34) and CDC assays (figure 35) using cytotoxic antibodies against HLA epitopes (HLA-a 2), non-HLA epitopes (CD 52, CD 3), blood group antigens (Rh (D)) and CAR receptors (anti-CD 19 scFv) were performed and CAR-T cells were killed in all assays. However, in ADCC (FIG. 36) and CDC assays (FIG. 37), CAR-T CD64t Antibody-mediated killing using all five antibodies can be avoided. CD64T expression does not affect the cytotoxicity of human CAR-T cells, but makes them resistant to antibodies, regardless of their specificity.
Example 6: engineered human NK cells evade ADCC and CDC killing
To show that antibody protection can be engineered into NK cells, human NK cells are transduced to express CD64t. Peripheral human NK cells did not express CD64t (fig. 38A). NK (Natural killer) CD64t Shows high CD64t expression (FIG. 38B).
Although NK cells did not bind any free IgG1 (using anti-TPO antibody, FIG. 39A), NK cells did not bind any free IgG1 CD64t Cells are able to capture and bind free IgG1F c (FIG. 39B).
ADCC (fig. 40) and CDC assays (fig. 41) using the anti-CD 52 antibody alemtuzumab were performed, and NK cells were rapidly killed. However, NK CD64t Cells were able to evade antibody-mediated killing in both ADCC (fig. 42) and CDC assays (fig. 43). CD64t expression enables NK cells to resist antibody-mediated killing.
Exemplary sequences:
SEQ ID NO: 1-human beta-2-microglobulin
MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDI
SEQ ID NO: 2-human CIITA protein, N-terminal 160 amino acids
MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPLCLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSRLLCDMEGDEETREAYANIAELDQYVFQDSQLEGLSKDIFKHIGPDEVIGESMEMPAEVGQKSQKRPFPEELPADLKHWKP
SEQ ID NO: 3-human CD47
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVIVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVE
SEQ ID NO: 4-herpes simplex virus thymidine kinase (HSV-tk)
MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN
SEQ ID NO: 5-Escherichia coli cytosine deaminase (EC-CD)
MSNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITENSLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQRAWQTLKWQIANGIQHVRTHVDVSDATLTALTALKAMLEVKQEVAPWIDLQIVAFPQEGILSYPNGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQSRFVETVAALAHHEGMARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNIHLQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQLMGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSVRGGKVIASTQPAQTTVYLEQPEAIDYKR
SEQ ID NO: 6-truncated human caspase 9
GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS
SEQ ID NO: 7-human CD64
NM_000566
MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHVLFYLAVGIMFLVNTVLWVTIRKELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGVHRKEPQGAT
SEQ ID NO: 8-human CD52
NM_001803
MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSSMSGGI FLFFVANAIIHLFCFS
SEQ ID NO: 9-human CD16FCGR3A
NP_000560.6
MAEGTLWQILCVSSDAQPQTFEGVKGADPPTLPPGSFLPGPVLWWGSLARLQTEKSDEVSRKGNWWVTEMGGGAGERLFTSSCLVGLVPLGLRISLVTCPLQCGIMWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK
SEQ ID NO: 10-human CD16FCGR3B
NP_001231682.2
MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYSVLEKDSVTLKCQGAYSPEDNSTQWFHNENLISSQASSYFIDAATVNDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKDRKYFHHNSDFHIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI
SEQ ID NO: 11-human CD32FCGR2A
>NP_001129691.1
MTMETQMSQNVCPRNLWLLQPLTVLLLLASADSQAAAPPKAVLKLEPPWINVLQEDSVTLTCQGARSPESDSIQWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIMLRCHSWKDKPLVKVTFFQNGKSQKFSHLDPTFSIPQANHSHSGDYHCTGNIGYTLFSSKPVTITVQVPSMGSSSPMGIIVAVVIATAVAAIVAAVVALIYCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDDKNIYLTLPPNDHVNSNN
SEQ ID NO: 12-human CD32FCGR2B
>NP_003992.3
MGILSFLPVLATESDWADCKSPQPWGHMLLWTAVLFLAPVAGTPAAPPKAVLKLEPQWINVLQEDSVTLTCRGTHSPESDSIQWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIVLRCHSWKDKPLVKVTFFQNGKSKKFSRSDPNFSIPQANHSHSGDYHCTGNIGYTLYSSKPVTITVQAPSSSPMGIIVAVVTGIAVAAIVAAVVALIYCRKKRISALPGYPECREMGETLPEKPANPTNPDEADKVGAENTITYSLLMHPDALEEPDDQNRI
SEQ ID NO: 13-human CD32FCGR2C
>NP_963857.3
MGILSFLPVLATESDWADCKSPQPWGHMLLWTAVLFLAPVAGTPAAPPKAVLKLEPQWINVLQEDSVTLTCRGTHSPESDSIPWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIVLRCHSWKDKPLVKVTFFQNGKSKKFSRSDPNFSIPQANHSHSGDYHCTGNIGYTLYSSKPVTITVQAPSSSPMGIIVAVVTGIAVAAIVAAVVALIYCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQPEETNNDYETADGGYMTLNPRAPTDDDDKNIYLTLPPNDHVNSNN
SEQ ID NO: 14-truncated human CD16
MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVSEQ ID NO: 15-truncated human CD32
MTMETQMSQNVCPRNLWLLQPLTVLLLLASADSQAAAPPKAVLKLEPPWINVLQEDSVTLTCQGARSPESDSIQWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIMLRCHSWKDKPLVKVTFFQNGKSQKFSHLDPTFSIPQANHSHSGDYHCTGNIGYTLFSSKPVTITVQVPSMGSSSPMGIIVAVVIATAVAAIVAAVVALIY
SEQ ID NO: 16-truncated human CD64
MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELQVLGLQLPTPVWFHVLFYLAVGIMFLVNTVLWVTI
SEQ ID NO:17-TfR1 cytoplasmic Domain (positions 1-67)
MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVD EEENADNNTKANVTKPKRCCSGSIC
SEQ ID NO:18-TfR1 transmembrane domain (positions 68-88)
YGTIAVIVFFLIGFMIGYLGY
SEQ ID NO:19-TfR1 extracellular domain (positions 89-760)
CKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF
SEQ ID NO:20-TfR2 cytoplasmic Domain (positions 1-83)
MERLWGLFQRAQQLSPRSSQTVYQRVEGPRKGHLEEEEEEDGE EGAETLAHFCPMELRGPEPLGSRPRQPNLIPWAAAGRRAAP
SEQ ID NO:21-TfR2 transmembrane domain (positions 84-104)
YLVLTALLIFTGAFLLGYVAF
SEQ ID NO:22-TfR2 extracellular domain (positions 105-801)
RGSCQACGDSVLVVSEDVNYEPDLDFHQGRLYWSDLQAMFLQFLGEGRLEDTIRQTSLRERVAGSAGMAALTQDIRAALSRQKLDHVWTDTHYVGLQFPDPAHPNTLHWVDEAGKVGEQLPLEDPDVYCPYSAIGNVTGELVYAHYGRPEDLQDLRARGVDPVGRLLLVRVGVISFAQKVTNAQDFGAQGVLIYPEPADFSQDPPKPSLSSQQAVYGHVHLGTGDPYTPGFPSFNQTQFPPVASSGLPSIPAQPISADIASRLLRKLKGPVAPQEWQGSLLGSPYHLGPGPRLRLVVNNHRTSTPINNIFGCIEGRSEPDHYVVIGAQRDAWGPGAAKSAVGTAILLELVRTFSSMVSNGFRPRRSLLFISWDGGDFGSVGSTEWLEGYLSVLHLKAVVYVSLDNAVLGDDKFHAKTSPLLTSLIESVLKQVDSPNHSGQTLYEQVVFTNPSWDAEVIRPLPMDSSAYSFTAFVGVPAVEFSFMEDDQAYPFLHTKEDTYENLHKVLQGRLPAVAQAVAQLAGQLLIRLSHDRLLPLDFGRYGDVVLRHIGNLNEFSGDLKARGLTLQWVYSARGDYIRAAEKLRQEIYSSEERDERLTRMYNVRIMRVEFYFLSQYVSPADSPFRHIFMGRGDHTLGALLDHLRLLRSNSSGTPGATSSTGFQESRFRRQLALLTWTLQGAANALSGDVWNIDNNF
All publications and patent documents disclosed or cited herein are incorporated by reference in their entirety. The foregoing description has been presented for purposes of illustration and description only. The description is not intended to limit the invention to the precise form disclosed. The scope of the invention is intended to be defined by the appended claims.
Claims (55)
1. A modified cell, wherein the modified cell expresses an Fc receptor protein comprising a truncation or modification, wherein the Fc receptor protein expression results in the modified cell being less sensitive to antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), wherein the truncation or modification reduces or eliminates intracellular signaling of the Fc receptor.
2. The modified cell of claim 1, wherein the Fc receptor protein is selected from the group consisting of truncated CD16 (CD 16 t), truncated CD32 (CD 32 t), and truncated CD64 (CD 64 t).
3. The modified cell of any one of claims 1 or 2, wherein the cell is a pluripotent cell.
4. The modified cell of any one of claims 1-3, wherein the Fc receptor protein is selected from the group consisting of SEQ ID NOs: 16, a CD64t protein having at least 90% sequence identity to SEQ ID NO:14 and a CD16t protein having at least 90% sequence identity to SEQ ID NO:15, a CD32t protein having at least 90% sequence identity.
5. The modified cell of claim 4, wherein the CD64t protein has the amino acid sequence of SEQ ID NO: 16.
6. The modified cell of any one of claims 1-5, wherein the modified cell is derived from a human low-immunogenicity pluripotent (HIP) cell.
7. The modified cell of any one of claims 1-6, wherein the modified cell is derived from a human low immunogenicity multipotent ABO blood group O rhesus factor negative (HIPO-) cell.
8. The modified cell of any one of claims 1-7, wherein the modified cell is derived from a human Induced Pluripotent Stem Cell (iPSC).
9. The modified cell of any one of claims 1-7, wherein the modified cell is derived from a human Embryonic Stem Cell (ESC).
10. The modified cell of any one of claims 1-9, wherein the modified cell is from a species selected from the group consisting of human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig.
11. The modified cell of any one of claims 1-10, further comprising a suicide gene that is activated by a trigger that causes death of the modified cell.
12. The modified cell of claim 11, wherein the suicide gene is a herpes simplex virus thymidine kinase gene (HSV-tk) and the trigger is ganciclovir.
13. The modified cell of claim 12, wherein the HSV-tk gene encodes a sequence identical to SEQ ID NO:4, a protein having at least 90% sequence identity.
14. The modified cell of claim 13, wherein the HSV-tk gene encodes a polypeptide comprising SEQ ID NO:4, and a protein of the sequence of 4.
15. The modified cell of claim 11, wherein the suicide gene is an e.coli cytosine deaminase gene (EC-CD) and the trigger is 5-fluorocytosine (5-FC).
16. The modified cell of claim 15, wherein the EC-CD gene encodes a nucleotide sequence that hybridizes to SEQ ID NO:5, a protein having at least 90% sequence identity.
17. The modified cell of claim 16, wherein the EC-CD gene encodes a polypeptide comprising SEQ ID NO:5, and a protein of the sequence of 5.
18. The modified cell of claim 11, wherein the suicide gene encodes an inducible caspase protein and the trigger is a dimerization Chemical Inducer (CID).
19. The modified cell of claim 18, wherein the suicide gene encodes a polypeptide that hybridizes to SEQ ID NO:6, an inducible caspase protein having at least 90% sequence identity.
20. The modified cell of claim 19, wherein the suicide gene encodes a polypeptide comprising SEQ ID NO:6 sequence of 6 is of the order of (a) and (b) caspase proteins.
21. The modified cell of any one of claims 18-20, wherein the CID is AP1903.
22. The modified cell of any one of claims 1-2 or 4-21, wherein the cell is selected from the group consisting of a Chimeric Antigen Receptor (CAR) cell, a T cell, an NK cell, an ILC, an endothelial cell, a dopaminergic neuron, a glial cell, an islet beta cell, a thyroid cell, an endocrine cell, a fibroblast cell, a liver cell, a cardiomyocyte, and a retinal pigment endothelial cell.
23. The modified cell of claim 22, wherein the CAR cell is a CAR-T cell.
24. The modified cell of claim 22, wherein the CAR cell is a CAR-NK cell.
25. A method comprising transplanting the cell of any one of claims 1-2, 4-24, or 44-49 into a subject, wherein the subject is a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, guinea pig.
26. The method of claim 25, wherein said cells derived from said modified cells are selected from the group consisting of Chimeric Antigen Receptor (CAR) cells, T cells, NK cells, ILC, endothelial cells, dopaminergic neurons, glial cells, islet cells, cardiomyocytes, hepatocytes, and retinal pigment endothelial cells.
27. A method of treating a disease comprising administering to a subject a cell derived from the modified cell of any one of claims 1-24 or 44-49.
28. The method of claim 27, wherein the derivative cell is selected from the group consisting of Chimeric Antigen Receptor (CAR) cells, T cells, NK cells, ILCs, endothelial cells, dopaminergic neurons, glial cells, islet beta cells, thyroid cells, fibroblasts, hepatocytes, cardiomyocytes, and retinal pigment endothelial cells.
29. The method of any one of claims 27 or 28, wherein the disease is selected from the group consisting of type I diabetes, heart disease, neurological disease, endocrine disease, cancer, ocular disease, and vascular disease.
30. A method for producing the modified cell of any one of claims 1-24 or 44-49, comprising expressing the CD16t, CD32t, or CD64t protein in a parent non-modified form of the cell.
31. The method of claim 30, wherein the modified cell has a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, or guinea pig origin.
32. The method of claim 30 or 31, wherein the modified cell is derived from a HIP cell.
33. The method of any one of claims 30-32, wherein the modified cell is derived from a HIPO-cell.
34. The method of any one of claims 30 or 31, wherein the modified cell is derived from an iPSC.
35. The method of any one of claims 30 or 31, wherein the modified cell is derived from ESC.
36. The method of any one of claims 30-35, wherein said CD16t, CD32t or CD64t expression is caused by introducing at least one copy of the human CD16t, CD32t or CD64t gene under the control of a promoter into said parental form of said modified cell.
37. The method of claim 36, wherein the promoter is a constitutive promoter.
38. A pharmaceutical composition for treating a disease comprising a cell derived from the modified cell of any one of claims 1-2, 4-24, or 44-49 and a pharmaceutically acceptable carrier.
39. The pharmaceutical composition of claim 38, wherein the cell is selected from the group consisting of Chimeric Antigen Receptor (CAR) cells, T cells, NK cells, ILCs, endothelial cells, dopaminergic neurons, glial cells, islet beta cells, thyroid cells, fibroblasts, hepatocytes, cardiomyocytes, and retinal pigment endothelial cells.
40. The pharmaceutical composition of any one of claims 38 or 39, wherein the disease is selected from the group consisting of type I diabetes, heart disease, neurological disease, endocrine disease, cancer, ocular disease, and vascular disease.
41. A medicament for treating a disease comprising cells derived from the modified cell of any one of claims 1-2, 4-24, or 44-49.
42. The agent of claim 42 wherein said derived cells are selected from the group consisting of Chimeric Antigen Receptor (CAR) cells, T cells, NK cells, ILCs, endothelial cells, dopaminergic neurons, glial cells, islet beta cells, thyroid cells, fibroblasts, hepatocytes, cardiomyocytes, and retinal pigment endothelial cells.
43. The medicament of any one of claims 41-42, wherein the diseases are selected from the group consisting of type I diabetes, heart disease, nervous system diseases, endocrine diseases, cancers, ocular diseases and vascular diseases.
44. A modified cell comprising an enhanced level of CD16t, CD32t, or CD64t protein produced by genetic engineering, wherein the protein expression results in the modified cell being less sensitive to antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
45. The modified cell of claim 44, wherein the cell is selected from the group consisting of a Chimeric Antigen Receptor (CAR) cell, a T cell, an NK cell, an ILC, an endothelial cell, a dopaminergic neuron, a glial cell, an islet beta cell, a thyroid cell, a fibroblast, a hepatocyte, a cardiomyocyte, and a retinal pigment endothelial cell.
46. The modified cell of any one of claims 1-2, 4-21, or 44-45, wherein the cell comprises an Fc receptor chimera comprising a cytoplasmic domain that does not mediate an Fc receptor signaling pathway, and wherein the cytoplasmic domain facilitates endocytosis of an antibody that binds to the extracellular domain of the Fc receptor chimera by its Fc.
47. The modified cell of claim 46, wherein the cytoplasmic domain is from a transferrin receptor.
48. The modified cell of claim 47, wherein the transferrin receptor is TfR1 or TfR2.
49. The modified cell of any one of claims 46-48, wherein the Fc receptor chimera comprises a CD16, CD32, or CD64 cell surface domain and a TfR1 or TfR2 cytoplasmic domain.
50. The modified cell of any one of claims 1-24 or 44-49, wherein the cell expresses a recombinant sirpa-adaptor protein.
51. The modified cell of claim 50, wherein the SIRPalpha junction protein comprises an immunoglobulin superfamily domain, an antibody Fab domain or a single chain variable fragment (scFV).
52. The modified cell of claim 51, wherein the SIRPalpha joining protein binds to SIRPalpha with an affinity measured by its dissociation constant (Kd), wherein the Kd is about 10 -7 To 10 -13 M。
53. The modified cell of any one of claims 22-25, wherein the cell comprises a B2M-/-phenotype, CIITA-/-phenotype, CD64t, and sirpa-junction molecule.
54. The modified cell of claim 53, wherein the SIRPalpha-binding protein is CD47.
55. The modified cell of any one of claims 53 or 54, wherein the cell is an engineered NK cell.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US63/168,225 | 2021-03-30 | ||
US202263305587P | 2022-02-01 | 2022-02-01 | |
US63/305,587 | 2022-02-01 | ||
PCT/US2022/022368 WO2022212393A1 (en) | 2021-03-30 | 2022-03-29 | Transplanted cell protection via modified fc receptors |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117279651A true CN117279651A (en) | 2023-12-22 |
Family
ID=89204950
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202280032962.8A Pending CN117279651A (en) | 2021-03-30 | 2022-03-29 | Transplantation cytoprotection via modified Fc receptor |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117279651A (en) |
-
2022
- 2022-03-29 CN CN202280032962.8A patent/CN117279651A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220213434A1 (en) | Modified pluripotent cells | |
US20210308183A1 (en) | Chimeric antigen receptor t cells derived from immunoengineered pluripotent stem cells | |
US20240091274A1 (en) | TRANSPLANTED CELL PROTECTION VIA Fc SEQUESTRATION | |
JP2023052079A (en) | Immunoengineered pluripotent cells | |
US20230062612A1 (en) | Safety switches for regulation of gene expression | |
KR20230074718A (en) | Methods of treating patients sensitized with hypoimmunogenic cells and related methods and compositions | |
US11162079B2 (en) | Blood type O Rh-hypo-immunogenic pluripotent cells | |
CA3201099A1 (en) | Innate immune cell silencing by sirp-alpha engager | |
US20230058557A1 (en) | Transplanted cell protection via inhibition of polymorphonuclear cells | |
CN117279651A (en) | Transplantation cytoprotection via modified Fc receptor | |
JP2024515037A (en) | Protection of transplanted cells by modified Fc receptors | |
KR20240073006A (en) | Genetically modified primary cells for allogeneic cell therapy | |
JP2024517864A (en) | Therapeutic engineered cells | |
KR20240095477A (en) | Immunoengineered pluripotent cells |
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 |