WO2020047527A2 - Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs - Google Patents
Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs Download PDFInfo
- Publication number
- WO2020047527A2 WO2020047527A2 PCT/US2019/049259 US2019049259W WO2020047527A2 WO 2020047527 A2 WO2020047527 A2 WO 2020047527A2 US 2019049259 W US2019049259 W US 2019049259W WO 2020047527 A2 WO2020047527 A2 WO 2020047527A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- cell
- reaction mixture
- genetically modified
- domain
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 325
- 210000004369 blood Anatomy 0.000 title claims abstract description 187
- 239000008280 blood Substances 0.000 title claims abstract description 187
- 210000004698 lymphocyte Anatomy 0.000 title abstract description 159
- 239000000203 mixture Substances 0.000 title abstract description 55
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 326
- 210000000822 natural killer cell Anatomy 0.000 claims abstract description 261
- 239000011541 reaction mixture Substances 0.000 claims abstract description 198
- 239000002245 particle Substances 0.000 claims abstract description 170
- 230000001177 retroviral effect Effects 0.000 claims abstract description 164
- 210000004027 cell Anatomy 0.000 claims description 242
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 219
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 205
- 229920001184 polypeptide Polymers 0.000 claims description 201
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 123
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 89
- 230000027455 binding Effects 0.000 claims description 87
- 230000010076 replication Effects 0.000 claims description 71
- 238000010361 transduction Methods 0.000 claims description 60
- 230000026683 transduction Effects 0.000 claims description 58
- 230000001589 lymphoproliferative effect Effects 0.000 claims description 54
- 239000012634 fragment Substances 0.000 claims description 47
- 230000002463 transducing effect Effects 0.000 claims description 45
- -1 miRl55 target Proteins 0.000 claims description 44
- 239000003146 anticoagulant agent Substances 0.000 claims description 42
- 229940127219 anticoagulant drug Drugs 0.000 claims description 42
- 230000004068 intracellular signaling Effects 0.000 claims description 39
- 238000012545 processing Methods 0.000 claims description 38
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 35
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 35
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 35
- 102000040430 polynucleotide Human genes 0.000 claims description 34
- 108091033319 polynucleotide Proteins 0.000 claims description 34
- 239000002157 polynucleotide Substances 0.000 claims description 34
- 210000000265 leukocyte Anatomy 0.000 claims description 32
- 210000000601 blood cell Anatomy 0.000 claims description 30
- 230000000284 resting effect Effects 0.000 claims description 27
- 238000004519 manufacturing process Methods 0.000 claims description 25
- 102100027207 CD27 antigen Human genes 0.000 claims description 21
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 21
- 230000002103 transcriptional effect Effects 0.000 claims description 20
- 102100027205 B-cell antigen receptor complex-associated protein alpha chain Human genes 0.000 claims description 17
- 230000006044 T cell activation Effects 0.000 claims description 16
- 230000002401 inhibitory effect Effects 0.000 claims description 16
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 15
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 15
- IJRKANNOPXMZSG-SSPAHAAFSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC(=O)CC(O)(C(O)=O)CC(O)=O IJRKANNOPXMZSG-SSPAHAAFSA-N 0.000 claims description 14
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 claims description 14
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 claims description 14
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 claims description 14
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 claims description 14
- 239000012528 membrane Substances 0.000 claims description 14
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 13
- 101000914489 Homo sapiens B-cell antigen receptor complex-associated protein alpha chain Proteins 0.000 claims description 13
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 claims description 13
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 13
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 claims description 13
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 claims description 11
- 102100039622 Granulocyte colony-stimulating factor receptor Human genes 0.000 claims description 11
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 claims description 11
- 101000746364 Homo sapiens Granulocyte colony-stimulating factor receptor Proteins 0.000 claims description 11
- 210000003714 granulocyte Anatomy 0.000 claims description 11
- 229920000669 heparin Polymers 0.000 claims description 11
- 102100027203 B-cell antigen receptor complex-associated protein beta chain Human genes 0.000 claims description 10
- 102100039061 Cytokine receptor common subunit beta Human genes 0.000 claims description 10
- 101000914491 Homo sapiens B-cell antigen receptor complex-associated protein beta chain Proteins 0.000 claims description 10
- 101001033280 Homo sapiens Cytokine receptor common subunit beta Proteins 0.000 claims description 10
- 101000799466 Homo sapiens Thrombopoietin receptor Proteins 0.000 claims description 10
- 102100034196 Thrombopoietin receptor Human genes 0.000 claims description 10
- 229960002897 heparin Drugs 0.000 claims description 10
- 102100026234 Cytokine receptor common subunit gamma Human genes 0.000 claims description 9
- 101001055227 Homo sapiens Cytokine receptor common subunit gamma Proteins 0.000 claims description 9
- 101001001420 Homo sapiens Interferon gamma receptor 1 Proteins 0.000 claims description 9
- 101001043817 Homo sapiens Interleukin-31 receptor subunit alpha Proteins 0.000 claims description 9
- 101000917821 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-c Proteins 0.000 claims description 9
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 claims description 9
- 101000679851 Homo sapiens Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 claims description 9
- 102100035678 Interferon gamma receptor 1 Human genes 0.000 claims description 9
- 102100021594 Interleukin-31 receptor subunit alpha Human genes 0.000 claims description 9
- 102100029206 Low affinity immunoglobulin gamma Fc region receptor II-c Human genes 0.000 claims description 9
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 claims description 9
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- 230000003612 virological effect Effects 0.000 claims description 9
- 101001037246 Homo sapiens Interleukin-27 receptor subunit alpha Proteins 0.000 claims description 8
- 101000599056 Homo sapiens Interleukin-6 receptor subunit beta Proteins 0.000 claims description 8
- 101000586302 Homo sapiens Oncostatin-M-specific receptor subunit beta Proteins 0.000 claims description 8
- 102100040066 Interleukin-27 receptor subunit alpha Human genes 0.000 claims description 8
- 102100037795 Interleukin-6 receptor subunit beta Human genes 0.000 claims description 8
- 102100021593 Interleukin-7 receptor subunit alpha Human genes 0.000 claims description 8
- 102100030098 Oncostatin-M-specific receptor subunit beta Human genes 0.000 claims description 8
- 102100028113 Granulocyte-macrophage colony-stimulating factor receptor subunit alpha Human genes 0.000 claims description 7
- 101000916625 Homo sapiens Granulocyte-macrophage colony-stimulating factor receptor subunit alpha Proteins 0.000 claims description 7
- 101001083151 Homo sapiens Interleukin-10 receptor subunit alpha Proteins 0.000 claims description 7
- 101001003149 Homo sapiens Interleukin-10 receptor subunit beta Proteins 0.000 claims description 7
- 101001003147 Homo sapiens Interleukin-11 receptor subunit alpha Proteins 0.000 claims description 7
- 101001003132 Homo sapiens Interleukin-13 receptor subunit alpha-2 Proteins 0.000 claims description 7
- 101001019588 Homo sapiens Interleukin-17 receptor D Proteins 0.000 claims description 7
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 claims description 7
- 101001055145 Homo sapiens Interleukin-2 receptor subunit beta Proteins 0.000 claims description 7
- 101000960936 Homo sapiens Interleukin-5 receptor subunit alpha Proteins 0.000 claims description 7
- 101000599048 Homo sapiens Interleukin-6 receptor subunit alpha Proteins 0.000 claims description 7
- 101001043809 Homo sapiens Interleukin-7 receptor subunit alpha Proteins 0.000 claims description 7
- 101001055219 Homo sapiens Interleukin-9 receptor Proteins 0.000 claims description 7
- 102100036157 Interferon gamma receptor 2 Human genes 0.000 claims description 7
- 108700003107 Interleukin-1 Receptor-Like 1 Proteins 0.000 claims description 7
- 102100036706 Interleukin-1 receptor-like 1 Human genes 0.000 claims description 7
- 102100030236 Interleukin-10 receptor subunit alpha Human genes 0.000 claims description 7
- 102100020788 Interleukin-10 receptor subunit beta Human genes 0.000 claims description 7
- 102100020787 Interleukin-11 receptor subunit alpha Human genes 0.000 claims description 7
- 102100020793 Interleukin-13 receptor subunit alpha-2 Human genes 0.000 claims description 7
- 102100035015 Interleukin-17 receptor D Human genes 0.000 claims description 7
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 claims description 7
- 102100026879 Interleukin-2 receptor subunit beta Human genes 0.000 claims description 7
- 102100039881 Interleukin-5 receptor subunit alpha Human genes 0.000 claims description 7
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 claims description 7
- 102100026244 Interleukin-9 receptor Human genes 0.000 claims description 7
- 102100031775 Leptin receptor Human genes 0.000 claims description 7
- 102100035891 T-cell surface glycoprotein CD3 delta chain Human genes 0.000 claims description 7
- 238000003776 cleavage reaction Methods 0.000 claims description 7
- 238000000249 far-infrared magnetic resonance spectroscopy Methods 0.000 claims description 7
- 238000012239 gene modification Methods 0.000 claims description 7
- 230000005017 genetic modification Effects 0.000 claims description 7
- 235000013617 genetically modified food Nutrition 0.000 claims description 7
- 108010085650 interferon gamma receptor Proteins 0.000 claims description 7
- 108010019813 leptin receptors Proteins 0.000 claims description 7
- 230000007017 scission Effects 0.000 claims description 7
- 102100020948 Growth hormone receptor Human genes 0.000 claims description 6
- 101000852870 Homo sapiens Interferon alpha/beta receptor 1 Proteins 0.000 claims description 6
- 101000960952 Homo sapiens Interleukin-1 receptor accessory protein Proteins 0.000 claims description 6
- 101001076418 Homo sapiens Interleukin-1 receptor type 1 Proteins 0.000 claims description 6
- 101001003138 Homo sapiens Interleukin-12 receptor subunit beta-2 Proteins 0.000 claims description 6
- 101001019602 Homo sapiens Interleukin-17 receptor C Proteins 0.000 claims description 6
- 101000961065 Homo sapiens Interleukin-18 receptor 1 Proteins 0.000 claims description 6
- 101000998120 Homo sapiens Interleukin-3 receptor subunit alpha Proteins 0.000 claims description 6
- 101001123448 Homo sapiens Prolactin receptor Proteins 0.000 claims description 6
- 101000648507 Homo sapiens Tumor necrosis factor receptor superfamily member 14 Proteins 0.000 claims description 6
- 102100036714 Interferon alpha/beta receptor 1 Human genes 0.000 claims description 6
- 102100039880 Interleukin-1 receptor accessory protein Human genes 0.000 claims description 6
- 102100026016 Interleukin-1 receptor type 1 Human genes 0.000 claims description 6
- 102100020792 Interleukin-12 receptor subunit beta-2 Human genes 0.000 claims description 6
- 102100035012 Interleukin-17 receptor C Human genes 0.000 claims description 6
- 102100039340 Interleukin-18 receptor 1 Human genes 0.000 claims description 6
- 108010017411 Interleukin-21 Receptors Proteins 0.000 claims description 6
- 102100030699 Interleukin-21 receptor Human genes 0.000 claims description 6
- 102100033493 Interleukin-3 receptor subunit alpha Human genes 0.000 claims description 6
- 101150113776 LMP1 gene Proteins 0.000 claims description 6
- 102100029000 Prolactin receptor Human genes 0.000 claims description 6
- 102100038497 Cytokine receptor-like factor 2 Human genes 0.000 claims description 5
- 101000956427 Homo sapiens Cytokine receptor-like factor 2 Proteins 0.000 claims description 5
- 101000852865 Homo sapiens Interferon alpha/beta receptor 2 Proteins 0.000 claims description 5
- 101000852965 Homo sapiens Interleukin-1 receptor-like 2 Proteins 0.000 claims description 5
- 101001003142 Homo sapiens Interleukin-12 receptor subunit beta-1 Proteins 0.000 claims description 5
- 101001003135 Homo sapiens Interleukin-13 receptor subunit alpha-1 Proteins 0.000 claims description 5
- 101001003140 Homo sapiens Interleukin-15 receptor subunit alpha Proteins 0.000 claims description 5
- 101001019600 Homo sapiens Interleukin-17 receptor B Proteins 0.000 claims description 5
- 101001019590 Homo sapiens Interleukin-17 receptor E Proteins 0.000 claims description 5
- 101001019615 Homo sapiens Interleukin-18 receptor accessory protein Proteins 0.000 claims description 5
- 101001044893 Homo sapiens Interleukin-20 receptor subunit alpha Proteins 0.000 claims description 5
- 101001044895 Homo sapiens Interleukin-20 receptor subunit beta Proteins 0.000 claims description 5
- 101001044883 Homo sapiens Interleukin-22 receptor subunit alpha-1 Proteins 0.000 claims description 5
- 101000853012 Homo sapiens Interleukin-23 receptor Proteins 0.000 claims description 5
- 101001042362 Homo sapiens Leukemia inhibitory factor receptor Proteins 0.000 claims description 5
- 102100036718 Interferon alpha/beta receptor 2 Human genes 0.000 claims description 5
- 102100036697 Interleukin-1 receptor-like 2 Human genes 0.000 claims description 5
- 102100020790 Interleukin-12 receptor subunit beta-1 Human genes 0.000 claims description 5
- 102100020791 Interleukin-13 receptor subunit alpha-1 Human genes 0.000 claims description 5
- 102100020789 Interleukin-15 receptor subunit alpha Human genes 0.000 claims description 5
- 102100035014 Interleukin-17 receptor B Human genes 0.000 claims description 5
- 102100035016 Interleukin-17 receptor E Human genes 0.000 claims description 5
- 102100035010 Interleukin-18 receptor accessory protein Human genes 0.000 claims description 5
- 102100022706 Interleukin-20 receptor subunit alpha Human genes 0.000 claims description 5
- 102100022705 Interleukin-20 receptor subunit beta Human genes 0.000 claims description 5
- 102100022723 Interleukin-22 receptor subunit alpha-1 Human genes 0.000 claims description 5
- 102100036672 Interleukin-23 receptor Human genes 0.000 claims description 5
- 102100021747 Leukemia inhibitory factor receptor Human genes 0.000 claims description 5
- 102100035794 T-cell surface glycoprotein CD3 epsilon chain Human genes 0.000 claims description 5
- 102100037911 T-cell surface glycoprotein CD3 gamma chain Human genes 0.000 claims description 5
- 230000034217 membrane fusion Effects 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 101000599613 Homo sapiens Interferon lambda receptor 1 Proteins 0.000 claims description 4
- 101000946863 Homo sapiens T-cell surface glycoprotein CD3 delta chain Proteins 0.000 claims description 4
- 102100037971 Interferon lambda receptor 1 Human genes 0.000 claims description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 4
- 229940122641 ABC transporter inhibitor Drugs 0.000 claims description 3
- 101150013553 CD40 gene Proteins 0.000 claims description 3
- 102100022132 High affinity immunoglobulin epsilon receptor subunit gamma Human genes 0.000 claims description 3
- 101001019598 Homo sapiens Interleukin-17 receptor A Proteins 0.000 claims description 3
- 101001033312 Homo sapiens Interleukin-4 receptor subunit alpha Proteins 0.000 claims description 3
- 101000934346 Homo sapiens T-cell surface antigen CD2 Proteins 0.000 claims description 3
- 101000946833 Homo sapiens T-cell surface glycoprotein CD8 beta chain Proteins 0.000 claims description 3
- 102100035018 Interleukin-17 receptor A Human genes 0.000 claims description 3
- 102100039078 Interleukin-4 receptor subunit alpha Human genes 0.000 claims description 3
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 claims description 3
- 102100034928 T-cell surface glycoprotein CD8 beta chain Human genes 0.000 claims description 3
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 3
- 238000013518 transcription Methods 0.000 claims description 3
- 230000035897 transcription Effects 0.000 claims description 3
- 101000946860 Homo sapiens T-cell surface glycoprotein CD3 epsilon chain Proteins 0.000 claims description 2
- 101000738413 Homo sapiens T-cell surface glycoprotein CD3 gamma chain Proteins 0.000 claims description 2
- 101150053046 MYD88 gene Proteins 0.000 claims description 2
- 102100024134 Myeloid differentiation primary response protein MyD88 Human genes 0.000 claims description 2
- 210000004700 fetal blood Anatomy 0.000 claims description 2
- 108020004999 messenger RNA Proteins 0.000 claims description 2
- 210000000581 natural killer T-cell Anatomy 0.000 claims description 2
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 2
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 claims 6
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 claims 6
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 claims 6
- 102100030751 Eomesodermin homolog Human genes 0.000 claims 5
- 101001064167 Homo sapiens Eomesodermin homolog Proteins 0.000 claims 5
- 102100025725 Mothers against decapentaplegic homolog 4 Human genes 0.000 claims 5
- 101710143112 Mothers against decapentaplegic homolog 4 Proteins 0.000 claims 5
- 101150089023 FASLG gene Proteins 0.000 claims 4
- 101000840551 Homo sapiens Hexokinase-2 Proteins 0.000 claims 3
- 101001050577 Homo sapiens Kinesin-like protein KIF2A Proteins 0.000 claims 3
- 101001026214 Homo sapiens Potassium voltage-gated channel subfamily A member 5 Proteins 0.000 claims 3
- 101001048456 Homo sapiens Protein Hook homolog 2 Proteins 0.000 claims 3
- 102100023426 Kinesin-like protein KIF2A Human genes 0.000 claims 3
- 108010003533 Viral Envelope Proteins Proteins 0.000 claims 3
- CDKIEBFIMCSCBB-UHFFFAOYSA-N 1-(6,7-dimethoxy-3,4-dihydro-1h-isoquinolin-2-yl)-3-(1-methyl-2-phenylpyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one;hydrochloride Chemical compound Cl.C1C=2C=C(OC)C(OC)=CC=2CCN1C(=O)C=CC(C1=CC=CN=C1N1C)=C1C1=CC=CC=C1 CDKIEBFIMCSCBB-UHFFFAOYSA-N 0.000 claims 2
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims 2
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 claims 2
- 102000003952 Caspase 3 Human genes 0.000 claims 2
- 108090000397 Caspase 3 Proteins 0.000 claims 2
- 101100126877 Dictyostelium discoideum ccbl gene Proteins 0.000 claims 2
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 claims 2
- 101001068133 Homo sapiens Hepatitis A virus cellular receptor 2 Proteins 0.000 claims 2
- 101001006892 Homo sapiens Krueppel-like factor 10 Proteins 0.000 claims 2
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 claims 2
- 101000761581 Homo sapiens Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B delta isoform Proteins 0.000 claims 2
- 101001068027 Homo sapiens Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform Proteins 0.000 claims 2
- 101000687808 Homo sapiens Suppressor of cytokine signaling 2 Proteins 0.000 claims 2
- 101000638161 Homo sapiens Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 claims 2
- 101000610604 Homo sapiens Tumor necrosis factor receptor superfamily member 10B Proteins 0.000 claims 2
- 101150026829 JUNB gene Proteins 0.000 claims 2
- 102000015617 Janus Kinases Human genes 0.000 claims 2
- 108010024121 Janus Kinases Proteins 0.000 claims 2
- 102100027798 Krueppel-like factor 10 Human genes 0.000 claims 2
- 102000017578 LAG3 Human genes 0.000 claims 2
- 102100025748 Mothers against decapentaplegic homolog 3 Human genes 0.000 claims 2
- 101710143111 Mothers against decapentaplegic homolog 3 Proteins 0.000 claims 2
- 102100024927 Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B delta isoform Human genes 0.000 claims 2
- 102100034464 Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform Human genes 0.000 claims 2
- 102100024784 Suppressor of cytokine signaling 2 Human genes 0.000 claims 2
- 102100040112 Tumor necrosis factor receptor superfamily member 10B Human genes 0.000 claims 2
- 102100026882 Alpha-synuclein Human genes 0.000 claims 1
- 102100025222 CD63 antigen Human genes 0.000 claims 1
- 102100027221 CD81 antigen Human genes 0.000 claims 1
- 102100027217 CD82 antigen Human genes 0.000 claims 1
- 102100037904 CD9 antigen Human genes 0.000 claims 1
- 101000741929 Caenorhabditis elegans Serine/threonine-protein phosphatase 2A catalytic subunit Proteins 0.000 claims 1
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 claims 1
- 102100035233 Furin Human genes 0.000 claims 1
- 108090001126 Furin Proteins 0.000 claims 1
- 101000834898 Homo sapiens Alpha-synuclein Proteins 0.000 claims 1
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 claims 1
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 claims 1
- 101000914469 Homo sapiens CD82 antigen Proteins 0.000 claims 1
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 claims 1
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 claims 1
- 101000824104 Homo sapiens High affinity immunoglobulin epsilon receptor subunit gamma Proteins 0.000 claims 1
- 101000980823 Homo sapiens Leukocyte surface antigen CD53 Proteins 0.000 claims 1
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 claims 1
- 101000652359 Homo sapiens Spermatogenesis-associated protein 2 Proteins 0.000 claims 1
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 claims 1
- 102100024221 Leukocyte surface antigen CD53 Human genes 0.000 claims 1
- 102000013010 Member 1 Subfamily G ATP Binding Cassette Transporter Human genes 0.000 claims 1
- 108010090314 Member 1 Subfamily G ATP Binding Cassette Transporter Proteins 0.000 claims 1
- 101710143123 Mothers against decapentaplegic homolog 2 Proteins 0.000 claims 1
- 102100025751 Mothers against decapentaplegic homolog 2 Human genes 0.000 claims 1
- 101100327112 Mus musculus Cbx1 gene Proteins 0.000 claims 1
- 101150045565 Socs1 gene Proteins 0.000 claims 1
- 108700027336 Suppressor of Cytokine Signaling 1 Proteins 0.000 claims 1
- 102100024779 Suppressor of cytokine signaling 1 Human genes 0.000 claims 1
- 101100215487 Sus scrofa ADRA2A gene Proteins 0.000 claims 1
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 claims 1
- 102000004060 Transforming Growth Factor-beta Type II Receptor Human genes 0.000 claims 1
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 claims 1
- 239000012190 activator Substances 0.000 claims 1
- 101150091914 cbx gene Proteins 0.000 claims 1
- 239000000427 antigen Substances 0.000 description 146
- 108091007433 antigens Proteins 0.000 description 145
- 102000036639 antigens Human genes 0.000 description 145
- 230000011664 signaling Effects 0.000 description 125
- 230000003834 intracellular effect Effects 0.000 description 115
- 235000001014 amino acid Nutrition 0.000 description 99
- 229940024606 amino acid Drugs 0.000 description 98
- 150000001413 amino acids Chemical class 0.000 description 98
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 77
- 108091008874 T cell receptors Proteins 0.000 description 76
- 230000003213 activating effect Effects 0.000 description 62
- 108090000623 proteins and genes Proteins 0.000 description 48
- 206010028980 Neoplasm Diseases 0.000 description 42
- 230000004913 activation Effects 0.000 description 40
- 102000004169 proteins and genes Human genes 0.000 description 40
- 235000018102 proteins Nutrition 0.000 description 36
- 125000003275 alpha amino acid group Chemical group 0.000 description 31
- 238000011534 incubation Methods 0.000 description 31
- 230000008569 process Effects 0.000 description 28
- 239000000306 component Substances 0.000 description 27
- 102000005962 receptors Human genes 0.000 description 26
- 108020003175 receptors Proteins 0.000 description 26
- 241000282414 Homo sapiens Species 0.000 description 25
- 239000002679 microRNA Substances 0.000 description 25
- 238000001727 in vivo Methods 0.000 description 23
- 102000001301 EGF receptor Human genes 0.000 description 22
- 108060006698 EGF receptor Proteins 0.000 description 22
- 230000000670 limiting effect Effects 0.000 description 21
- 150000007523 nucleic acids Chemical class 0.000 description 19
- 102000004127 Cytokines Human genes 0.000 description 18
- 108090000695 Cytokines Proteins 0.000 description 18
- 102000039446 nucleic acids Human genes 0.000 description 17
- 108020004707 nucleic acids Proteins 0.000 description 17
- 239000000725 suspension Substances 0.000 description 17
- 239000003446 ligand Substances 0.000 description 16
- 210000000440 neutrophil Anatomy 0.000 description 16
- 230000004083 survival effect Effects 0.000 description 16
- 210000000170 cell membrane Anatomy 0.000 description 15
- 238000000338 in vitro Methods 0.000 description 15
- 230000035755 proliferation Effects 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 230000000638 stimulation Effects 0.000 description 15
- 102100038717 TYRO protein tyrosine kinase-binding protein Human genes 0.000 description 14
- 239000003795 chemical substances by application Substances 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 14
- 230000001976 improved effect Effects 0.000 description 14
- 108700011259 MicroRNAs Proteins 0.000 description 13
- 210000001772 blood platelet Anatomy 0.000 description 13
- 201000011510 cancer Diseases 0.000 description 13
- 201000010099 disease Diseases 0.000 description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 13
- 210000003743 erythrocyte Anatomy 0.000 description 13
- 210000003527 eukaryotic cell Anatomy 0.000 description 13
- 239000000523 sample Substances 0.000 description 13
- 230000009870 specific binding Effects 0.000 description 13
- 239000000872 buffer Substances 0.000 description 12
- 230000004927 fusion Effects 0.000 description 12
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 11
- 108091070501 miRNA Proteins 0.000 description 11
- 238000004806 packaging method and process Methods 0.000 description 11
- 241001430294 unidentified retrovirus Species 0.000 description 11
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 10
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 description 10
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 description 10
- 108010076504 Protein Sorting Signals Proteins 0.000 description 10
- 239000012503 blood component Substances 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- 230000004936 stimulating effect Effects 0.000 description 10
- 102000035160 transmembrane proteins Human genes 0.000 description 10
- 108091005703 transmembrane proteins Proteins 0.000 description 10
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 9
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 9
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 9
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 9
- 238000011467 adoptive cell therapy Methods 0.000 description 9
- 210000003651 basophil Anatomy 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 230000008030 elimination Effects 0.000 description 9
- 238000003379 elimination reaction Methods 0.000 description 9
- 238000001914 filtration Methods 0.000 description 9
- 210000004379 membrane Anatomy 0.000 description 9
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 9
- 210000002381 plasma Anatomy 0.000 description 9
- 210000000130 stem cell Anatomy 0.000 description 9
- 230000008685 targeting Effects 0.000 description 9
- 101000809875 Homo sapiens TYRO protein tyrosine kinase-binding protein Proteins 0.000 description 8
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 8
- 230000008901 benefit Effects 0.000 description 8
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 8
- 102000003675 cytokine receptors Human genes 0.000 description 8
- 108010057085 cytokine receptors Proteins 0.000 description 8
- 230000001472 cytotoxic effect Effects 0.000 description 8
- 238000001802 infusion Methods 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 7
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 7
- 102100029360 Hematopoietic cell signal transducer Human genes 0.000 description 7
- 101000990188 Homo sapiens Hematopoietic cell signal transducer Proteins 0.000 description 7
- 101000818543 Homo sapiens Tyrosine-protein kinase ZAP-70 Proteins 0.000 description 7
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 7
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 102100037906 T-cell surface glycoprotein CD3 zeta chain Human genes 0.000 description 7
- 102100021125 Tyrosine-protein kinase ZAP-70 Human genes 0.000 description 7
- 230000010261 cell growth Effects 0.000 description 7
- 210000003979 eosinophil Anatomy 0.000 description 7
- 239000005090 green fluorescent protein Substances 0.000 description 7
- 210000001616 monocyte Anatomy 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 229920000642 polymer Polymers 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 210000003705 ribosome Anatomy 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 239000002699 waste material Substances 0.000 description 7
- 229920001917 Ficoll Polymers 0.000 description 6
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 6
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 6
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 description 6
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 6
- 101710187864 TYRO protein tyrosine kinase-binding protein Proteins 0.000 description 6
- 238000007792 addition Methods 0.000 description 6
- 238000002617 apheresis Methods 0.000 description 6
- 238000002659 cell therapy Methods 0.000 description 6
- 238000006471 dimerization reaction Methods 0.000 description 6
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 5
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 5
- 239000004471 Glycine Substances 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 101100508818 Mus musculus Inpp5k gene Proteins 0.000 description 5
- 101100366438 Rattus norvegicus Sphkap gene Proteins 0.000 description 5
- 108091008605 VEGF receptors Proteins 0.000 description 5
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 210000002865 immune cell Anatomy 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 230000000717 retained effect Effects 0.000 description 5
- 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 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 description 4
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 4
- 101710095183 B-cell antigen receptor complex-associated protein alpha chain Proteins 0.000 description 4
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 4
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 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 4
- 108010015776 Glucose oxidase Proteins 0.000 description 4
- 239000004366 Glucose oxidase Substances 0.000 description 4
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 4
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 4
- 108010002586 Interleukin-7 Proteins 0.000 description 4
- 102100021592 Interleukin-7 Human genes 0.000 description 4
- 108010038498 Interleukin-7 Receptors Proteins 0.000 description 4
- 102100027670 Islet amyloid polypeptide Human genes 0.000 description 4
- 102100036735 Prostate stem cell antigen Human genes 0.000 description 4
- 101710120463 Prostate stem cell antigen Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 4
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 4
- 235000004279 alanine Nutrition 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000015271 coagulation Effects 0.000 description 4
- 238000005345 coagulation Methods 0.000 description 4
- 231100000433 cytotoxic Toxicity 0.000 description 4
- 230000000447 dimerizing effect Effects 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000009368 gene silencing by RNA Effects 0.000 description 4
- 229940116332 glucose oxidase Drugs 0.000 description 4
- 235000019420 glucose oxidase Nutrition 0.000 description 4
- 239000003102 growth factor Substances 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 102000006495 integrins Human genes 0.000 description 4
- 108010044426 integrins Proteins 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 230000010412 perfusion Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 239000013589 supplement Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 3
- 108010052500 Calgranulin A Proteins 0.000 description 3
- 108010052495 Calgranulin B Proteins 0.000 description 3
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101000738335 Homo sapiens T-cell surface glycoprotein CD3 zeta chain Proteins 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 206010021143 Hypoxia Diseases 0.000 description 3
- 102000009438 IgE Receptors Human genes 0.000 description 3
- 108010073816 IgE Receptors Proteins 0.000 description 3
- 108090000172 Interleukin-15 Proteins 0.000 description 3
- 102000003812 Interleukin-15 Human genes 0.000 description 3
- 102000010782 Interleukin-7 Receptors Human genes 0.000 description 3
- 102000015696 Interleukins Human genes 0.000 description 3
- 108010063738 Interleukins Proteins 0.000 description 3
- 108010041872 Islet Amyloid Polypeptide Proteins 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 102100032442 Protein S100-A8 Human genes 0.000 description 3
- 102100032420 Protein S100-A9 Human genes 0.000 description 3
- 108091027967 Small hairpin RNA Proteins 0.000 description 3
- 101710146340 T-cell surface glycoprotein CD3 epsilon chain Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 3
- 230000000712 assembly Effects 0.000 description 3
- 238000000429 assembly Methods 0.000 description 3
- 238000011130 autologous cell therapy Methods 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000023555 blood coagulation Effects 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 230000032823 cell division Effects 0.000 description 3
- 238000003693 cell processing method Methods 0.000 description 3
- 229960005395 cetuximab Drugs 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000000139 costimulatory effect Effects 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 238000000432 density-gradient centrifugation Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000000799 fusogenic effect Effects 0.000 description 3
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000002147 killing effect Effects 0.000 description 3
- 235000005772 leucine Nutrition 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 230000002093 peripheral effect Effects 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 235000019419 proteases Nutrition 0.000 description 3
- 230000010410 reperfusion Effects 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- HWXBTNAVRSUOJR-GSVOUGTGSA-N (R)-2-hydroxyglutaric acid Chemical compound OC(=O)[C@H](O)CCC(O)=O HWXBTNAVRSUOJR-GSVOUGTGSA-N 0.000 description 2
- RSGFPIWWSCWCFJ-VAXZQHAWSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;phosphoric acid Chemical compound OP(O)(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC(=O)CC(O)(C(O)=O)CC(O)=O RSGFPIWWSCWCFJ-VAXZQHAWSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102000006306 Antigen Receptors Human genes 0.000 description 2
- 108010083359 Antigen Receptors Proteins 0.000 description 2
- 208000028564 B-cell non-Hodgkin lymphoma Diseases 0.000 description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 2
- 102100032912 CD44 antigen Human genes 0.000 description 2
- 108010028326 Calbindin 2 Proteins 0.000 description 2
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 2
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 2
- 102100021849 Calretinin Human genes 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- 102400001368 Epidermal growth factor Human genes 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 102000053171 Glial Fibrillary Acidic Human genes 0.000 description 2
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 2
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 2
- 101710154606 Hemagglutinin Proteins 0.000 description 2
- 108091010847 High affinity immunoglobulin epsilon receptor subunit gamma Proteins 0.000 description 2
- 102100037907 High mobility group protein B1 Human genes 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 2
- 101100112778 Homo sapiens CD247 gene Proteins 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 101001075287 Homo sapiens Growth hormone receptor Proteins 0.000 description 2
- 101001025337 Homo sapiens High mobility group protein B1 Proteins 0.000 description 2
- 101001042104 Homo sapiens Inducible T-cell costimulator Proteins 0.000 description 2
- 101000628547 Homo sapiens Metalloreductase STEAP1 Proteins 0.000 description 2
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 description 2
- 101000589307 Homo sapiens Natural cytotoxicity triggering receptor 3 Proteins 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 102100021317 Inducible T-cell costimulator Human genes 0.000 description 2
- 102000008070 Interferon-gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 239000000232 Lipid Bilayer Substances 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 108050008953 Melanoma-associated antigen Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000003735 Mesothelin Human genes 0.000 description 2
- 108090000015 Mesothelin Proteins 0.000 description 2
- 206010027406 Mesothelioma Diseases 0.000 description 2
- 102100026712 Metalloreductase STEAP1 Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 2
- 230000006051 NK cell activation Effects 0.000 description 2
- 102100032852 Natural cytotoxicity triggering receptor 3 Human genes 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 2
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 102000038030 PI3Ks Human genes 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 2
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 102100035703 Prostatic acid phosphatase Human genes 0.000 description 2
- 101710176177 Protein A56 Proteins 0.000 description 2
- 108010029485 Protein Isoforms Proteins 0.000 description 2
- 102000001708 Protein Isoforms Human genes 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 description 2
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- 102000018210 Recoverin Human genes 0.000 description 2
- 108010076570 Recoverin Proteins 0.000 description 2
- 108020004422 Riboswitch Proteins 0.000 description 2
- 108700028909 Serum Amyloid A Proteins 0.000 description 2
- 102000054727 Serum Amyloid A Human genes 0.000 description 2
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 2
- 206010041067 Small cell lung cancer Diseases 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 230000006052 T cell proliferation Effects 0.000 description 2
- 102100027208 T-cell antigen CD7 Human genes 0.000 description 2
- 108050005496 T-cell surface glycoprotein CD3 delta chains Proteins 0.000 description 2
- 101710131569 T-cell surface glycoprotein CD3 gamma chain Proteins 0.000 description 2
- 101710156660 T-cell surface glycoprotein CD3 zeta chain Proteins 0.000 description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 2
- 102100026497 Zinc finger protein 654 Human genes 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 229960005305 adenosine Drugs 0.000 description 2
- 125000003295 alanine group Chemical class N[C@@H](C)C(=O)* 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 239000003792 electrolyte Substances 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 229940116977 epidermal growth factor Drugs 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 108010021843 fluorescent protein 583 Proteins 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000000185 hemagglutinin Substances 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 229920002674 hyaluronan Polymers 0.000 description 2
- 229940099552 hyaluronan Drugs 0.000 description 2
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 description 2
- 230000007954 hypoxia Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229960003130 interferon gamma Drugs 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 230000000329 lymphopenic effect Effects 0.000 description 2
- 235000018977 lysine Nutrition 0.000 description 2
- 150000002669 lysines Chemical class 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 239000007800 oxidant agent Substances 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000026792 palmitoylation Effects 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 108060006184 phycobiliprotein Proteins 0.000 description 2
- 108091007428 primary miRNA Proteins 0.000 description 2
- 108010043671 prostatic acid phosphatase Proteins 0.000 description 2
- 238000003380 quartz crystal microbalance Methods 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000004055 small Interfering RNA Substances 0.000 description 2
- 208000000587 small cell lung carcinoma Diseases 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000009987 spinning Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 235000002374 tyrosine Nutrition 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 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 2
- RJBDSRWGVYNDHL-XNJNKMBASA-N (2S,4R,5S,6S)-2-[(2S,3R,4R,5S,6R)-5-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2-[(2R,3S,4R,5R,6R)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(E,2R,3S)-3-hydroxy-2-(octadecanoylamino)octadec-4-enoxy]oxan-3-yl]oxy-3-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-5-amino-6-[(1S,2R)-2-[(2S,4R,5S,6S)-5-amino-2-carboxy-4-hydroxy-6-[(1R,2R)-1,2,3-trihydroxypropyl]oxan-2-yl]oxy-1,3-dihydroxypropyl]-4-hydroxyoxane-2-carboxylic acid Chemical compound CCCCCCCCCCCCCCCCCC(=O)N[C@H](CO[C@@H]1O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO)[C@H](O[C@@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3NC(C)=O)[C@H](O[C@@]3(C[C@@H](O)[C@H](N)[C@H](O3)[C@H](O)[C@@H](CO)O[C@@]3(C[C@@H](O)[C@H](N)[C@H](O3)[C@H](O)[C@H](O)CO)C(O)=O)C(O)=O)[C@H]2O)[C@H](O)[C@H]1O)[C@@H](O)\C=C\CCCCCCCCCCCCC RJBDSRWGVYNDHL-XNJNKMBASA-N 0.000 description 1
- JARGNLJYKBUKSJ-KGZKBUQUSA-N (2r)-2-amino-5-[[(2r)-1-(carboxymethylamino)-3-hydroxy-1-oxopropan-2-yl]amino]-5-oxopentanoic acid;hydrobromide Chemical compound Br.OC(=O)[C@H](N)CCC(=O)N[C@H](CO)C(=O)NCC(O)=O JARGNLJYKBUKSJ-KGZKBUQUSA-N 0.000 description 1
- SPFMQWBKVUQXJV-BTVCFUMJSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;hydrate Chemical compound O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O SPFMQWBKVUQXJV-BTVCFUMJSA-N 0.000 description 1
- KUHSEZKIEJYEHN-BXRBKJIMSA-N (2s)-2-amino-3-hydroxypropanoic acid;(2s)-2-aminopropanoic acid Chemical compound C[C@H](N)C(O)=O.OC[C@H](N)C(O)=O KUHSEZKIEJYEHN-BXRBKJIMSA-N 0.000 description 1
- WKBPZYKAUNRMKP-UHFFFAOYSA-N 1-[2-(2,4-dichlorophenyl)pentyl]1,2,4-triazole Chemical compound C=1C=C(Cl)C=C(Cl)C=1C(CCC)CN1C=NC=N1 WKBPZYKAUNRMKP-UHFFFAOYSA-N 0.000 description 1
- WLKSPGHQGFFKGE-UHFFFAOYSA-N 1-chloropropan-2-yl n-(3-chlorophenyl)carbamate Chemical compound ClCC(C)OC(=O)NC1=CC=CC(Cl)=C1 WLKSPGHQGFFKGE-UHFFFAOYSA-N 0.000 description 1
- UFBJCMHMOXMLKC-UHFFFAOYSA-N 2,4-dinitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O UFBJCMHMOXMLKC-UHFFFAOYSA-N 0.000 description 1
- YMHOBZXQZVXHBM-UHFFFAOYSA-N 2,5-dimethoxy-4-bromophenethylamine Chemical compound COC1=CC(CCN)=C(OC)C=C1Br YMHOBZXQZVXHBM-UHFFFAOYSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- ILAYIAGXTHKHNT-UHFFFAOYSA-N 4-[4-(2,4,6-trimethyl-phenylamino)-pyrimidin-2-ylamino]-benzonitrile Chemical compound CC1=CC(C)=CC(C)=C1NC1=CC=NC(NC=2C=CC(=CC=2)C#N)=N1 ILAYIAGXTHKHNT-UHFFFAOYSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 1
- 108010055851 Acetylglucosaminidase Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 1
- 108010011170 Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Proteins 0.000 description 1
- 102100024321 Alkaline phosphatase, placental type Human genes 0.000 description 1
- 102100027165 Alpha-2-macroglobulin receptor-associated protein Human genes 0.000 description 1
- 101710126837 Alpha-2-macroglobulin receptor-associated protein Proteins 0.000 description 1
- 241000242757 Anthozoa Species 0.000 description 1
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 1
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 1
- 102100021663 Baculoviral IAP repeat-containing protein 5 Human genes 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 108700012439 CA9 Proteins 0.000 description 1
- 102100038078 CD276 antigen Human genes 0.000 description 1
- 101710185679 CD276 antigen Proteins 0.000 description 1
- 108010058905 CD44v6 antigen Proteins 0.000 description 1
- 102100025221 CD70 antigen Human genes 0.000 description 1
- 108060001253 CD99 Proteins 0.000 description 1
- 102000024905 CD99 Human genes 0.000 description 1
- 102000016838 Calbindin 1 Human genes 0.000 description 1
- 108010028310 Calbindin 1 Proteins 0.000 description 1
- 102000004631 Calcineurin Human genes 0.000 description 1
- 108010042955 Calcineurin Proteins 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 108010032088 Calpain Proteins 0.000 description 1
- 102000007590 Calpain Human genes 0.000 description 1
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 102100024423 Carbonic anhydrase 9 Human genes 0.000 description 1
- 102100026550 Caspase-9 Human genes 0.000 description 1
- 108090000566 Caspase-9 Proteins 0.000 description 1
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 1
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 108091005944 Cerulean Proteins 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 241000579895 Chlorostilbon Species 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 102100028757 Chondroitin sulfate proteoglycan 4 Human genes 0.000 description 1
- 108010007718 Chromogranins Proteins 0.000 description 1
- 102000007345 Chromogranins Human genes 0.000 description 1
- 108091005960 Citrine Proteins 0.000 description 1
- PTOAARAWEBMLNO-KVQBGUIXSA-N Cladribine Chemical compound C1=NC=2C(N)=NC(Cl)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 PTOAARAWEBMLNO-KVQBGUIXSA-N 0.000 description 1
- 102100035167 Coiled-coil domain-containing protein 54 Human genes 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 108091005943 CyPet Proteins 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102100036912 Desmin Human genes 0.000 description 1
- 108010044052 Desmin Proteins 0.000 description 1
- 101100421450 Drosophila melanogaster Shark gene Proteins 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 108010055196 EphA2 Receptor Proteins 0.000 description 1
- 102100030340 Ephrin type-A receptor 2 Human genes 0.000 description 1
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 1
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 1
- 102100031940 Epithelial cell adhesion molecule Human genes 0.000 description 1
- 241000214054 Equine rhinitis A virus Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 108010008177 Fd immunoglobulins Proteins 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 241001251094 Formica Species 0.000 description 1
- 101710113436 GTPase KRas Proteins 0.000 description 1
- 241001663880 Gammaretrovirus Species 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 108010058940 Glutamyl Aminopeptidase Proteins 0.000 description 1
- 102100025783 Glutamyl aminopeptidase Human genes 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 241000534124 Glyptothorax panda Species 0.000 description 1
- 102000009465 Growth Factor Receptors Human genes 0.000 description 1
- 108010009202 Growth Factor Receptors Proteins 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 108010035452 HLA-A1 Antigen Proteins 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 101001023784 Heteractis crispa GFP-like non-fluorescent chromoprotein Proteins 0.000 description 1
- 108010050763 Hippocalcin Proteins 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 102100027893 Homeobox protein Nkx-2.1 Human genes 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
- 101100112772 Homo sapiens CD3G gene Proteins 0.000 description 1
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 description 1
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 1
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 1
- 101000916489 Homo sapiens Chondroitin sulfate proteoglycan 4 Proteins 0.000 description 1
- 101000737052 Homo sapiens Coiled-coil domain-containing protein 54 Proteins 0.000 description 1
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 1
- 101000920667 Homo sapiens Epithelial cell adhesion molecule Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101001103039 Homo sapiens Inactive tyrosine-protein kinase transmembrane receptor ROR1 Proteins 0.000 description 1
- 101001015004 Homo sapiens Integrin beta-3 Proteins 0.000 description 1
- 101000697493 Homo sapiens Large proline-rich protein BAG6 Proteins 0.000 description 1
- 101000991061 Homo sapiens MHC class I polypeptide-related sequence B Proteins 0.000 description 1
- 101001008874 Homo sapiens Mast/stem cell growth factor receptor Kit Proteins 0.000 description 1
- 101000620359 Homo sapiens Melanocyte protein PMEL Proteins 0.000 description 1
- 101000578784 Homo sapiens Melanoma antigen recognized by T-cells 1 Proteins 0.000 description 1
- 101000623901 Homo sapiens Mucin-16 Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101001024605 Homo sapiens Next to BRCA1 gene 1 protein Proteins 0.000 description 1
- 101001103036 Homo sapiens Nuclear receptor ROR-alpha Proteins 0.000 description 1
- 101001091538 Homo sapiens Pyruvate kinase PKM Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000824971 Homo sapiens Sperm surface protein Sp17 Proteins 0.000 description 1
- 101100046560 Homo sapiens TNFRSF14 gene Proteins 0.000 description 1
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 description 1
- 101000851018 Homo sapiens Vascular endothelial growth factor receptor 1 Proteins 0.000 description 1
- 101000851030 Homo sapiens Vascular endothelial growth factor receptor 3 Proteins 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102100039615 Inactive tyrosine-protein kinase transmembrane receptor ROR1 Human genes 0.000 description 1
- 102100032999 Integrin beta-3 Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 108010017515 Interleukin-12 Receptors Proteins 0.000 description 1
- 102000004560 Interleukin-12 Receptors Human genes 0.000 description 1
- 108010017511 Interleukin-13 Receptors Proteins 0.000 description 1
- 102000004559 Interleukin-13 Receptors Human genes 0.000 description 1
- 108010017535 Interleukin-15 Receptors Proteins 0.000 description 1
- 102000004556 Interleukin-15 Receptors Human genes 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 241000283953 Lagomorpha Species 0.000 description 1
- 102100028047 Large proline-rich protein BAG6 Human genes 0.000 description 1
- 108010092277 Leptin Proteins 0.000 description 1
- 102000016267 Leptin Human genes 0.000 description 1
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 206010025327 Lymphopenia Diseases 0.000 description 1
- 208000030289 Lymphoproliferative disease Diseases 0.000 description 1
- 108090000362 Lymphotoxin-beta Proteins 0.000 description 1
- 102100030300 MHC class I polypeptide-related sequence B Human genes 0.000 description 1
- 102100027754 Mast/stem cell growth factor receptor Kit Human genes 0.000 description 1
- 208000000172 Medulloblastoma Diseases 0.000 description 1
- 102100022430 Melanocyte protein PMEL Human genes 0.000 description 1
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 108091033773 MiR-155 Proteins 0.000 description 1
- 102100023123 Mucin-16 Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 108010067385 Myosin Light Chains Proteins 0.000 description 1
- 102000016349 Myosin Light Chains Human genes 0.000 description 1
- 108010001657 NK Cell Lectin-Like Receptor Subfamily K Proteins 0.000 description 1
- 208000021320 Nasu-Hakola disease Diseases 0.000 description 1
- 102100029527 Natural cytotoxicity triggering receptor 3 ligand 1 Human genes 0.000 description 1
- 101710201161 Natural cytotoxicity triggering receptor 3 ligand 1 Proteins 0.000 description 1
- 108010069196 Neural Cell Adhesion Molecules Proteins 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 102400000058 Neuregulin-1 Human genes 0.000 description 1
- 108090000556 Neuregulin-1 Proteins 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 102000010751 Neurocalcin Human genes 0.000 description 1
- 108010077960 Neurocalcin Proteins 0.000 description 1
- 108010088373 Neurofilament Proteins Proteins 0.000 description 1
- 102000008763 Neurofilament Proteins Human genes 0.000 description 1
- 102100028669 Neuron-specific calcium-binding protein hippocalcin Human genes 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000036673 PRAME Human genes 0.000 description 1
- 108060006580 PRAME Proteins 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 108060005874 Parvalbumin Proteins 0.000 description 1
- 102000001675 Parvalbumin Human genes 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 102100033237 Pro-epidermal growth factor Human genes 0.000 description 1
- 102100030122 Protein O-GlcNAcase Human genes 0.000 description 1
- 101710150593 Protein beta Proteins 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 102000013009 Pyruvate Kinase Human genes 0.000 description 1
- 108020005115 Pyruvate Kinase Proteins 0.000 description 1
- 102100034911 Pyruvate kinase PKM Human genes 0.000 description 1
- 101100425901 Rattus norvegicus Tpm1 gene Proteins 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 241000242739 Renilla Species 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 102000012738 S100 Calcium Binding Protein G Human genes 0.000 description 1
- 108010079423 S100 Calcium Binding Protein G Proteins 0.000 description 1
- 102000014400 SH2 domains Human genes 0.000 description 1
- 108050003452 SH2 domains Proteins 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 102000005886 STAT4 Transcription Factor Human genes 0.000 description 1
- 108010019992 STAT4 Transcription Factor Proteins 0.000 description 1
- 102000001712 STAT5 Transcription Factor Human genes 0.000 description 1
- 108010029477 STAT5 Transcription Factor Proteins 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102100037253 Solute carrier family 45 member 3 Human genes 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 101800001707 Spacer peptide Proteins 0.000 description 1
- 108010002687 Survivin Proteins 0.000 description 1
- 102000004874 Synaptophysin Human genes 0.000 description 1
- 108090001076 Synaptophysin Proteins 0.000 description 1
- 108010092262 T-Cell Antigen Receptors Proteins 0.000 description 1
- 101710085551 T-cell surface glycoprotein CD3 delta chain Proteins 0.000 description 1
- 102100036407 Thioredoxin Human genes 0.000 description 1
- 241001648840 Thosea asigna virus Species 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 108010057966 Thyroid Nuclear Factor 1 Proteins 0.000 description 1
- 101001023030 Toxoplasma gondii Myosin-D Proteins 0.000 description 1
- 102100023132 Transcription factor Jun Human genes 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 102100034030 Transient receptor potential cation channel subfamily M member 8 Human genes 0.000 description 1
- 102000013534 Troponin C Human genes 0.000 description 1
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 1
- 102100039094 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 1
- 102100033179 Vascular endothelial growth factor receptor 3 Human genes 0.000 description 1
- 241000545067 Venus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 102100038287 Visinin-like protein 1 Human genes 0.000 description 1
- 101710194459 Visinin-like protein 1 Proteins 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 101000979710 Xenopus laevis Neuronal calcium sensor 1 Proteins 0.000 description 1
- UYRDHEJRPVSJFM-VSWVFQEASA-N [(1s,3r)-3-hydroxy-4-[(3e,5e,7e,9e,11z)-11-[4-[(e)-2-[(1r,3s,6s)-3-hydroxy-1,5,5-trimethyl-7-oxabicyclo[4.1.0]heptan-6-yl]ethenyl]-5-oxofuran-2-ylidene]-3,10-dimethylundeca-1,3,5,7,9-pentaenylidene]-3,5,5-trimethylcyclohexyl] acetate Chemical compound C[C@@]1(O)C[C@@H](OC(=O)C)CC(C)(C)C1=C=C\C(C)=C\C=C\C=C\C=C(/C)\C=C/1C=C(\C=C\[C@]23[C@@](O2)(C)C[C@@H](O)CC3(C)C)C(=O)O\1 UYRDHEJRPVSJFM-VSWVFQEASA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000009676 acute homeostatic proliferation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 229960001456 adenosine triphosphate Drugs 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 108010004469 allophycocyanin Proteins 0.000 description 1
- SRHNADOZAAWYLV-XLMUYGLTSA-N alpha-L-Fucp-(1->2)-beta-D-Galp-(1->4)-[alpha-L-Fucp-(1->3)]-beta-D-GlcpNAc Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@H]2[C@@H]([C@@H](NC(C)=O)[C@H](O)O[C@@H]2CO)O[C@H]2[C@H]([C@H](O)[C@H](O)[C@H](C)O2)O)O[C@H](CO)[C@H](O)[C@@H]1O SRHNADOZAAWYLV-XLMUYGLTSA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000006368 anti-apoptosis response Effects 0.000 description 1
- 230000001745 anti-biotin effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 150000001484 arginines Chemical class 0.000 description 1
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- WZSDNEJJUSYNSG-UHFFFAOYSA-N azocan-1-yl-(3,4,5-trimethoxyphenyl)methanone Chemical compound COC1=C(OC)C(OC)=CC(C(=O)N2CCCCCCC2)=C1 WZSDNEJJUSYNSG-UHFFFAOYSA-N 0.000 description 1
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 108010068032 caltractin Proteins 0.000 description 1
- 101150058049 car gene Proteins 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 108091006090 chromatin-associated proteins Proteins 0.000 description 1
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 1
- 239000011035 citrine Substances 0.000 description 1
- 229960002436 cladribine Drugs 0.000 description 1
- 238000010960 commercial process Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 150000004775 coumarins Chemical class 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 229950006497 dapivirine Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 108010017271 denileukin diftitox Proteins 0.000 description 1
- 229960002923 denileukin diftitox Drugs 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 210000005045 desmin Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 229960002542 dolutegravir Drugs 0.000 description 1
- RHWKPHLQXYSBKR-BMIGLBTASA-N dolutegravir Chemical compound C([C@@H]1OCC[C@H](N1C(=O)C1=C(O)C2=O)C)N1C=C2C(=O)NCC1=CC=C(F)C=C1F RHWKPHLQXYSBKR-BMIGLBTASA-N 0.000 description 1
- 239000010976 emerald Substances 0.000 description 1
- 229910052876 emerald Inorganic materials 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000004955 epithelial membrane Anatomy 0.000 description 1
- 229940082789 erbitux Drugs 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 229960000390 fludarabine Drugs 0.000 description 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000009454 functional inhibition Effects 0.000 description 1
- 108010044804 gamma-glutamyl-seryl-glycine Proteins 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 108700026078 glutathione trisulfide Proteins 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000003284 homeostatic effect Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000009675 homeostatic proliferation Effects 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000037189 immune system physiology Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000017730 intein-mediated protein splicing Effects 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001535 kindling effect Effects 0.000 description 1
- 229940039781 leptin Drugs 0.000 description 1
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 1
- 108020001756 ligand binding domains Proteins 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229940127215 low-molecular weight heparin Drugs 0.000 description 1
- 238000011469 lymphodepleting chemotherapy Methods 0.000 description 1
- 231100001023 lymphopenia Toxicity 0.000 description 1
- 230000005741 malignant process Effects 0.000 description 1
- 229950008001 matuzumab Drugs 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- QCAWEPFNJXQPAN-UHFFFAOYSA-N methoxyfenozide Chemical compound COC1=CC=CC(C(=O)NN(C(=O)C=2C=C(C)C=C(C)C=2)C(C)(C)C)=C1C QCAWEPFNJXQPAN-UHFFFAOYSA-N 0.000 description 1
- 108091007426 microRNA precursor Proteins 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000007193 modulation by symbiont of host erythrocyte aggregation Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000004682 monohydrates Chemical class 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000007498 myristoylation Effects 0.000 description 1
- 102000042628 natural cytotoxicity receptor (NCR) family Human genes 0.000 description 1
- 108091053394 natural cytotoxicity receptor (NCR) family Proteins 0.000 description 1
- 229960000513 necitumumab Drugs 0.000 description 1
- 210000005044 neurofilament Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000009635 nitrosylation Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229960001972 panitumumab Drugs 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- UTIQDNPUHSAVDN-UHFFFAOYSA-N peridinin Natural products CC(=O)OC1CC(C)(C)C(=C=CC(=CC=CC=CC=C2/OC(=O)C(=C2)C=CC34OC3(C)CC(O)CC4(C)C)C)C(C)(O)C1 UTIQDNPUHSAVDN-UHFFFAOYSA-N 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229940085991 phosphate ion Drugs 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 108010031345 placental alkaline phosphatase Proteins 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 208000031334 polycystic lipomembranous osteodysplasia with sclerosing leukoencephaly Diseases 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 230000013823 prenylation Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 108010079891 prostein Proteins 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 108010014186 ras Proteins Proteins 0.000 description 1
- 102000016914 ras Proteins Human genes 0.000 description 1
- 108700015048 receptor decoy activity proteins Proteins 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 102200137588 rs121913614 Human genes 0.000 description 1
- QSHGUCSTWRSQAF-FJSLEGQWSA-N s-peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C1=CC=C(OS(O)(=O)=O)C=C1 QSHGUCSTWRSQAF-FJSLEGQWSA-N 0.000 description 1
- 108091008601 sVEGFR Proteins 0.000 description 1
- 229910052594 sapphire Inorganic materials 0.000 description 1
- 239000010980 sapphire Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000003319 supportive effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 101150047061 tag-72 gene Proteins 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 238000013169 thromboelastometry Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 239000011031 topaz Substances 0.000 description 1
- 229910052853 topaz Inorganic materials 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 108010072106 tumstatin (74-98) Proteins 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 230000000982 vasogenic effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 108010079528 visinin Proteins 0.000 description 1
- 229940019333 vitamin k antagonists Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000004572 zinc-binding Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4631—Chimeric Antigen Receptors [CAR]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
- A61K39/464411—Immunoglobulin superfamily
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16041—Use of virus, viral particle or viral elements as a vector
- C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- This disclosure relates to the field of immunology, or more specifically, to the genetic modification of T lymphocytes or other immune cells, and methods of controlling proliferation of such cells.
- Lymphocytes isolated from a subject can be activated in vitro and genetically modified to express synthetic proteins that enable redirected engagement with other cells and
- TCRs recombinant T cell receptors
- CARs chimeric antigen receptors
- One CAR that is currently used is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a
- transmembrane domain and one or more intracellular signaling domains encoded by a replication incompetent recombinant retrovirus.
- the intracellular signaling portion of the CAR can initiate an activation-related response in an immune cell and release of cytolytic molecules to induce target cell death.
- CAR therapies further cannot be controlled for propagation rate in vivo once introduced into the body, nor safely directed towards targets that are also expressed outside the tumor.
- CAR therapies today are typically infused from cells expanded ex vivo from 12 to 28 days using doses from 1 x 10 5 to 1 x 10 8 cells/kg and are directed towards targets, for example tumor targets, for which off tumor on target toxicity is generally acceptable.
- kits that help overcome issues related to the effectiveness and safety of methods for transducing and/or genetically modifying lymphocytes such as T cells and/or NK cells. Certain embodiments of such methods are useful for performing adoptive cell therapy with these cells. Accordingly, in some aspects, provided herein are methods, compositions, and kits for genetically modifying lymphocytes, especially T cell and/or NK cells, and/or for regulating the activity of transduced and/or genetically modified T cells and/or NK cells.
- T cells and/or NK cells that express recombinant T cell receptors (TCRs), chimeric antigen receptors (CARs), and in illustrative embodiments microenvironment restricted biologic (“MRB”) CARs.
- Transduced and/or genetically modified T cells and/or NK cells that are produced by and/or used in methods provided herein, include functionality and combinations of functionality, in illustrative embodiments delivered from retroviral (e.g. lentiviral) genomes via retroviral (e.g. lentiviral) particles, that provide improved features for such cells and for methods that utilize such cells, such as research methods, commercial production methods, and adoptive cellular therapy. For example, such cells can be produced in less time ex vivo, and that have improved growth properties that can be better regulated.
- methods are provided for transducing and/or genetically modifying lymphocytes such as T cells and/or NK cells, and in illustrative embodiments, ex vivo methods for transducing and/or genetically modifying resting T cells and/or NK cells.
- Some of these aspects can be performed much more quickly than previous methods, which can facilitate more efficient research, more effective commercial production, and improved methods of patient care.
- Methods, compositions, and kits provided herein, can be used as research tools, in commercial production, and in adoptive cellular therapy with transduced and/or genetically modified T cells and/or NK cells expressing a TCR or a CAR.
- lymphocytes such as T cells and/or NK cells
- methods, and associated uses and compositions are provide herein that include transduction reactions of enriched PBMCs or transduction reactions without prior PBMC enrichment, such as in whole blood that are simplified and quicker methods for performing ex- vivo cell processing, for example for CAR-T therapy.
- Such methods require less specialized
- RNAs include certain target inhibitory RNAs, polypeptide lymphoproliferative elements, and pseudotyping elements that can be optionally be combined with any other aspects provided herein to provide powerful methods, uses, and compositions for driving expansion of lymphocytes, especially T cells and/or NK cells in vitro, ex vivo, and in vivo.
- FIGs. 1A-1B are flowcharts of non-limiting exemplary cell processing workflows.
- FIG. 1A is a flow chart of a process that uses a system with PBMC enrichment before the contacting of T cells and NK cells in the PBMCs with retroviral particles.
- FIG. IB is a flow chart of a process in which no blood cell fractionation or enrichment is performed before T cells and NK cells in the whole blood are contacted with retroviral particles, and a PBMC enrichment is performed after transduction.
- FIG. 2 is a diagram of a non-limiting exemplary leukodepletion filter assembly (200) with associated blood processing bags, tubes, valves, and filter enclosure (210) comprising a leukodepletion filter set.
- FIGs. 3A and 3B show histograms of experimental results with different pseudotyping elements.
- FIG. 3A shows a histogram of the total number of live cells per well on Day 6 following transduction.
- FIG. 3B shows a histogram of the percent of CD3+ cells transduced as measured by eTAG expression.
- FIGs. 4A and 4B show histograms of experimental results with transduction reaction mixtures that include whole blood, lentiviral particles, and anti-coagulants EDTA or heparin, without PBMC enrichment before the reaction mixture was formed.
- the process was performed by contacting whole blood for 4 hours with the indicated lentiviral particle F1-3-23G or F1-3-23GU followed by a density gradient centrifugation-based PBMC enrichment procedure.
- FIG. 4A shows a histogram of the absolute cell number per uL of the live lymphocyte population.
- FIG. 4B shows a histogram of the percentage (%) CD3+eTag+ cells in the live lymphocyte population at Day 6 post-transduction.
- FIG. 5 is a histogram showing the CD3+FLAG+ cell number per pi of culture at Day 6 after transduction of unstimulated PBMCs by the different recombinant lentiviral particles at an MOI of lfor the indicated period of time.
- Fl-3-253 encoded an anti-CD19 CAR
- Fl-3-451 encoded a CLE in addition to the same CAR.
- the lentiviral particles were pseudotyped with VSV-G [VSV-G] and optionally displayed UCFITlScFvFc-GPI [VSV-G + U] as indicated.
- Samples were treated with dapivirine, an inhibitor of reverse transcription (RT inb) or dolutegravir, an inhibitor to integration (INT Inb), as indicated.
- FIG. 6 is a schematic of a non-limiting, exemplary transgene expression cassette containing a polynucleotide sequence encoding a CAR and a candidate CLE of Libraries analyzed in Example 6.
- chimeric antigen receptor or “CAR” or “CARs” refers to engineered receptors, which graft an antigen specificity onto cells, for example T cells, NK cells, macrophages, and stem cells.
- the CARs of the invention include at least one antigen-specific targeting region (ASTR), a transmembrane domain (TM), and an intracellular activating domain (I AD) and can include a stalk, and one or more co-stimulatory domains (CSDs).
- ASTR antigen-specific targeting region
- TM transmembrane domain
- I AD intracellular activating domain
- the CAR is a bispecific CAR, which is specific to two different antigens or epitopes. After the ASTR binds specifically to a target antigen, the IAD activates intracellular signaling.
- the IAD can redirect T cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of antibodies.
- the non-MHC-restricted antigen recognition gives T cells expressing the CAR the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.
- CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
- the term "microenvironment” means any portion or region of a tissue or body that has constant or temporal, physical, or chemical differences from other regions of the tissue or regions of the body.
- a tumor microenvironment refers to the environment in which a tumor exists, which is the non-cellular area within the tumor and the area directly outside the tumorous tissue but does not pertain to the intracellular compartment of the cancer cell itself.
- the tumor microenvironment can refer to any and all conditions of the tumor milieu including conditions that create a structural and or functional environment for the malignant process to survive and/or expand and/or spread.
- the tumor microenvironment can include alterations in conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, levels of adenosine, levels of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and/or presence of oxidants, reductants, or co-factors, as well as other conditions a skilled artisan will understand.
- conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, levels of adenosine, levels of R-2-hydroxyglutar
- polynucleotide and “nucleic acid” refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
- this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
- antibody includes polyclonal and monoclonal antibodies, including intact antibodies and fragments of antibodies which retain specific binding to antigen.
- the antibody fragments can be, but are not limited to, fragment antigen binding (Fab) fragments, Fab' fragments,
- F(ab')2 fragments Fv fragments, Fab'-SFl fragments, (Fab')2 Fv fragments, Fd fragments, recombinant IgG (rlgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), divalent scFv's, trivalent scFv's, and single domain antibody fragments (e.g., sdAb, sdFv, nanobody).
- scFv single-chain variable fragments
- divalent scFv's divalent scFv's
- trivalent scFv's single domain antibody fragments
- the term includes genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, single-chain antibodies, fully human antibodies, humanized antibodies, fusion proteins including an antigen-specific targeting region of an antibody and a non antibody protein, heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv's, and tandem tri-scFv's.
- antibody should be understood to include functional antibody fragments thereof.
- the term also includes intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub classes thereof, IgM, IgE, IgA, and IgD.
- antibody fragment includes a portion of an intact antibody, for example, the antigen binding or variable region of an intact antibody.
- antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.
- the terms "single-chain Fv,” “scFv,” or “sFv” antibody fragments include the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
- the Fv polypeptide further includes a polypeptide linker or spacer between the V H and V L domains, which enables the sFv to form the desired structure for antigen binding.
- VF1 and VL domains refer to VF1 and VL domains that have been isolated from a host without further molecular evolution to change their affinities when generated in an scFv format under specific conditions such as those disclosed in US patent 8709755 B2 and application WO/2017/033331A1.
- affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd).
- Kd dissociation constant
- Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20- fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least lOO-fold greater, or at least 1000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences.
- Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more.
- nM nanomolar
- pM picomolar
- fM femtomolar
- the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.
- the terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and/or antigen-binding fragments.
- binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
- Non-specific binding would refer to binding with an affinity of less than about 10 7 M, e.g., binding with an affinity of 10 6 M, 10 5 M, 10 4 M, etc.
- cell surface expression system or “cell surface display system” refers to the display or expression of a protein or portion thereof on the surface of a cell.
- a cell is generated that expresses proteins of interest fused to a cell-surface protein.
- a protein is expressed as a fusion protein with a transmembrane domain.
- the term “element” includes polypeptides, including fusions of polypeptides, regions of polypeptides, and functional mutants or fragments thereof and polynucleotides, including microRNAs and shRNAs, and functional mutants or fragments thereof.
- region is any segment of a polypeptide or polynucleotide.
- a "domain” is a region of a polypeptide or polynucleotide with a functional and/or structural property.
- the terms "stalk” or “stalk domain” refer to a flexible polypeptide connector region providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides.
- a stalk can be derived from a hinge or hinge region of an
- immunoglobulin e.g., IgGl
- IgGl immunoglobulin
- Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions.
- S-S inter-heavy chain disulfide
- the stalk may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region, as disclosed in U.S. Pat. No. 5,677,425.
- the stalk can include a complete hinge region derived from an antibody of any class or subclass.
- the stalk can also include regions derived from CD8, CD28, or other receptors that provide a similar function in providing flexibility and spacing to flanking regions.
- isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
- a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
- Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
- a“polypeptide” is a single chain of amino acid residues linked by peptide bonds. A polypeptide does not fold into a fixed structure nor does it have any posttranslational modification.
- a “protein” is a polypeptide that folds into a fixed structure.“Polypeptides” and“proteins” are used interchangeably herein.
- a polypeptide may be“purified” to remove contaminant components of a polypeptide’s natural environment, e.g. materials that would interfere with diagnostic or therapeutic uses for the polypeptide such as, for example, enzymes, hormones, and other proteinaceous or
- a polypeptide can be purified (1) to greater than 90%, greater than 95%, or greater than 98%, by weight of antibody as determined by the Lowry method, for example, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions using Coomassie blue or silver stain.
- SDS-PAGE sodium dodecyl sulfate- polyacrylamide gel electrophoresis
- immune cells generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow.
- HSC hematopoietic stem cells
- Immune cells includes, e.g., lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).
- T cell includes all types of immune cells expressing CD3 including T-helper cells (CD4 + cells), cytotoxic T cells (CD8 + cells), T-regulatory cells (Treg) and gamma-delta T cells.
- a "cytotoxic cell” includes CD8 + T cells, natural -killer (NK) cells, NK-T cells, gd T cells, a subpopulation of CD4 + cells, and neutrophils, which are cells capable of mediating cytotoxicity responses.
- stem cell generally includes pluripotent or multipotent stem cells.
- stem cells includes, e.g., embryonic stem cells (ES); mesenchymal stem cells (MSC); induced- pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSC); bone marrow derived cells, etc.).
- ES embryonic stem cells
- MSC mesenchymal stem cells
- iPS induced- pluripotent stem cells
- HSC hematopoietic stem cells
- bone marrow derived cells etc.
- treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
- the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
- Treatment covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
- the terms “individual”, “subject”, “host”, and “patient” refer to a mammal, including, but not limited to, humans, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.
- murines e.g., rats, mice
- lagomorphs e.g., rabbits
- non human primates humans
- canines felines
- ungulates e.g., equines, bovines, ovines, porcines, caprines
- the terms “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease.
- the “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.
- the term “evolution” or “evolving” refers to using one or more methods of mutagenesis to generate a different polynucleotide encoding a different polypeptide, which is itself an improved biological molecule and/or contributes to the generation of another improved biological molecule.
- Physiological or "normal” or “normal physiological” conditions are conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, levels of adenosine, levels of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and/or presence of oxidants, reductants, or co-factors, as well as other conditions, that would be considered within a normal range at the site of administration, or at the tissue or organ at the site of action, to a subject.
- a“genetically modified cell” is a cell that contain an exogenous nucleic acid(s) regardless of whether the exogenous nucleic acid(s) is integrated into the genome of the cell.
- a“transduced cell” is a cell that contains an exogenous nucleic acid(s) that is integrated into the genome of the cell.
- A“polypeptide” as used herein can include part of or an entire protein molecule as well as any posttranslational or other modifications.
- a pseudotyping element as used herein can include a "binding polypeptide” that includes one or more polypeptides, typically glycoproteins, that identify and bind the target host cell, and one or more "fusogenic polypeptides” that mediate fusion of the retroviral and target host cell membranes, thereby allowing a retroviral genome to enter the target host cell.
- The“binding polypeptide” as used herein can also be referred to as a“T cell and/or NK cell binding polypeptide” or a“target engagement element,” and the“fusogenic polypeptide” can also be referred to as a“fusogenic element”.
- A“resting” lymphocyte such as for example, a resting T cell, is a lymphocyte in the GO stage of the cell cycle that does not express activation markers such as Ki-67. Resting lymphocytes can include naive T cells that have never encountered specific antigen and memory T cells that have been altered by a previous encounter with an antigen. A“resting” lymphocyte can also be referred to as a“quiescent” lymphocyte.
- lymphodepletion involves methods that reduce the number of lymphocytes in a subject, for example by administration of a lymphodepletion agent. Lymphodepletion can also be attained by partial body or whole body fractioned radiation therapy.
- a lymphodepletion agent can be a chemical compound or composition capable of decreasing the number of functional lymphocytes in a mammal when administered to the mammal.
- One example of such an agent is one or more chemotherapeutic agents.
- Such agents and dosages are known, and can be selected by a treating physician depending on the subject to be treated.
- lymphodepletion agents include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin diftitox, or combinations thereof.
- RNA interference is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted RNA molecules.
- the RNA target may be mRNA, or it may be any other RNA susceptible to functional inhibition by RNAi.
- an“inhibitory RNA molecule” refers to an RNA molecule whose presence within a cell results in RNAi and leads to reduced expression of a transcript to which the inhibitory RNA molecule is targeted.
- An inhibitory RNA molecule as used herein has a 5’ stem and a 3’ stem that is capable of forming an RNA duplex.
- the inhibitory RNA molecule can be, for example, a miRNA (either endogenous or artificial) or a shRNA, a precursor of a miRNA (i.e. a Pri-miRNA or Pre-miRNA) or shRNA, or a dsRNA that is either transcribed or introduced directly as an isolated nucleic acid, to a cell or subject.
- a miRNA either endogenous or artificial
- a shRNA a precursor of a miRNA (i.e. a Pri-miRNA or Pre-miRNA) or shRNA
- a dsRNA that is either transcribed or introduced directly as an isolated nucleic acid, to a cell or subject.
- double stranded RNA or“dsRNA” or“RNA duplex” refers to RNA molecules that are comprised of two strands. Double-stranded molecules include those comprised of two RNA strands that hybridize to form the duplex RNA structure or a single RNA strand that doubles back on itself to form a duplex structure. Most, but not necessarily all of the bases in the duplex regions are base- paired. The duplex region comprises a sequence complementary to a target RNA.
- the sequence complementary to a target RNA is an antisense sequence, and is frequently from 18 to 29, from 19 to 29, from 19 to 21, or from 25 to 28 nucleotides long, or in some embodiments between 18, 19, 20, 21, 22, 23, 24, 25 on the low end and 21, 22, 23, 24, 25, 26, 27, 28 29, or 30 on the high end, where a given range always has a low end lower than a high end.
- Such structures typically include a 5’ stem, a loop, and a 3’ stem connected by a loop which is contiguous with each stem and which is not part of the duplex.
- the loop comprises, in certain embodiments, at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
- the loop comprises from 2 to 40, from 3 to 40, from 3 to 21, or from 19 to 21 nucleotides, or in some embodiments between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 on the low end and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 on the high end, where a given range always has a low end lower than a high end.
- microRNA flanking sequence refers to nucleotide sequences including microRNA processing elements.
- MicroRNA processing elements are the minimal nucleic acid sequences which contribute to the production of mature microRNA from precursor microRNA. Often these elements are located within a 40 nucleotide sequence that flanks a microRNA stem-loop structure. In some instances the microRNA processing elements are found within a stretch of nucleotide sequences of between 5 and 4,000 nucleotides in length that flank a microRNA stem-loop structure.
- linker when used in reference to a multiplex inhibitory RNA molecule refers to a connecting means that joins two inhibitory RNA molecules.
- a“recombinant retrovirus” refers to a non-replicable, or“replication
- retrovirus incompetent
- retroviral particle a retrovirus
- retrovirus/retroviral particle can be any type of retroviral particle including, for example, gamma retrovirus, and in illustrative embodiments, lentivirus.
- retroviral particles for example lenti viral particles
- such retroviral particles typically are formed in packaging cells by transfecting the packing cells with plasmids that include packaging components such as Gag, Pol and Rev, an envelope or pseudotyping plasmid that encodes a pseudotyping element, and a transfer, genomic, or retroviral (e.g. lentiviral) expression vector, which is typically a plasmid on which a gene(s) or other coding sequence of interest is encoded.
- a retroviral (e.g. lentiviral) expression vector includes sequences (e.g. a 5’ LTR and a 3’ LTR flanking e.g. a psi packaging element and a target heterologous coding sequence) that promote expression and packaging after transfection into a cell.
- sequences e.g. a 5’ LTR and a 3’ LTR flanking e.g. a psi packaging element and a target heterologous coding sequence
- lentivirus and“lentiviral particle” are used interchangeably herein.
- A“framework” of a miRNA consists of“5’ microRNA flanking sequence” and/or“3’ microRNA flanking sequence” surrounding a miRNA and, in some cases, a loop sequence that separates the stems of a stem-loop structure in a miRNA.
- the“framework” is derived from naturally occurring miRNAs, such as, for example, miR-155.
- the terms“5’ microRNA flanking sequence” and“5’ arm” are used interchangeably herein.
- the terms“3’ microRNA flanking sequence” and“3’ arm” are used interchangeably herein.
- miRNA precursor refers to an RNA molecule of any length which can be enzymatically processed into an miRNA, such as a primary RNA transcript, a pri-miRNA, or a pre- rniRNA.
- construct refers to an isolated polypeptide or an isolated
- polynucleotide encoding a polypeptide.
- a polynucleotide construct can encode a polypeptide, for example, a lymphoproliferative element.
- a skilled artisan will understand whether a construct refers to an isolated polynucleotide or an isolated polypeptide depending on the context.
- MOI Multiplicity of Infection ratio where the MOI is equal to the ratio of the number of virus particles used for infection per number of cells. Functional titering of the number of virus particles can be performed using FACS and reporter expression.
- PBMCs peripheral blood mononuclear cells
- lymphocytes e.g. T cells, NK cells, and B cells
- monocytes e.g. T cells, NK cells, and B cells
- red blood cells e.g. platelets and granulocytes (i.e. neutrophils, eosinophils, and basophils).
- the present disclosure overcomes prior art challenges by providing improved methods and compositions for genetically modifying lymphocytes, for example NK cells and in illustrative embodiments, T cells.
- Some of the methods and compositions herein provide simplified and more rapid processes for transducing lymphocytes that avoid some steps that require specialized devices.
- the methods provide better control of post-transduction processing since any such processing is done ex vivo, which therefore allows the option of removing various unwanted cells.
- the methods provide an important step toward democratization of cell therapy methods.
- compositions for genetically modifying lymphocytes are performed in less time than prior methods. Furthermore, compositions that have many uses, including their use in these improved methods, are provided. Some of these compositions are genetically modified lymphocytes that have improved proliferative and survival qualities, including in in vitro culturing, for example in the absence of growth factors. Such genetically modified lymphocytes will have utility for example, as research tools to better understand factors that influence T cell proliferation and survival, and for commercial production, for example for the production of certain factors, such as growth factors and immunomodulatory agents, that can be harvested and tested or used in commercial products.
- a method of transducing and/or genetically modifying a lymphocyte such as a (typically a population of) peripheral blood mononuclear cell (PBMC), typically a T cell and/or an NK cell, and in certain illustrative embodiments a resting T cell and/or resting NK cell, that includes contacting the lymphocyte with a (typically a population of) replication incompetent recombinant retroviral particle, wherein the replication incompetent recombinant retroviral particle typically comprises a pseudotyping element on its surface, wherein said contacting (and incubation under contacting conditions) facilitates membrane association, membrane fusion, and optionally transduction of the resting T cell and/or NK cell by the replication incompetent recombinant retroviral particle, thereby producing the genetically modified T cell and/or NK cell.
- PBMC peripheral blood mononuclear cell
- NK cell typically comprises a pseudotyping element on its surface
- pre-activation of the T cell and/or NK cell is not required, and an activation element, which can be any activation element provided herein, is present in a reaction mixture in which the contacting takes place.
- the activation element is present on a surface of the replication incompetent recombinant retroviral particle.
- the activation element is anti-CD3, such as anti-CD3 scFv, or anti-CD3 scFvFc.
- the contacting step and an optional incubation thereafter which includes a step to remove retroviral particles not associated with cells, in a method provided herein of transducing and/or genetically modifying a PBMC or a lymphocyte, typically a T cell and/or an NK cell, can be performed (or can occur), for 72, 48, or 24 hours or less or for any of the contacting time ranges provided herein.
- the contacting is performed for less than 2 hours, less than 1 hour, less than 30 minutes or less than 15 minutes, but in each case there is at least an initial contacting step in which retroviral particles and cells are brought into contact in suspension in a transduction reaction mixture.
- This contacting typically includes an initial step in which retroviral particles that are not associated with a cell of the reaction mixture are separated from the cells, which are then further processed.
- Such suspension can include allowing cells and retroviral particles to settle or causing such settling through application of a force, such as a centrifugal force, to the bottom of a vessel or chamber, as discussed in further detail herein.
- a force such as a centrifugal force
- such g force is lower than the g forces used successfully in spinoculation procedures.
- the contacting is performed for between an initial contacting step only (without any further incubating in the reaction mixture including the retroviral particles free in suspension and cells in suspension) without any further incubation in the reaction mixture, or a 5 minute, 10 minute, 15 minute, 30 minute, or 1 hour incubation in the reaction mixture, which can be a step of separating free retroviral particles in a reaction mixture from those associated with cells.
- a T cell can include any of the embodiments of replication incompetent recombinant retroviral particles provided herein, including those that include one or more lymphoproliferative element, CAR, pseudotyping element, riboswitch, activation element, membrane-bound cyto
- the retroviral particle is a lentiviral particle.
- a method for genetically modifying and/or transducing a PBMC or a lymphocyte, such as a T cell and/or NK cell can be performed in vitro or ex vivo.
- a skilled artisan will recognize that details provided herein for transducing and/or genetically modifying PBMCs or lymphocytes, such as T cell(s) and/or NK cell(s) can apply to any aspect that includes such step(s).
- the cell is genetically modified and/or transduced without requiring prior activation or stimulation, whether in vivo, in vitro, or ex vivo.
- the cell is activated during the contacting and is not activated at all or for more than 15 minutes, 30 minutes, 1, 2, 4, or 8 hours before the contacting.
- activation by elements that are not present on the retroviral particle surface is not required for genetically modifying and/or transducing the cell. Accordingly, such activation or stimulation elements are not required other than on the retroviral particle, before, during, or after the contacting.
- these illustrative embodiments that do not require pre-activation or stimulation provide the ability to rapidly perform in vitro experiments aimed at better understanding T cells and the biologicals mechanisms, therein. Furthermore, such methods provide for much more efficient commercial production of biological products produced using PBMCs, lymphocytes, T cells, or NK cells, and development of such commercial production methods. Finally, such methods provide for more rapid ex vivo processing of PBMCs for adoptive cell therapy, fundamentally simplifying the delivery of such therapies, for example by providing point of care methods. COMPOSITIONS AND METHODS FOR TRANSDUCING LYMPHOCYTES IN WHOLE BLOODLYMPHOCYTES IN WHOLE BLOOD
- PBMCs peripheral blood mononuclear cells
- lymphocytes typically T cells and/or NK cells
- resting T cells and/or resting NK cells in a reaction mixture comprising blood, or a component thereof, and/or an anticoagulant, that includes contacting the lymphocytes with replication incompetent recombinant retroviral particles in the reaction mixture that itself represents a separate aspect provided herein
- the reaction mixture in illustrative embodiments comprises the lymphocytes and the replication incompetent recombinant retroviral particles, a T cell activation element and one or more additional blood components set out below that in illustrative embodiments are present because the reaction mixture comprises at least 10% whole blood, wherein the replication incompetent recombinant retroviral particles typically comprises a pseudotyping element on its surface.
- the contacting facilitates association of the lymphocytes with the replication incompetent recombinant retroviral particles, wherein the recombinant retroviral particles genetically modify and/or transduce the lymphocytes.
- the reaction mixture of this aspect comprises at least 10% whole blood (e.g. at least 10%, 20%, 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% whole blood) and optionally an effective amount of an anticoagulant, or the reaction mixture further comprises at least one additional blood or blood preparation component that is not a PBMC, for example the reaction mixture comprises an effective amount of an anti-coagulant and one or more blood preparation component that is not a PBMC.
- such blood or blood preparation component that is not a PBMC is one or more (e.g. at least one, two, three, four, or five) or all of the following additional components:
- erythrocytes wherein the erythrocytes comprise between 1 and 60% of the volume of the reaction mixture;
- neutrophils comprise at least 10% of the white blood cells in the reaction mixture, or wherein the reaction mixture comprises at least 10% as many neutrophils as T cells;
- basophils wherein the basophils comprise at least 0.05% of the white blood cells in the reaction mixture;
- reaction mixture comprises at least 0.1% of the white blood cells in the reaction mixture
- plasma wherein the plasma comprises at least 1% of the volume of the reaction mixture; and f) an anti-coagulant (such blood or blood preparation components a-f above referred to herein as (“Noteworthy Non- PBMC Blood or Blood Preparation Components”)).
- an anti-coagulant such blood or blood preparation components a-f above referred to herein as (“Noteworthy Non- PBMC Blood or Blood Preparation Components”)
- the one or more additional blood components are present in certain illustrative embodiments of the reaction mixture (including related use, genetically modified T cell or NK cell, or method for genetically modifying T cells and/or NK cells aspects provided herein) because in these illustrative embodiments the reaction mixture comprises at least 10% whole blood, and in certain illustrative embodiments, at least 25%, 50%, 75%, 90%, or 95% whole blood, or for example between 25% and 95% whole blood.
- such reaction mixtures are formed by combining whole blood with an anticoagulant (for example by collecting whole blood into a blood collection tube comprising an anti-coagulant), and adding a solution of recombinant retroviruses to the blood with anticoagulant.
- the reaction mixture comprises an anti-coagulant as set out in more detail herein.
- the whole blood is not, or does not comprise, cord blood.
- the reaction mixture in these aspects typically does not include a PBMC enrichment procedure before the transduction reaction mixture is formed.
- typically such reaction mixtures include additional components listed in a)-f) above, which are not PBMCs.
- the reaction mixture comprises all of the additional components listed in a) to e) above, because the reaction mixture comprises substantially whole blood, or whole blood.“Substantially whole blood” is blood that was isolated from an individual(s), has not been subjected to a PBMC enrichment procedure, and is diluted by less than 50% with other solutions. For example, this dilution can be from addition of an anti-coagulant as well as addition of a volume of fluid comprising retroviral particles. Further reaction mixture embodiments for methods and compositions that relate to transducing lymphocytes in whole blood, are provided herein.
- kits for genetically modifying lymphocytes in illustrative embodiments T cells and/or NK cells of a subject, wherein the use of the kit comprises the above method of transducing and/or genetically modifying lymphocytes in whole blood.
- kits for administering genetically modified lymphocytes to a subject, wherein the genetically modified lymphocytes are produced by the above method of transducing and/or genetically modifying lymphocytes in whole blood.
- methods for transducing and/or genetically modifying lymphocytes in whole blood uses of such a method in the manufacture of a kit, reaction mixtures formed in such a method, genetically modified lymphocytes made by such a method, and methods for administering a genetically modified lymphocyte made by such a method, are referred to herein as“composition and method aspects for transducing lymphocytes in whole blood.”
- composition and method aspects for transducing lymphocytes in whole blood are referred to herein as“composition and method aspects for transducing lymphocytes in whole blood.”
- embodiments of any of the composition and method aspects for transducing lymphocytes in whole blood can include any of the embodiments of replication incompetent recombinant retroviral particles provided herein, including those that include one or more polypeptide lymphoproliferative element, inhibitory RNA, CAR, pseudotyping element, riboswitch, activation element, membrane-bound cytokine, miRNA, Kozak-type sequence, WPRE element, triple stop codon, and/or other element disclosed herein, and can be combined with methods herein for producing retroviral particles using a packaging cell.
- the pseudotyping element is typically capable of binding lymphocytes (e.g. T cells and/or NK cells) in illustrative embodiments resting T cells and/or resting NK cells and facilitating membrane fusion on its own or in conjunction with other protein(s) of the replication incompetent recombinant retroviral particles.
- the retroviral particle is a lentiviral particle.
- Anticoagulants are included in reaction mixtures for certain embodiments of the composition and method aspects for transducing lymphocytes in whole blood provided herein.
- blood is collected with the anti-coagulant present in the collection vessel (e.g. tube or bag), for example using standard blood collection protocols known in the art.
- Anticoagulants that can be used in composition and method aspects for transducing lymphocytes in whole blood provided herein include compounds or biologies that block or limit the thrombin blood clotting cascade.
- the anti-coagulants include: metal chelating agents, preferably calcium ion chelating agents, such as citrate (e.g.
- citrate containing free citrate ion
- solutions of citrate that contain one or more components such as citric acid, sodium citrate, phosphate, adenine and mono or polysaccharides, for example dextrose, oxalate, and EDTA; heparin and heparin analogues, such as unfractionated heparin, low molecular weight heparins, and other synthetic saccharides; and vitamin K antagonists such as coumarins.
- Exemplary citrate compositions include: acid citrate dextrose (ACD) (also called anticoagulant citrate dextrose solution A and solution B (United States Pharmacopeia 26, 2002, pp 158)); and a citrate phosphate dextrose (CPD) solution, which can also be prepared as CPD-A1 as is known in the art.
- ACD acid citrate dextrose
- CPD citrate phosphate dextrose
- the anticoagulant composition may also include phosphate ions or monobasic phosphate ion, adenine, and mono or polysaccharides.
- Such anti-coagulants can be present in a reaction mixture at concentrations that are effective for preventing coagulation of blood (i.e. effective amounts) as known in the art, or at a concentration that is, for example, 2 times, 1.5 times, 1.25 times, 1.2 times, 1.1 times, or 9/10, 4/5, 7/10, 3/5, 1 ⁇ 2, 2/5, 3/10, 1/5, or 1/10 the effective concentration.
- concentrations of many different anticoagulants is known and can be readily determined empirically by analyzing different concentrations for their ability to prevent blood coagulation, which can be physically observed.
- the effective concentration includes the concentration of any commercially available anti-coagulant in a commercially available tube or bag after the anti-coagulant is diluted in the volume of blood intended for the tube or bag.
- the concentration of acid citrate dextrose (ACD) in a reaction mixture in certain embodiments of the composition and method aspects for transducing lymphocytes in whole blood provided herein can be between 0.1 and 5X, or between 0.25 and 2.5X, between 0.5 and 2X, between 0.75 and 1.5X, between 0.8 and 1.2X, between 0.9 and 1.1X, about IX, or IX the concentration of ACD in a commercially available ACD blood collection tube or bag.
- blood can be collected into tubes or bags containing 3.2% (109 mM) sodium citrate (109 mM) at a ratio of 9 parts blood and 1 part anticoagulant.
- the citrate concentration can be between for example, .25% to .4%, or .30% to .35%.
- 15 mls of ACD Solution A are present in a blood bag for collecting 100 mL of blood.
- the ACD before addition of blood contains Citric acid (anhydrous) 7.3 g/F (0.73%), Sodium citrate (dihydrate) 22.0 g/F (2.2 %), and Dextrose (monohydrate) 24.5 g/F [USP] (2.4%).
- the concentration of ACD components in a reaction mixture can be between .05 and 0.1%, or 0.06 and 0.08% Citric acid (anhydrous), 0.17 and 0.27, or 0.20 and 0.24 Sodium citrate (dihydrate), 0.2 and 0.3, or 0.20 and 0.28, or 0.22 and 0.26% Dextrose (monohydrate).
- sodium citrate is used at a concentration of between .001 and .02 M in the reaction mixture.
- heparin is present in the reaction mixtures, for example at a concentration between 0.1 and 5X, or between 0.25 and 2.5X, between 0.5 and 2X, between 0.75 and 1.5X, between 0.8 and 1.2X, between 0.9 and 1.1X, about IX, or IX the concentration of heparin in a commercially available heparin blood collection tube.
- Heparin is a glycosaminoglycan anticoagulant with a molecular weight ranging from 5,000-30,000 daltons.
- heparin is used at a concentration of about 1.5 to 45, 5 to 30, 10 to 20, or 15 USP units/ml of reaction mixture.
- the effective concentration for EDTA, for example as K2EDTA, in the reaction mixtures herein can be between 0.15 and 5 mg/ml, between 1 and 3 mg/ml between 1.5-2.2 mg/ml of blood, or between 1 and 2 mg/ml, or about 1.5 mg/ml.
- the reaction mixtures in composition and method aspects for transducing lymphocytes in whole blood provided herein can include two or more anticoagulants whose combined effective dose prevents coagulation of the blood prior to formation of the reaction mixture and/or of the reaction mixture itself.
- the anti-coagulant can be administered to a subject before blood is collected from the subject for ex vivo transduction, such that coagulation of the blood when it is collected in inhibited, at least partially and at least through a contacting step and optional incubation period thereafter.
- acid citrate dextrose can be administered to the subject at between 80 mg/kg/day and 5 mg/kg/day (mg refer to the mg of citric acid and kg applies to the mammal to be treated).
- Heparin can be delivered for example, at a dose of between 5 units/kg/hr to 30 units/kg/hr.
- composition and method aspects for transducing lymphocytes in whole blood can include at least one additional component selected from one or more of the following components:
- erythrocytes wherein the erythrocytes comprise between 0.1 and 75% of the volume of the reaction mixture
- neutrophils comprise at least 10% of the white blood cells in the reaction mixture, or wherein the reaction mixture comprises at least 10% as many neutrophils as T cells;
- basophils wherein the basophils comprise at least 0.05% of the white blood cells in the reaction mixture
- reaction mixture comprises at least 0.1% of the white blood cells in the reaction mixture
- erythrocytes can comprise between 0.1, 0.5, 1, 5, 10, 25, 35 or 40% of the volume of the reaction mixture on the low end of the range, and between 25, 50, 60, or 75% of the volume of the reaction mixture on the high end of the range.
- erythrocytes comprise between 1 and 60%, between 10 and 60%, between 20 and 60%, between 30 and 60%, between 40 and 60%, between 40 and 50%, between 42 and 48%, between 44 and 46%, about 45% or 45%.
- neutrophils can comprise between 0.1, 0.5, 1, 5, 10, 20, 25, 35 or 40% of the white blood cells of the reaction mixture on the low end of the range, and between 25, 50, 60, 70, 75 and 80% of the white blood cells of the reaction mixture on the high end of the range, for example between 25% and 70%, or between 30% and 60%, or between 40% and 60% of the white blood cells of the reaction mixture.
- more neutrophils are present than T cells and/or NK cells, in reaction mixtures herein.
- eosinophils can comprise between 0.05, 0.1 , 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 % of the white blood cells of the reaction mixture on the low end of the range, and between 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.5, 4, 5, 6, 8 and 10 % of the white blood cells of the reaction mixture on the high end of the range.
- eosinophils comprise between 0.05 and 10.0 %, between 0.1 and 9%, between 0.2 and 8%, between 0.2 and 6%, between 0.5 and 4%, between 0.8 and 4%, or between 1 and 4% of the white blood cells of the reaction mixture.
- basophils can comprise between 0.05, 0.1, 0.2, 0.4, 0.45 and .5 % of the white blood cells of the reaction mixture on the low end of the range, and between 0.8, 0.9, 1.0, 1.1, 1.2, 1.5 and 2.0% of the white blood cells of the reaction mixture on the high end of the range.
- basophils comprise between 0.05 and 1.4%, between 0.1 and 1.4%, between 0.2 and 1.4%, between 0.3 and 1.4%, between 0.4 and 1.4%, between 0.5 and 1.4%, between 0.5 and 1.2%, between 0.5 and 1.1%, or between 0.5 and 1.0 % of the white blood cells of the reaction mixture.
- plasma can comprise between 0.1, 0.5, 1, 5, 10, 25, 35 or 45% of the volume of the reaction mixture on the low end of the range, and between 25, 50, 60, 70 and 80% of the volume of the reaction mixture on the high end of the range.
- plasma comprise between 0.1 and 80%, between 1 and 80%, between 5 and 80%, between 10 and 80%, between 30 and 80%, between 40 and 80%, between 45 and 70%, between 50 and 60%, between 52 and 58%, between 54 and 56%, about 55% or 55% of the reaction mixture.
- platelets can comprise between lxlO 5 , lxlO 6 , lxlO 7 , or lxlO 8 platelets/mL of the reaction mixture on the low end of the range, and between lxlO 9 , lxlO 10 , lxlO 11 , lxlO 12 , 2xl0 13 , or 2xl0 14 platelets /mL of the reaction mixture on the high end of the range.
- platelets comprise between lxlO 5 and lxlO 12 platelets, between lxlO 6 and lxlO 11 platelets, between lxlO 7 and lxlO 10 platelets, between lxlO 8 , and lxl0 9 platelets/mL, or between lxlO 8 and 5xl0 8 platelets/ml of the reaction mixture.
- some embodiments of methods for genetically modifying provided herein do not include a step of collecting blood from a subject.
- some of the methods provided herein include a step where blood is collected (110) from a subject.
- Blood can be collected or obtained from a subject by any suitable method known in the art as discussed in more detail herein.
- the blood can be collected by venipuncture or any other blood collection method by which a sample of blood is collected.
- the volume of blood collected is between 25 ml and 250 ml, for example, between 25ml and 60 ml, between 50 ml and 90 ml, between 75 ml and 125 ml, or between 90 ml and 120 ml, or between 95 and 110 ml.
- lymphocytes are contacted with replication incompetent retroviral particles in a reaction mixture.
- this contacting, and the reaction mixture in which the contacting occurs takes place within a closed cell processing system, as discussed in more detail herein.
- PBMC enrichment(s), washing(s), cell activation, transduction, expansion, collection, and optionally reintroduction occur over days, such as PBMC enrichment(s), washing(s), cell activation, transduction, expansion, collection, and optionally reintroduction.
- the T cell and/or NK cell activation element is associated with surfaces of retroviral particles present in the reaction mixture.
- such a method is used in a point of care autologous cell therapy method.
- PBMC enrichment step/procedure 120
- cell counting, transfer and media addition which takes at least around 45 additional minutes before lymphocytes are contacted with retroviral particles to form a transduction reaction mixture (130A).
- lymphocytes are typically washed away from retroviral particles that remain in suspension (140A), for example using a Sepax, and collected (150A), with the final product typically in an infusion bag for reinfusion or cryopreservation vial for storage (160A).
- 140A retroviral particles that remain in suspension
- 150A the final product typically in an infusion bag for reinfusion or cryopreservation vial for storage
- 160A cryopreservation vial for storage
- centripetal forces or use leukophoresis to enrich PBMCs.
- lymphocytes e.g. T cells and/or NK cells
- lymphocytes can be contacted with replication incompetent retroviral particles in a reaction mixture of whole blood that contains an anti-coagulant, and a significant percentage of the lymphocytes can be genetically modified and transduced.
- effective genetic modification of lymphocytes by recombinant retroviral particles can be carried out in the presence of blood components and blood cells in addition to PBMCs.
- lymphocytes are genetically modified and/or transduced by adding replication incompetent retroviral particles directly to whole blood to form a reaction mixture (130B), and cells in the whole blood are contacted by the replication incompetent retroviral particles for contacting times with optional incubations provided herein.
- a further improved method in this illustrative embodiment thus includes no lymphocyte enrichment steps before lymphocytes in whole blood, typically containing an anti-coagulant, are contacted with retroviral particles.
- This further improved method is typically carried out within a closed cell processing system and can include no or minimal preactivation before lymphocytes are contacted with retroviral particles.
- lymphocytes in whole blood can be contacted with retroviral particles directly in a blood bag.
- lymphocytes that were contacted with retroviral particles are washed and concentrated using a PBMC enrichment procedure (135B), which also reduces neutrophils to facilitate reintroduction into a subject.
- PBMC enrichment procedure (135B)
- no lymphocyte-enriching filtration is performed before cells in whole blood, and typically comprising an anticoagulant, are contacted with recombinant retroviral particles.
- lymphocytes optionally can be washed further away from any retroviral particles that remain (140B), for example using a Sepax, and collected (150B), with the final product typically in an infusion bag for reinfusion or cryopreservation vial for storage (160B).
- the illustrative leukodepletion filtration assembly (200) which in illustrative embodiments is a single -use filtration assembly, comprises a leukocyte depletion media (e.g. filter set) within a filter enclosure (210), that has an inlet (225), and an outlet (226), and a configuration of bags, valves and/or channels/tubes that provide the ability to concentrate, enrich, wash and collect retained white blood cells or nucleated blood cells using perfusion and reverse perfusion (see e.g.
- the leukodepletion filtration assembly (200) is a commercially available FlemaTrate filter (Cook Regenetec, Indianapolis, IN).
- Leukodepletion filtration assemblies can be used, to concentrate total nucleated cells (TNC) including granulocytes, which are removed in PBMC enrichment procedures in a closed cell processing system. Since a filter assembly comprising leukocyte depletion media of EP2602315A1 such as a HemaTrate filter and the illustrative leukodepletion filter assembly of FIG. 2 do not remove granulocytes, they are not considered PBMC enrichment assemblies or filters herein, and methods that incorporate them are not considered PBMC enrichment procedures or steps herein.
- the leukodepletion filter assembly (200) of FIG. 2 is a single-use sterile assembly that includes various tubes and valves, typically needle-free valves, that allow isolation of white blood cells from whole blood and blood cell preparations that include leukocytes, as well as rapid washing and concentrating of white blood cells.
- a blood bag (215) for example a 500 ml PVC bag containing about 120 ml of a transduction/contacting reaction mixture comprising whole blood, an anti-coagulant, and retroviral particles is connected to the assembly (200) at a first assembly opening (217) of an inlet tubing (255), after the reaction mixture is subjected to a contacting step with optional incubation, as disclosed in detail herein.
- Lymphocytes including some T cells and/or NK cells with associated retroviral particles, and some that could be genetically modified at this point, as well as other blood cells and components in the whole blood reaction mixture as well as the anti-coagulant enter the inlet tubing (255) through the first assembly opening (217) by gravitational force when a clamp on the first inlet tubing (255) is released.
- the genetically modified T cells and/or NK cells pass through a inlet valve (247) and a collection valve (245), to enter a filter enclosure (210) through a filter enclosure inlet (225) to contact a leukodepletion IV filter set (e.g. SKU J1472A Jorgensen Labs) within the filter enclosure (210).
- a leukodepletion IV filter set e.g. SKU J1472A Jorgensen Labs
- Nucleated blood cells including leukocytes are retained by the filter, but other blood components pass through the filter and out the filter enclosure outlet (226) into the outlet tubing (256), then through an outlet valve (247) and are collected in a waste collection bag (216), which for example can be a 2L PVC waste collection bag.
- An optional buffer wash step can be performed by switching inlet valve (247) to a wash position.
- a buffer bag (219) for example a 500 ml saline wash bag, is connected to a second assembly opening (218) of inlet tubing (255). The buffer moves into the inlet tubing (255) through the second assembly opening (218) by gravitational force when a clamp on the inlet tubing (255) is released.
- the buffer passes through inlet valve (247) and collection valve (245), to enter filter enclosure (210) through the filter enclosure inlet (225) and passes through the leukodepletion filter set within the filter enclosure (210) to rinse the lymphocytes retained on the filter.
- the buffer moves out the filter enclosure outlet (226) into the outlet tubing (256), then through an outlet valve (247) and is collected in a waste collection bag (216), which can be the same waste collection bag as used to collect reaction mixture components that passed through the filter in the previous step, or a new waste collection bag swapped in place of the first waste collection bag before the buffer was allowed to enter the second assembly opening (218).
- the optional wash step can be optionally performed multiple times by repeating the above process with additional buffer.
- a reverse perfusion process is initiated to move fluid in an opposite direction in the assembly (200) to collect lymphocytes retained on the filter set within the filter enclosure (210).
- Illustrative embodiments of leukodepletion filter assemblies herein are adaptable for reperfusion.
- the outlet valve (247) is switched to a reperfusion position and the collection valve (245) is switched to a collection position.
- a buffer e.g.
- PBS in syringe (266) which for example can be a 25 ml syringe, is passed into outlet tubing (256) by injection using syringe (266).
- the buffer then enters the filter enclosure (210) through the filter enclosure outlet (226) and moves lymphocytes retained on the filter set out of the filter enclosure (210) through the filter enclosure inlet (225) and into the inlet tubing (255).
- lymphocytes including some T cells and/or NK cells with associated retroviral particles, some of which could be genetically modified and/or transduced at this point, are collected in a cell sample collection bag (265), which for example can be a 25 ml cryopreservation bag, after the pass through the collection valve (245).
- kits for genetically modifying NK cells and/or in illustrative embodiments, T cells includes a leukodepletion filtration assembly and any of the replication incompetent retroviral vector embodiments disclosed herein, typically contained in a tube or vial.
- the leukodepletion filtration assembly in such a kit typically includes a leukodepletion filter or a
- kits optionally includes a blood collection bag, in illustrative embodiments comprising an anti-coagulant, a blood processing buffer bag, a blood processing waste collection bag, a blood processing cell sample collection bag, and a sterile syringe.
- the kit includes a T cell activation element as disclosed in detail herein, for example anti-CD3.
- Such activation element can be provided in solution in the tube or vial containing the retroviral particle, or in a separate tube or vial.
- the activation element is an anti-CD3 associated with a surface of the replication incompetent retroviral particle.
- the replication incompetent recombinant retroviral particles in the kit comprise a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and optionally a lymphoproliferative element, according to any of the embodiments provided herein.
- CAR chimeric antigen receptor
- Some embodiments of any methods used in any aspects provided herein can include a step of collecting blood from a subject.
- the blood includes blood components including blood cells such as lymphocytes (e.g. T cells and NK cells) that can be used in methods and compositions provided herein.
- the subject is a human subject afflicted with cancer (i.e. a human cancer subject). It is noteworthy that certain embodiments, do not include such a step.
- blood can be collected or obtained from a subject by any suitable method known in the art as discussed in more detail herein.
- the blood can be collected by venipuncture or any other blood collection method by which a sample of blood is collected.
- the volume of blood collected is between 50 ml and 250 ml, for example, between 75 ml and 125 ml, or between 90 ml and 120 ml, or between 95 and 110 ml.
- the volume of blood collected can be between 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, or 900 ml on the low end of the range and 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300,
- lymphocytes e.g. T cells and/or NK cells
- the volume of blood taken and processed during apheresis can be between 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, or 1.5 total blood volumes of a subject on the low end of the range and 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, 1.5 1.75, 2, 2.25, or 2.5 total blood volumes of a subject on the high end of the range.
- the total blood volume of a human typically ranges from 4.5 to 6 L and thus much more blood is taken and processed during apheresis than if the blood is collected and then lymphocytes therein are genetically modified and/or transduced, as in illustrative embodiments herein.
- lymphocytes are contacted with replication incompetent retroviral particles in a reaction mixture.
- the contacting in any embodiment provided herein, can be performed for example in a chamber of a closed system adapted for processing of blood cells, for example within a blood bag, as discussed in more detail herein.
- the transduction reaction mixture can include one or more buffers, ions, and a culture media.
- lentiviral particles in certain exemplary reaction mixtures provided herein, between 0.1 and 50, 0.5 and 50, 0.5 and 20, 0.5 and 10, 1 and 25, 1 and 15, 1 and 10, 1 and 5, 2 and 15, 2 and 10, 2 and 7, 2 and 3, 3 and 10, 3 and 15, or 5 and 15, multiplicity of infection (MOI); or at least 1 and less than 6, 11, or 51 MOI; or in some embodiments, between 5 and 10 MOI units of replication incompetent recombinant retroviral particles are present.
- the MOI can be at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10 or 15.
- compositions and methods for transducing lymphocytes in blood in certain embodiments higher MOI can be used than in methods wherein PBMCs are isolated and used in the reaction mixtures.
- illustrative embodiments of compositions and methods for transducing lymphocytes in whole blood assuming lxlO 6 PBMCs/ml of blood, can use retroviral particles with an MOI of between 1 and 50, 2 and 25, 2.5 and 20, 2.5 and 10, 4 and 6, or about 5, and in some embodiments between 5 and 20, 5 and 15, 10 and 20, or 10 and 15.
- this contacting, and the reaction mixture in which the contacting occurs takes place within a closed cell processing system, as discussed in more detail herein.
- a packaging cell and in illustrative embodiments a packaging cell line, and in particularly illustrative embodiments a packaging cell provided in certain aspects herein, can be used to produce the replication incompetent recombinant retroviral particles.
- the lymphocytes in the reaction mixture can be PBMCs, or in aspects herein that provide compositions and methods for transducing lymphocytes in whole blood, an anti-coagulant and/or an additional blood component, including additional types of blood cells that are not PBMCs, as discussed herein.
- the reaction mixture can essentially be whole blood, and typically an anti-coagulant, retroviral particles, and a small amount of the solution in which the retroviral particles were delivered to the whole blood.
- T-cells can be present for example, between 10, 20,
- T-cells comprise between 10 and 90%, between 20 and 90%, between 30 and 90%, between 40 and 90%, between 40 and 80%, between 45% to 75% or of the lymphocytes.
- NK cells can be present at between 1, 2, 3, 4, or 5 % of the lymphocytes of the reaction mixture on the low end of the range, and between 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14% of the lymphocytes of the reaction mixture on the high end of the range.
- T-cells comprise between 1 and 14%, between 2 and 14%, between 3 and 14%, between 4 and 14%, between 5 and 14%, between 5 to 13%, between 5 to 12% , between 5 to 11% or , between 5 to 10% of the lymphocytes of the reaction mixture.
- lymphocytes including NK cells and T cells
- lymphocytes can be present at a lower percent of blood cells, and at a lower percentage of white blood cells, in the reaction mixture than methods that involve a PBMC enrichment procedure before forming the reaction mixture.
- more granulocytes or neutrophils are present in the reaction mixture than NK cells or even T cells.
- composition and method aspects for transducing lymphocytes in whole blood typically do not involve a PBMC enrichment step of a blood sample, before lymphocytes from the blood sample are contacted with retroviral particles in the reaction mixtures disclosed herein for those aspects.
- neutrophils/granulocytes are separated away from other blood cells before the cells are contacted with replication incompetent recombinant retroviral particles.
- peripheral blood mononuclear cells (PBMCs) including peripheral blood lymphocytes (PBLs) such as T cell and/or NK cells are isolated away from other components of a blood sample using for example, a PBMC enrichment procedure, before they are combined into a reaction mixture with retroviral particles.
- a PBMC enrichment procedure is a procedure in which PBMCs are enriched at least 25 -fold, and typically at least 50-fold from other blood cell types. For example, it is believed that PBMCs make up less than 1% of blood cells in whole blood. After a PBMC enrichment procedure, at least 30%, and in some examples as many as 70% of cells isolated in the PBMC fraction are PBMCs. It is possible that even higher enrichment of PBMCs is achieved using some PBMC enrichment procedures. Various different PBMC enrichment procedures are known in the art.
- a PBMC enrichment procedure is a ficoll density gradient centrifugation process that separates the main cell populations, such as lymphocytes, monocytes, granulocytes, and red blood cells, throughout a density gradient medium.
- the aqueous medium includes ficoll, a hydrophilic polysaccharide that forms the high density solution. Layering of whole blood over or under a density medium without mixing of the two layers followed by centrifugation will disperse the cells according to their densities with the PBMC fraction forming a thin white layer at the interface between the plasma and the density gradient medium (see e.g. Panda and Ravindran (2013) Isolation of Human PBMCs. BioProtoc. Vol. 3(3)).
- centripetal forces can be used to separate PBMCs from other blood components, in ficoll using the spinning force of a Sepax cell processing system.
- an automated leukapheresis collection system (such as SPECTRA OPTIA® APHERESIS SYSTEM form TERUMO BCT, INC. Lakewood CO 80215, USA) is used to separate the inflow of whole blood from the target PBMC fraction using high-speed centrifugation while typically returning the outflow material, such as plasma, red blood cells, and granulocytes, back to the donor, although this returning would be optional in methods provided herein. Further processing may be necessary to remove residual red blood cells and granulocytes. Both methods include a time intensive purification of the PBMCs, and the leukapheresis method requires the presence and participation of the patient during the PBMC enrichment step.
- PBMCs are isolated using a Sepax or Sepax 2 cell processing system (BioSafe).
- the PBMCs are isolated using a CliniMACS Prodigy cell processor (Miltenyi Biotec).
- an automated apheresis separator is used which takes blood from the subject, passes the blood through an apparatus that sorts out a particular cell type (such as, for example, PBMCs), and returns the remainder back into the subject. Density gradient centrifugation can be performed after apheresis.
- the PBMCs are isolated using a leukodepletion filter assembly.
- magnetic bead activated cell sorting is then used for purifying a specific cell population from PBMCs, such as, for example, PBLs or a subset thereof, according to a cellular phenotype (i.e. positive selection), before they are used in a reaction mixture herein.
- Other methods for purification can also be used, such as, for example, substrate adhesion, which utilizes a substrate that mimics the environment that a T cell encounters during recruitment, to purify T cells before adding them to a reaction mixture, or negative selection can be used, in which unwanted cells are targeted for removal with antibody complexes that target the unwanted cells for removal before a reaction mixture for a contacting step is formed.
- substrate adhesion which utilizes a substrate that mimics the environment that a T cell encounters during recruitment
- negative selection can be used, in which unwanted cells are targeted for removal with antibody complexes that target the unwanted cells for removal before a reaction mixture for a contacting step is formed.
- red blood cell rosetting can be used to remove red blood cells before forming a reaction mixture.
- hematopoietic stem cells can be removed before a contacting step, and thus in these embodiments, are not present during the contacting step.
- an ABC transporter inhibitor and/or substrate is not present before, during, or both before and during the contacting (i.e. not present in the reaction mixture in which contacting takes place) with or without optional incubating, or any step of the method.
- lymphocytes are genetically modified and/or transduced without prior activation or stimulation, and/or without requiring prior activation or stimulation, whether in vivo, in vitro, or ex-vivo and/or furthermore, in some embodiments, without ex vivo or in vitro activation or stimulation after an initial contacting with or without an optional incubation, or without requiring ex vivo or in vitro activation or stimulation after an initial contacting with or without an optional incubation.
- lymphocytes are resting when they are combined with retroviral particles to form a reaction mixture, and typically are resting when they are contacted with retroviral viral particles in a reaction mixture.
- lymphocytes can be contacted in the typically resting state they were in when present in the collected blood in vivo immediately before collection.
- the T cells and/or NK cells consist of between 95 and 100% resting cells (Ki-67 ).
- the T cell and/or NK cells that are contacted by replication incompetent recombinant retroviral particles include between 90, 91, 92, 93, 94, and 95% resting cells on the low end of the range and 96, 97, 98, 99, or 100% resting cells on the high end of the range.
- the T cells and/or NK cells include naive cells.
- the subembodiments in this paragraph are included in composition and method aspects for transducing lymphocytes in whole blood.
- the replication incompetent recombinant retroviral particles can facilitate transduction of the T cells and/or NK cells by the replication incompetent recombinant retroviral particles.
- the replication incompetent recombinant retroviral particles identify and bind to T cells and/or NK cells at which point the retroviral and host cell membranes start to fuse.
- genetic material from the replication incompetent recombinant retroviral particles enters the T cells and/or NK cells at which time the T cells and/or NK cells are“genetically modified” as the phrase is used herein.
- any method for genetically modifying lymphocytes e.g. T cells and/or NK cells
- a method for transducing lymphocytes e.g. T cells and/or NK cells. It is believed that by day 6 in vivo or ex vivo, after contacting is initiated, the vast majority of genetically modified cells have been transduced.
- a transduced T cell and/or NK cell includes progeny of ex vivo transduced cells that retain at least some of the nucleic acids or polynucleotides that are incorporated into the genome of a cell during the ex vivo transduction.
- methods herein that recite“reintroducing” a transduced cell it will be understood that such cell is typically not in a transduced state when it is collected from the blood of a subject.
- Many of the methods provided herein include genetic modification and transduction of T cells and/or NK cells. Methods are known in the art for genetically modifying and transducing T cells and/or NK cells ex vivo with replication incompetent recombinant retroviral particles, such as replication incompetent recombinant lentiviral particles. Methods provided herein, in illustrative embodiments, do not require ex vivo stimulation or activation. Thus, this common step in prior methods can be avoided in the present method, although ex vivo stimulatory molecule(s) such as anti-CD3 and/or anti-CD28 beads, can be present during the contacting and optional incubation thereafter. However, with illustrative methods provided herein, ex vivo stimulation is not required.
- the blood cells such as lymphocytes, and especially T cells and/or NK cells are activated during the contacting or an optional incubation thereafter, and are not activated at all or for more than 15 minutes, 30 minutes, 1, 2, 4, or 8 hours before the contacting.
- activation by elements that are not present on the retroviral particle surface is not required for genetically modifying the lymphocytes. Accordingly, such activation or stimulation elements are not required other than on the retroviral particle, before, during, or after the contacting.
- these illustrative embodiments that do not require pre-activation or stimulation provide the ability to rapidly perform in vitro experiments aimed at better understanding T cells and the biologicals mechanisms, therein. Furthermore, such methods provide for much more efficient commercial production of biological products produced using PBMCs, lymphocytes, T cells, or NK cells, and development of such commercial production methods. Finally, such methods provide for more rapid ex vivo processing of lymphocytes (e.g. NK cells and especially T cells) for adoptive cell therapy, fundamentally simplifying the delivery of such therapies, for example by providing point of care methods.
- lymphocytes e.g. NK cells and especially T cells
- a T cell activation element in illustrative embodiments is present in the reaction mixture where initial contacting of a recombinant retrovirus and lymphocytes occurs.
- T cell activation element can be in solution in the reaction mixture.
- soluble anti-CD3 antibodies can be present in the reaction mixture during the contacting and optional incubation thereafter, at 25-200, 50-150, 75-125, or 100 ng/ml.
- the T cell activation element is associated with the retroviral surface.
- the T cell activation element can be any T cell activation element provided herein.
- the T cell activation element can be anti-CD3, such as anti-CD3 scFv, or anti-CD3 scFvFc.
- the replication incompetent recombinant retroviral particle can further include a T cell activation element, which in further illustrative examples is associated with the external side of the surface of the retrovirus.
- the contacting step of a method for transducing and/or a method for genetically modifying lymphocytes in whole blood typically includes an initial step in which the retroviral particle, typically a population of retroviral particles, are brought into contact with blood cells, typically a population of blood cells that includes an anti-coagulant and/or additional blood components other than PBMCs, that are not present after a PBMC enrichment procedure, while in suspension in a liquid buffer and/or media to form a transduction reaction mixture.
- This contacting can be followed by an optional incubating period in this reaction mixture that includes the retroviral particles and the blood cells comprising lymphocytes (e.g. T cells and/or NK cells) in suspension.
- the reaction mixture can include at least one, two, three, four, five, or all additional blood components as disclosed herein, and in illustrative embodiments includes one or more anticoagulants.
- the transduction reaction mixture in any of the aspects provided herein can be incubated at between 23 and 39 °C, and in some illustrative embodiments at 37 °C, in an optional incubation step after the initial contacting of retroviral particles and lymphocytes.
- the transduction reaction can be carried out at 37-39 °C for faster fusion/transduction.
- the cells and retroviral particles when brought into contact in the transduction reaction mixture can be immediately processed to remove the retroviral particles that remain free in suspension and not associated with cells, from the cells.
- the cells in suspension and retroviral particles whether free in suspension or associated with the cells in suspension can be incubated for various lengths of time, as provided herein for a contacting step in a method provided herein. Before further steps, a wash can be performed, regardless of whether such cells will be studied in vitro, ex vivo or introduced into a subject.
- Illustrative methods are disclosed herein for genetically modifying lymphocytes, especially NK cells and in illustrative embodiments, T cells, that are much shorter and simpler than prior methods. Accordingly, in some embodiments, the contacting step in any method provided herein of transducing and/or genetically modifying a PBMC or a lymphocyte, typically a T cell and/or an NK cell, can be performed (or can occur) for any of the time periods provided in this specification, included, but not limited to those provided in the Exemplary Embodiments section.
- said contacting can be for less than 24 hours, for example, less than 12 hours, less than 8 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes or less than 15 minutes, but in each case there is at least an initial contacting step in which retroviral particles and cells come into contact in suspension in a transduction reaction mixture before retroviral particles that remain in suspension not associated with a cell, are separated from cells and typically discarded, as discussed in further detail herein. It should be noted, but not intending to be limited by theory, that it is believed that contacting begins at the time that retroviral particles and lymphocytes are combined together, typically by adding a solution containing the retroviral particles into a solution containing lymphocytes (e.g. T cells and/or NK cells).
- a solution containing the retroviral particles e.g. T cells and/or NK cells
- the contacting can be performed (or can occur) (where as indicated in general herein the low end of a selected range is less than the high end of the selected range) for between 30 seconds or 1, 2, 5, 10, 15, 30 or 45 minutes, or 1, 2, 3, 4, 5, 6, 7, or 8 hours on the low end of the range, and between 10 minutes, 15 minutes, 30 minutes, or 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72 hours on the high end of the range.
- the contacting step can be performed for between 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, or 30 minutes on the low end of the range and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours on the high end of the range. In some embodiments, the contacting step is performed for between 30 seconds, 1 minute, and 5 minutes on the low end of the range, and 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours on the high end of the range.
- the reaction mixture can be incubated for between 5 minutes and 12 hours, between 5 minutes and 10 hours, between 5 minutes and 8 hours, between 5 minutes and 6 hours, between 5 minutes and 4 hours, between 5 minutes and 2 hours, between 5 minutes and 1 hour, between 5 minutes and 30 minutes, or between 5 minutes and 15 minutes.
- the reaction mixture can be incubated for between 15 minutes and 12 hours, between 15 minutes and 10 hours, between 15 minutes and 8 hours, between 15 minutes and 6 hours, between 15 minutes and 4 hours, between 15 minutes and 2 hours, between 15 minutes and 1 hour, between 15 minutes and 45 minutes, or between 15 minutes and 30 minutes.
- the reaction mixture can be incubated for between 30 minutes and 12 hours, between 30 minutes and 10 hours, between 30 minutes and 8 hours, between 30 minutes and 6 hours, between 30 minutes and 4 hours, between 30 minutes and 2 hours, between 30 minutes and 1 hour, between 30 minutes and 45 minutes. In other embodiments, the reaction mixture can be incubated for between 1 hour and 12 hours, between 1 hour and 8 hours, between 1 hour and 4 hours, or between lhour and 2 hours.
- the contacting is performed for between an initial contacting step only (without any further incubating in the reaction mixture including the retroviral particles free in suspension and cells in suspension) without any further incubation in the reaction mixture, or a 5 minute, 10 minute, 15 minute, 30 minute, or 1 hour incubation in the reaction mixture.
- blood cells or a T cell and/or NK cell-containing fraction thereof in the reaction mixture are separated from retroviral particles that are not associated with such cells.
- a PBMC enrichment procedure e.g. a Ficoll gradient in a Sepax unit
- this can be performed using a PBMC enrichment procedure (e.g. a Ficoll gradient in a Sepax unit), or in certain illustrative embodiments provided herein, by filtering the reaction mixture over a leukocyte depletion filter set assembly, and then collecting the leukocytes, which include T cells and NK cells.
- this can be performed by centrifugation of the reaction mixture at a relative centrifugal force less than 500 g, for example 400g, or between 300 and 490 g, or between 350 and 450 g.
- centrifugation to separate retroviral particles from cells can be performed for example, for between 5 minutes and 15 minutes, or between 5 minutes and 10 minutes.
- g force is typically lower than the g forces used successfully in spinoculation procedures.
- a method provided herein in any aspect does not involve performing a spinoculation.
- spinoculation is included as part of a contacting step.
- when spinoculation is performed there is no additional incubating as part of the contacting as the time of the spinoculation provides the incubation time of the optional incubation discussed above.
- the spinoculation can be performed for example, for 30 minutes to 120 minutes, typically for at least 60 minutes, for example for 60 minutes to 180 minutes, or 60 minutes to 90 minutes.
- the spinoculation is typically performed in a centrifuge with a relative centrifugal force of at least 800g, and more typically at least l,200g, for example between 800g and 2400g, or between 800 g and 1800g, or between 1200 g and 2400 g, or between 1200 g and 1800g.
- a relative centrifugal force typically at least 800g, and more typically at least l,200g, for example between 800g and 2400g, or between 800 g and 1800g, or between 1200 g and 2400 g, or between 1200 g and 1800g.
- such methods typically involve an additional step of resuspending the pelleted cells and retroviral particles, and then removing retroviral particles that are not associated with cells according to steps discussed above when spinoculation is not performed.
- the contacting step including the optional incubation therein, and the spinoculation, in embodiments that include spinoculation, can be performed at between 4C and 42C, or between 20C and 37C. In certain illustrative embodiments, spinoculation is not performed and the contacting and associated optional incubation are carried out at 20-25C for 4 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, or 15 minutes to 2 hours, 15 minutes to 1 hour, or 15 minutes to 30 minutes.
- between 5% and 85% of the total lymphocytes collected from the blood are genetically modified.
- the percent of lymphocytes that are genetically modified and/or transduced is between 1, 5, and 10% on the low end of the range, and 15, 20, 25, 30, 40, 50, 60, 70, 80, and 85% on the high end of the range.
- the percent of T cells and NK cells that are genetically modified and/or transduced is at least 5%, at least 10%, at least 15%, or at least 20%.
- lymphocytes are genetically modified and/or transduced.
- Methods of genetically modifying lymphocytes typically include insertion into the cell, of a polynucleotide comprising one or more transcriptional units encoding a CAR or a lymphoproliferative element, or in illustrative embodiments encoding both a CAR and a lymphoproliferative element according to any of the CAR and lymphoproliferative element embodiments provided herein.
- Such CAR and lymphoproliferative elements can be provided to support the shorter and more simplified methods provided herein, which can support expansion of genetically modified and/or transduced T cells and/or NK cells after the contacting and optional incubation.
- lymphoproliferative elements can be delivered from the genome of the retroviral particles inside genetically modified and/or transduced T cells and/or NK cells, such that those cells have the characteristics of increased proliferation and/or survival disclosed in the Lymphoproliferative Elements section herein.
- the genetically modified T cell or NK cell is capable of engraftment in vivo in mice and/or enrichment in vivo in mice for at least 7, 14, or 28 days.
- mice may be treated or otherwise genetically modified so that any immunological differences between the genetically modified T cell and/or NK cell do not result in an immune response being elicited in the mice against any component of the lymphocyte transduced by the replication incompetent recombinant retroviral particle.
- Media that can be included in a contacting step for example when the cells and retroviral particles are initially brought into contact, or in any aspects provided herein, during optional incubation periods with the reaction mixture thereafter that include retroviral particles and cells in suspension in the media, or media that can be used during cell culturing and/or during various wash steps in any aspects provided herein, can include base media such as commercially available media for ex vivo T cell and/or NK cell culture.
- Non-limiting examples of such media include, X-VIVOTM 15 Chemically Defined, Serum-free Hematopoietic Cell Medium (Lonza) (2018 catalog numbers BE02-060F, BE02-00Q, BE-02- 061Q, 04-744Q, or 04-418Q), ImmunoCulfTM-XF T Cell Expansion Medium (STEMCELL
- Thermo Fisher (Thermo Fisher) (2018 catalog numbers A10221-01 (basal media (bottle)), and A10484-02 (supplement), A10221-03 (basal media (bag)), A1048501 (basal media and supplement kit (bottle)) and, A1048503 (basal media and supplement kit (bag)).
- Such media can be a chemically defined, serum-free formulation manufactured in compliance with cGMP.
- the media can be xeno-free and complete.
- the base media has been cleared by regulatory agencies for use in ex vivo cell processing, such as an FDA 5l0(k) cleared device.
- the media is the basal media with or without the supplied T cell expansion supplement of 2018 catalog number A1048501 (CTSTM OpTmizerTM T Cell Expansion SFM, bottle format) or A1048503 (CTSTM OpTmizerTM T Cell Expansion SFM, bag format) both available from Thermo Fisher (Waltham, MA).
- Additives such as human serum albumin, human AB+ serum, and/or serum derived from the subject can be added to the transduction reaction mixture.
- Supportive cytokines can be added to the transduction reaction mixture, such as IL2, IL7, or IL15, or those found in human sera.
- dGTP can be added to the transduction reaction in certain embodiments.
- the cells can be contacted with a retroviral particle without prior activation.
- the T cells and/or NK cells have not been incubated on a substrate that adheres to monocytes for more than 4 hours in one embodiment, or for more than 6, hours in another embodiment, or for more than 8 hours in another embodiment before the transduction.
- the T cells and/or NK cells have been incubated overnight on an adherent substrate to remove monocytes before the transduction.
- the method can include incubating the T cells and/or NK cells on an adherent substrate that binds monocytes for no more than 30 minutes, 1 hour, or 2 hours before the transduction.
- the T cells and/or NK cells are exposed to no step of removing monocytes by an incubation on an adherent substrate before said transduction step.
- the T cells and/or NK cells are not incubated with or exposed to a bovine serum, such as a cell culturing bovine serum, for example fetal bovine serum before or during a contacting step and/or a genetically modifying and/or transduction step.
- a closed system is a cell processing system that is generally closed or fully closed to an environment, such as an environment within a room or even the environment within a hood, outside of the conduits such as tubes, and chambers, of the system in which cells are processed and/or transported.
- an environment such as an environment within a room or even the environment within a hood, outside of the conduits such as tubes, and chambers, of the system in which cells are processed and/or transported.
- Such closed system methods can be performed with commercially available devices. Different closed system devices can be used at different steps within a method and the cells can be transferred between these devices using tubing and connections such as welded, luer, spike, or clave ports to prevent exposure of the cells or media to the environment.
- blood can be collected into an IV bag or syringe, optionally including an anti-coagulant, and transferred to a Sepax 2 device (Biosafe) for PBMC enrichment and isolation.
- whole blood can be filtered to collect leukocytes using a leukodepletion filter assembly.
- the isolated PBMCs or isolated leukocytes can be transferred to a chamber of a G-Rex device for an optional activation, a transduction and optional expansion.
- collected blood can be transduced in a blood bag, for example, the bag in which it was collected.
- the cells can be harvested and collected into another bag using a Sepax 2 device.
- the methods can be carried out in any device or combination of devices adapted for closed system T cell and/or NK cell production.
- Non-limiting examples of such devices include G-Rex devices (Wilson Wolf), GatheRex (Wilson Wolf), Sepax 2 (Biosafe), WAVE Bioreactors (General Electric), a CultiLife Cell Culture bag (Takara), a PermaLife bag (OriGen), CliniMACS Prodigy (Miltenyi Biotec), and VueLife bags (Saint-Gobain).
- the optional activating, the transducing and optional expanding can be performed in the same chamber or vessel in the closed system.
- the chamber can be a chamber of a G-Rex device and PBMCs or leukocytes can be transferred to the chamber of the G-Rex device after they are enriched and isolated, and can remain in the same chamber of the G-Rex device until harvesting.
- Methods provided herein can include transferring blood and cells therein and/or fractions thereof, as well as lymphocytes before or after they are contacted with retroviral particles, between vessels within a closed system, which thus is without environmental exposure.
- Vessels used in the closed system can be a tube, bag, syringe, or other container.
- the vessel is a vessel that is used in a research facility.
- the vessel is a vessel used in commercial production.
- the vessel can be a collection vessel used in a blood collection process.
- Methods for genetically modifying herein typically involve a contacting step wherein lymphocytes are contacted with a replication incompetent recombinant retroviral particle.
- the contacting in some embodiments, can be performed in the vessel, for example, within a blood bag. Blood and various lymphocyte-containing fractions thereof, can be transferred from the vessel to another vessel (for example from a first vessel to a second vessel) within the closed system for the contacting.
- the second vessel can be a cell processing compartment of a closed device, such as a G-Rex device.
- the genetically modified (e.g. transduced) cells can be transferred to a different vessel within the closed system (i.e. without exposure to the environment). Either before or after this transfer the cells are typically washed within the closed system to remove substantially all or all of the retroviral particles.
- a process disclosed herein, from collection of blood, to contacting (e.g. transduction), optional incubating, and post-incubation isolation and optional washing, is performed for between 15 minutes, 30 minutes, or 1, 2, 3, or 4 hours on the low end of the range, and 4, 8, 10, or 12 hours on the high end of the range.
- the delivery of a polynucleotide encoding a lymphoproliferative element, to a resting T cell and/or NK cell ex vivo, which can integrate into the genome of the T cell or NK cell provides that cell with a driver for in vivo expansion without the need for lymphodepleting the host.
- the subject is not exposed to a lymphodepleting agent within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months or 6 months of performing the contacting, during the contacting, and/or within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months or 6 months after the modified T cells and/or NK cells are reintroduced back into the subject.
- methods provided herein can be performed without exposing the subject to a lymphodepleting agent within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months or 6 months of performing the contacting, during the contacting, and/or within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months or 6 months after the modified T cells and/or NK cells are reintroduced back into the subject.
- methods provided herein can be performed without exposing the subject to a lymphodepleting agent within 1, 2, 3, 4, 5, 6, 7,
- methods of expanding genetically modified T cells and/or NK cells in a subject in vivo is a feature of some embodiments of the present disclosure. In illustrative embodiments, such methods are ex vivo propagation-free or substantially propagation-free.
- This entire method/process from blood draw from a subject to reintroduction of blood back into the subject after ex vivo transduction of T cells and/or NK cells can occur over a time period less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, 2 hours, or less than 2 hours.
- the entire method/process from blood draw/collection from a subject to reintroduction of blood back into the subject after ex vivo transduction of T cells and/or NK cells occurs over a time period between 1 hour and 12 hours, or between 2 hours and 8 hours, or between 1 hour and 3 hours, or between 2 hours and 4 hours, or between 2 hours and 6 hours, or between 4 hours and 12 hours, or between 4 hours and 24 hours, or between 8 hours and 24 hours, or between 8 hours and 36 hours, or between 8 hours and 48 hours, or between 12 hours and 24 hours, or between 12 hours and 36 hours, or between 12 hours and 48 hours, or over a time period between 15, 30, 60, 90, 120, 180, and 240 minutes on the low end of the range, and 120, 180, and 240, 300, 360, 420, and 480 minutes on the high end of the range.
- the entire method/process from blood draw/collection from a subject to reintroduction occurs over a time period between 1 hour and 12 hours, or between 2 hours and 8 hours, or
- the genetically modified T cells and/or NK cells are separated from the replication incompetent recombinant retroviral particles after the time period in which contact occurs.
- methods provided herein for genetically modifying lymphocytes, and associated methods for performing adoptive cell therapy can be performed in significantly less time than prior methods, fundamental improvements in patient care and safety as well as product manufacturability are made possible. Therefore, such processes are expected to be favorable in the view of regulatory agencies responsible for approving such processes when carried out in vivo for therapeutic purposes.
- the subject in non-limiting examples of any aspects provided herein that include a subject can remain in the same building (e.g. infusion clinic) or room as the instrument processing their blood or sample for the entire time that the sample is being processed before modified T cells and/or NK cells are reintroduced into the patient.
- a subject remains within line of site and/or within 100, 50, 25, or 12 feet or arm’s distance of their blood or cells that are being processed, for the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells.
- a subject remains awake and/or at least one person can continue to monitor the blood or cells of the subject that are being processed, throughout and/or continuously for the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells. Because of improvements provided herein, the entire
- method/process for adoptive cell therapy and/or for transducing resting T cells and/or NK cells from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells can be performed with continuous monitoring by a human.
- the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells are blood cells incubated in a room that does not have a person present.
- the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells is performed next to the subject and/or in the same room as the subject and/or next to the bed or chair of the subject.
- sample identity mix-ups can be avoided, as well as long and expensive incubations over periods of days or weeks.
- methods provided herein are readily adaptable to closed and automated blood processing systems, where a blood sample and its components that will be reintroduced into the subject, only make contact with disposable, single-use components.
- Methods for genetically modifying and/or transducing lymphocytes such as T cells and/or NK cells provided herein, can be part of a method for performing adoptive cell therapy.
- methods for performing adoptive cell therapy include steps of collecting blood from a subject, and returning genetically modified and/or transduced lymphocytes (e.g T cells and/or NK cells) to the subject.
- the present disclosure provides various treatment methods using a CAR.
- a CAR of the present disclosure when present in a T lymphocyte or an NK cell, can mediate cytotoxicity toward a target cell.
- a CAR of the present disclosure binds to an antigen present on a target cell, thereby mediating killing of a target cell by a T lymphocyte or an NK cell genetically modified to produce the CAR.
- the ASTR of the CAR binds to an antigen present on the surface of a target cell.
- the present disclosure provides methods of killing, or inhibiting the growth of, a target cell, the method involving contacting a cytotoxic immune effector cell (e.g., a cytotoxic T cell, or an NK cell) that is genetically modified to produce a subject CAR, such that the T lymphocyte or NK cell recognizes an antigen present on the surface of a target cell, and mediates killing of the target cell.
- a cytotoxic immune effector cell e.g., a cytotoxic T cell, or an NK cell
- the target cell can be a cancer cell, for example, and autologous cell therapy methods herein, can be methods for treating cancer, in some illustrative embodiments.
- the subject can be a an animal or human suspected of having cancer, or more typically, a subject that is known to have cancer.
- the genetically modified and/or transduced lymphocyte e.g. T cell and/or NK cell
- introduction or reintroduction of the genetically modified lymphocytes into a subject can be via any route known in the art.
- introduction or reintroduction can be delivery via infusion into a blood vessel of the subject.
- the genetically modified and/or transduced lymphocyte e.g. T cell and/or NK cell or population thereof, undergo 4 or fewer cell divisions ex vivo prior to being introduced or reintroduced into the subject.
- the lymphocyte(s) used in such a method are resting T cells and/or resting NK cells that are in contact with the replication incompetent recombinant retroviral particles for between 1 hour and 12 hours. In some embodiments, no more than 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour pass(es) between the time blood is collected from the subject and the time the genetically modified T cells and/or NK cells are reintroduced into the subject. In some embodiments, all steps after the blood is collected and before the blood is reintroduced, are performed in a closed system in which a person monitors the closed system throughout the processing.
- the genetically modified T cells and/or NK cells are introduced back, reintroduced, reinfused or otherwise delivered into the subject without additional ex vivo manipulation, such as stimulation and/or activation of T cells and/or NKs.
- ex vivo manipulation is used for stimulation/activation of T cells and/or NK cells and for expansion of genetically modified T cells and/or NK cells prior to introducing the genetically modified T cells and/or NK cells into the subject.
- this generally takes days or weeks and requires a subject to return to a clinic for a blood infusion days or weeks after an initial blood draw.
- T cells and/or NK cells are not stimulated ex vivo by exposure to anti-CD3/anti-CD28 solid supports such as, for example, beads coated with anti-CD3/anti-CD28, prior to contacting the T cells and/or NK cells with the replication incompetent recombinant retroviral particles.
- anti-CD3/anti-CD28 solid supports such as, for example, beads coated with anti-CD3/anti-CD28
- genetically modified T cells and/or NK cells are not expanded ex vivo, or only expanded for a small number of cell divisions (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds of cell division), but are rather expanded, or predominantly expanded, in vivo, i.e. within the subject.
- no additional media is added to allow for further expansion of the cells.
- no cell manufacturing of the primary blood lymphocytes occurs while the PBLs are contacted with the replication incompetent recombinant retroviral particles.
- no cell manufacturing of the PBLs occurs while the PBLs are ex vivo.
- subjects are lymphodepleted prior to reinfusion with genetically modified T cells and or NK cells.
- patients or subjects are not lymphodepleted prior to blood being withdrawn.
- patients or subjects are not lymphodepleted prior to reinfusion with genetically modified T cells and or NK cells.
- T cells and/or NK cells can be stimulated ex vivo by exposure to anti-CD3/anti-CD28 solid supports prior to contacting the T cells and/or NK cells with the replication incompetent recombinant retroviral particles.
- the T cells and/or NK cells can be exposed to anti-CD3/anti- CD28 solid supports for less than 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, or 24 hours, including no exposure, before the T cells and/or NK cells are contacted the replication incompetent recombinant retroviral particles.
- the T cells and/or NK cells can be exposed to anti-CD3/anti-CD28 solid supports for less than 1, 2, 3, 4, 6, or 8 hours before the T cells and/or NK cells are contacted the replication incompetent recombinant retroviral particles.
- cells are introduced or reintroduced into the subject by infusion into a vein or artery.
- the number of T cells and/or NK cells to be reinfused into a subject can be between 1 x 10 3 , 2.5 x 10 3 , 5 x 10 3 , 1 x 10 4 , 2.5 x 10 4 , 5 x 10 4 , 1 x 10 5 , 2.5 x 10 5 , 5 x 10 5 , 1 x 10 6 , 2.5 x 10 6 , 5 x 10 6 , and 1 x 10 7 cells/kg on the low end of the range and 5 x 10 4 ,
- the number of T cells and/or NK cells to be reinfused or otherwise delivered into a subject can be between 1 x 10 4 , 2.5 x 10 4 , 5 x 10 4 , and 1 x 10 5 cells/kg on the low end of the range and 2.5 x 10 4 , 5 x 10 4 , 1 x 10 5 , 2.5 x 10 5 , 5 x 10 5 , and 1 x 10 6 cells/kg on the high end of the range.
- the number of PBLs to be reinfused or otherwise delivered into a subject can be fewer than 5 x 10 5 , 1 x 10 6 , 2.5 x 10 6 , 5 x 10 6 , 1 x 10 7 , 2.5 x 10 7 , 5 x 10 7 , and 1 x 10 8 cells and the low end of the range and 2.5 x 10 6 , 5 x 10 6 , 1 x 10 7 , 2.5 x 10 7 , 5 x 10 7 , 1 x 10 8 ,
- the number of T cells and/or NK cells available for infusion or reinfusion into a 70 kg subject or patient is between 7 x 10 5 and 2.5 x 10 8 cells. In other embodiments, the number of T cells and/or NK cells available for transduction is approximately 7 x 10 6 plus or minus 10%.
- the replication incompetent recombinant retroviral particles used to contact T cells and/or NK cells have a polynucleotide or nucleic acid having one or more transcriptional units that encode one or more engineered signaling polypeptides.
- an engineered signaling polypeptide includes any combination of an extracellular domain (e.g. an antigen-specific targeting region or ASTR), a stalk and a transmembrane domain, combined with one or more intracellular activating domains, optionally one or more modulatory domains (such as a co-stimulatory domain), and optionally one or more T cell survival motifs.
- At least one, two, or all of the engineered signaling polypeptides is a chimeric antigen receptor (CAR) or a lymphoproliferative element (LE) such as a chimeric lymphoproliferative element (CLE).
- at least one, two, or all of the engineered signaling polypeptides is a recombinant T cell receptor (TCR).
- one when two signaling polypeptides are utilized, one encodes a lymphoproliferative element and the other encodes a chimeric antigen receptor (CAR) that includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
- CAR chimeric antigen receptor
- ASTR antigen-specific targeting region
- transmembrane domain a transmembrane domain
- intracellular activating domain exemplary sequences can be found in
- engineered polypeptides can also be referred to as recombinant polypeptides.
- the engineered signaling polypeptides such as CARs, recombinant TCRs, LEs, and CLEs provided herein, are typically transgenes with respect to lymphocytes, especially T cells and NK cells, and most especially T cells and/or NK cells that are engineered using methods and compositions provided herein, to express such signaling polypeptides.
- an engineered signaling polypeptide includes an extracellular domain that is a member of a specific binding pair.
- the extracellular domain can be the extracellular domain of a cytokine receptor, or a mutant thereof, or a hormone receptor, or a mutant thereof.
- Such mutant extracellular domains in some embodiments have been reported to be constitutively active when expressed at least in some cell types.
- such extracellular and transmembrane domains do not include a ligand binding region. It is believed that such domains do not bind a ligand when present in an engineered signaling polypeptide and expressed in B cells, T cells, and/or NK cells.
- the extracellular domain comprises a dimerizing motif.
- the dimerizing motif comprises a leucine zipper.
- the leucine zipper is from a jun polypeptide, for example c-jun.
- extracellular domains that comprise a dimerizing motif can be found, for example, in the Lymphoproliferative Element section herein.
- the extracellular domain is an antigen-specific targeting region (ASTR), sometimes called an antigen binding domain herein.
- ASTR antigen-specific targeting region
- Specific binding pairs include, but are not limited to, antigen-antibody binding pairs; ligand-receptor binding pairs; and the like.
- a member of a specific binding pair suitable for use in an engineered signaling polypeptide of the present disclosure includes an ASTR that is an antibody, an antigen, a ligand, a receptor binding domain of a ligand, a receptor, a ligand binding domain of a receptor, and an affibody.
- An ASTR suitable for use in an engineered signaling polypeptide of the present disclosure can be any antigen-binding polypeptide.
- the ASTR is an antibody such as a full-length antibody, a single-chain antibody, an Fab fragment, an Fab' fragment, an (Fab')2 fragment, an Fv fragment, and a divalent single-chain antibody or a diabody.
- the ASTR is a single chain Fv (scFv).
- the heavy chain is positioned N-terminal of the light chain in the engineered signaling polypeptide.
- the light chain is positioned N-terminal of the heavy chain in the engineered signaling polypeptide.
- the heavy and light chains can be separated by a linker as discussed in more detail herein.
- the heavy or light chain can be at the N-terminus of the engineered signaling polypeptide and is typically C-terminal of another domain, such as a signal sequence or peptide.
- cAb VF1F1 camelid antibody variable domains
- IgNAR VF1 shk antibody variable domains
- sdAb VF1 single domain antibody variable domains
- camelized antibody variable domains are suitable for use with the engineered signaling polypeptides and methods using the engineered signaling polypeptides of the present disclosure.
- T cell receptor (TCR) based recognition domains are suitable for use with the engineered signaling polypeptides and methods using the engineered signaling polypeptides of the present disclosure.
- TCR T cell receptor
- a CAR include CARs having extracellular domains engineered to co-opt the endogenous TCR signaling complex and CD3Z signaling pathway.
- a chimeric antigen receptor ASTR is fused to one of the endogenous TCR complex chains (e.g. TCR alpha, CD3E etc) to promote incorporation into the TCR complex and signaling through the endogenous CD3Z chains.
- a CAR contains a first scFv or protein that binds to the TCR complex and a second scFv or protein that binds to the target antigen (e.g. tumor antigen).
- the TCR can be a single chain TCR (scTv, single chain two-domain TCR containing nanb).
- scFv single chain two-domain TCR containing nanb.
- scFv single chain two-domain TCR containing nanb.
- scFv single chain two-domain TCR containing nanb.
- scFv single chain two-domain TCR containing nanb
- scFv may also be generated to recognize the specific MFlC/peptide complex, thereby acting as a surrogate TCR.
- Such peptide/MF!C scFv-binders may be used in many similar configurations as CAR’s.
- the ASTR can be multispecific, e.g. bispecific antibodies.
- Multispecific antibodies have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one target antigen and the other is for another target antigen.
- bispecific antibodies may bind to two different epitopes of a target antigen. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a target antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.
- An ASTR suitable for use in an engineered signaling polypeptide of the present disclosure can have a variety of antigen-binding specificities.
- the antigen-binding domain is specific for an epitope present in an antigen that is expressed by (synthesized by) a target cell.
- the target cell is a cancer cell associated antigen.
- the cancer cell associated antigen can be an antigen associated with, e.g., a breast cancer cell, a B cell lymphoma, a Flodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, etc.
- a cancer cell associated antigen may also be expressed by
- Non-limiting examples of antigens to which an ASTR of an engineered signaling polypeptide can bind include, e.g., CD19, CD20, CD38, CD30, ERBB2, CA125, MUC-l, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-l3R-a2, GD2, Axl, Ror2, and the like.
- PSMA prostate-specific membrane antigen
- CEA carcinoembryonic antigen
- EGFR epidermal growth factor receptor
- EGFRvIII vascular endothelial growth factor receptor-2
- HMW-MAA high molecular weight-melanoma associated antigen
- MAGE-A1
- a member of a specific binding pair suitable for use in an engineered signaling polypeptide is an ASTR that is a ligand for a receptor.
- Ligands include, but are not limited to, hormones (e.g. erythropoietin, growth hormone, leptin, etc.); cytokines (e.g., interferons, interleukins, certain hormones, etc.); growth factors (e.g., heregulin; vascular endothelial growth factor (VEGF); and the like); an integrin-binding peptide (e.g., a peptide comprising the sequence Arg-Gly-Asp (SEQ ID NO:l); and the like.
- hormones e.g. erythropoietin, growth hormone, leptin, etc.
- cytokines e.g., interferons, interleukins, certain hormones, etc.
- growth factors e.g., heregulin; vascular end
- the engineered signaling polypeptide can be activated in the presence of a second member of the specific binding pair, where the second member of the specific binding pair is a receptor for the ligand.
- the second member of the specific binding pair can be a VEGF receptor, including a soluble VEGF receptor.
- the member of a specific binding pair that is included in an engineered signaling polypeptide is an ASTR that is a receptor, e.g., a receptor for a ligand, a co-receptor, etc.
- the receptor can be a ligand-binding fragment of a receptor.
- Suitable receptors include, but are not limited to, a growth factor receptor (e.g., a VEGF receptor); a killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptide (receptor for MICA, MICB, and ULB6); a cytokine receptor (e.g., an IL-13 receptor; an IL-2 receptor; etc.); CD27; a natural cytotoxicity receptor (NCR) (e.g., NKP30 (NCR3/CD337) polypeptide (receptor for F1LA-B -associated transcript 3 (BAT3) and B7-F16); etc.); etc.
- a growth factor receptor e.g., a VEGF receptor
- a killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptide receptor for MICA, MICB, and ULB6
- a cytokine receptor e.g., an IL-13 receptor; an IL-2 receptor;
- the ASTR can be directed to an intermediate protein that links the ASTR with a target molecule expressed on a target cell.
- the intermediate protein may be endogenously expressed or introduced exogenously and may be natural, engineered, or chemically modified.
- the ASTR can be an anti-tag ASTR such that at least one tagged intermediate, typically an antibody-tag conjugate, is included between a tag recognized by the ASTR and a target molecule, typically a protein target, expressed on a target cell. Accordingly, in such embodiments, the ASTR binds a tag and the tag is conjugated to an antibody directed against an antigen on a target cell, such as a cancer cell.
- Non-limiting examples of tags include fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein.
- FITC fluorescein isothiocyanate
- streptavidin biotin
- biotin histidine
- dinitrophenol dinitrophenol
- peridinin chlorophyll protein complex green fluorescent protein
- PE phycoerythrin
- horse radish peroxidase palmitoylation
- nitrosylation alkaline phosphatase
- glucose oxidase glucose oxidase
- maltose binding protein a binds the tag.
- the engineered signaling polypeptide includes a stalk which is located in the portion of the engineered signaling polypeptide lying outside the cell and interposed between the ASTR and the transmembrane domain.
- the stalk has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD 8 stalk region (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVF1TRGLDFA (SEQ ID NO:2), has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD28 stalk region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NOG), or has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type immunoglobulin heavy chain stalk region.
- the stalk employed allows the antigen-specific targeting region, and typically the entire engineered signaling polypeptide, to retain increased binding to a target antigen.
- the stalk region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa.
- the stalk of an engineered signaling polypeptide includes at least one cysteine.
- the stalk can include the sequence Cys-Pro-Pro-Cys (SEQ ID NO:4). If present, a cysteine in the stalk of a first engineered signaling polypeptide can be available to form a disulfide bond with a stalk in a second engineered signaling polypeptide.
- Stalks can include immunoglobulin hinge region amino acid sequences that are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779.
- an immunoglobulin hinge region can include a domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the following amino acid sequences: DKTHT (SEQ ID NO:5); CPPC (SEQ ID NO:4); CPEPKSCDTPPPCPR (SEQ ID NO:6) (see, e.g., Glaser et al. (2005) J. Biol. Chem.
- ELKTPLGDTTHT SEQ ID NO:7
- KSCDKTHTCP SEQ ID NO:8
- KCCVDCP SEQ ID NO:9
- KYGPPCP SEQ ID NO: 10
- EPKSCDKTHTCPPCP SEQ ID NO: 11
- ERKCCVECPPCP SEQ ID NO: 12
- ELKTPLGDTTHTCPRCP SEQ ID NO: 13
- SPNMVPHAHHAQ SEQ ID NO: 14
- the stalk can include a hinge region with an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region.
- the stalk can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally-occurring) hinge region.
- Flis229 of human IgG 1 hinge can be substituted with Tyr, so that the stalk includes the sequence EPKSCDKTYTCPPCP (SEQ ID NO:15), (see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891).
- the stalk can include an amino acid sequence derived from human CD8; e.g., the stalk can include the amino acid sequence:
- TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 16
- An engineered signaling polypeptide of the present disclosure can include transmembrane domains for insertion into a eukaryotic cell membrane.
- the transmembrane domain can be interposed between the ASTR and the co-stimulatory domain.
- the transmembrane domain can be interposed between the stalk and the co-stimulatory domain, such that the chimeric antigen receptor includes, in order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus): an ASTR; a stalk; a transmembrane domain; and an activating domain.
- Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use in aspects and embodiments disclosed herein.
- Non-limiting examples of TM domains suitable for any of the aspects or embodiments provided herein, include a domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the following TM domains or combined stalk and TM domains: a) CD8 alpha TM (SEQ ID NO:l7); b) CD8 beta TM (SEQ ID NO:l8); c) CD4 stalk (SEQ ID NO: 19); d) CD3Z TM (SEQ ID NO:20); e) CD28 TM (SEQ ID NO:2l); f) CD134 (0X40) TM: (SEQ ID NO:22); g) CD7 TM (SEQ ID NO:23); h) CD 8 stalk and TM (SEQ ID NO:24); and i) CD28 stalk and TM (SEQ ID NO:25).
- a transmembrane domain of an aspect of the invention can have at least 80%, 90%, or 95% or can have 100% sequence identity to the SEQ ID NO: 17 transmembrane domain, or can have 100% sequence identity to any of the transmembrane domains from the following genes respectively: the CD8 beta transmembrane domain, the CD4 transmembrane domain, the CD3 zeta transmembrane domain, the CD28 transmembrane domain, the CD 134 transmembrane domain, or the CD7 transmembrane domain.
- Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure when activated typically induce the production of one or more cytokines; increase cell death; and/or increase proliferation of CD8 + T cells, CD4 + T cells, NKT cells, gd T cells, and/or neutrophils.
- Activating domains can also be referred to as activation domains herein.
- Activating domains can be used in CARs or in lymphoproliferative elements provided herein.
- the intracellular activating domain includes at least one (e.g., one, two, three, four, five, six, etc.) IT AM motifs as described below.
- an intracellular activating domain of an aspect of the invention can have at least 80%, 90%, or 95% or can have 100% sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70 domains as described below.
- Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides.
- ITAM immunoreceptor tyrosine-based activation motif
- An IT AM motif is YX1X2L/I, where Xi and X2 are independently any amino acid.
- the intracellular activating domain of an engineered signaling polypeptide includes 1, 2, 3, 4, or 5 IT AM motifs.
- an IT AM motif is repeated twice in an intracellular activating domain, where the first and second instances of the IT AM motif are separated from one another by 6 to 8 amino acids, e.g., (YXiX 2 L/I)(X 3 ) n (YXiX 2 L/I), where n is an integer from 6 to 8, and each of the 6-8 X 3 can be any amino acid.
- the intracellular activating domain of an engineered signaling polypeptide includes 3 IT AM motifs.
- a suitable intracellular activating domain can be an IT AM motif-containing portion that is derived from a polypeptide that contains an IT AM motif.
- a suitable intracellular activating domain can be an IT AM motif-containing domain from any IT AM motif-containing protein.
- a suitable intracellular activating domain need not contain the entire sequence of the entire protein from which it is derived.
- IT AM motif-containing polypeptides include, but are not limited to: CD3Z (CD3 zeta); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD79A (antigen receptor complex-associated protein alpha chain); CD79B (antigen receptor complex-associated protein beta chain)DAPl2; and FCER1G (Fc epsilon receptor I gamma chain).
- the intracellular activating domain is derived from T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3F1, CD3Q, T3Z, TCRZ, etc.).
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences (2 isoforms):
- a suitable intracellular activating domain polypeptide can include an ITAM motif- containing a portion of the full length CD3 zeta amino acid sequence.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences:
- RVKFSRS AD AP A Y QQGQN QL [ YNELNLGRREE YD VL] DKRRGRDPEMGGKPRRKNPQEGL [ YNE LQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO:28);
- RVKFSRS AD AP AY QQGQN QL [YNELNLGRREE YD VL] DKRRGRDPEMGGKPQRRKNPQEGL [ YN ELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO:29); N QL [YNELNLGRREE YD VL] DKR SEQ ID NO:30); EGL[ YNELQKDKMAEAY SEI] GMK (SEQ ID NO:3l); or DGL [ Y QGLST ATKDT YD AL] F1MQ (SEQ ID NO:32), where the IT AM motifs are set out in brackets.
- the intracellular activating domain is derived from T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.).
- T cell surface glycoprotein CD3 delta chain also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences:
- a suitable intracellular activating domain polypeptide can comprise an IT AM motif- containing portion of the full length CD3 delta amino acid sequence.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: DQV[Y QPLRDRDD AQYSF1L] GGN (SEQ ID NO:35), where the ITAM motifs are set out in brackets.
- the intracellular activating domain is derived from T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3/Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.).
- T cell surface glycoprotein CD3 epsilon chain also known as CD3e, T cell surface antigen T3/Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of the following amino acid sequence:
- a suitable intracellular activating domain polypeptide can comprise an IT AM motif- containing portion of the full length CD3 epsilon amino acid sequence.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: NPD [ YEPIRKGQRDL Y SGL]N QR (SEQ ID NO:37), where the ITAM motifs are set out in brackets.
- the intracellular activating domain is derived from T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.).
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of the following amino acid sequence:
- a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length CD3 gamma amino acid sequence.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: DQL [ Y QPLKDREDD Q Y SHL]QGN (SEQ ID NO:39), where the ITAM motifs are set out in brackets.
- the intracellular activating domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin- associated alpha); MB-l membrane glycoprotein; Ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.).
- CD79A also known as B-cell antigen receptor complex-associated protein alpha chain
- CD79a antigen immunoglobulin- associated alpha
- MB-l membrane glycoprotein Ig-alpha
- membrane-bound immunoglobulin-associated protein surface IgM-associated protein; etc.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences:
- a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length CD79A amino acid sequence.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: ENL[YEGLNLDDCSMYEDI]SRG (SEQ ID NO:42), where the ITAM motifs are set out in brackets.
- the intracellular activating domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.).
- DAP12 also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences (4 isoforms):
- a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length DAP12 amino acid sequence.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: ESP[YQELQGQRSDVYSDL]NTQ (SEQ ID NO:47), where the ITAM motifs are set out in brackets.
- the intracellular activating domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma;
- fcRgamma fceRI gamma
- high affinity immunoglobulin epsilon receptor subunit gamma
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 50 amino acids to about 60 amino acids (aa), from about 60 aa to about 70 aa, from about 70 aa to about 80 aa, or from about 80 aa to about 88 aa, of the following amino acid sequence:
- a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length FCER1G amino acid sequence.
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: DGV[YTGLSTRNQETYETL]KHE (SEQ ID NO:49), where the ITAM motifs are set out in brackets.
- Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure include a DAP10/CD28 type signaling chain.
- a suitable intracellular activating domain includes a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in SEQ ID NO:50.
- a CD28 signaling chain is the amino acid sequence is SEQ ID NO:5l.
- a suitable intracellular domain includes a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids of SEQ ID NO:5l.
- Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure include a ZAP70 polypeptide
- a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 300 amino acids to about 400 amino acids, from about 400 amino acids to about 500 amino acids, or from about 500 amino acids to 619 amino acids, of SEQ ID NO:52.
- Modulatory domains can change the effect of the intracellular activating domain in the engineered signaling polypeptide, including enhancing or dampening the downstream effects of the activating domain or changing the nature of the response.
- Modulatory domains suitable for use in an engineered signaling polypeptide of the present disclosure include co-stimulatory domains.
- a modulatory domain suitable for inclusion in the engineered signaling polypeptide can have a length of from about 30 amino acids to about 70 amino acids (aa), e.g., a modulatory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
- modulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.
- Co-stimulatory domains typically enhance and/or change the nature of the response to an activation domain.
- Co-stimulatory domains suitable for use in an engineered signaling polypeptide of the present disclosure are generally polypeptides derived from receptors. In some embodiments, co stimulatory domains homodimerize.
- a subject co-stimulatory domain can be an intracellular portion of a transmembrane protein (i.e., the co-stimulatory domain can be derived from a transmembrane protein).
- co-stimulatory polypeptides include, but are not limited to, 4-1BB (CD 137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, 0X40, BTLA, CD27, CD30, GITR, and HVEM.
- a co-stimulatory domain of an aspect of the invention can have at least 80%, 90%, or 95% sequence identity to the co-stimulatory domain of 4-1BB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, 0X40, BTLA, CD27, CD30, GITR, or HVEM.
- a co stimulatory domain of an aspect of the invention can have at least 80%, 90%, or 95% sequence identity to the co-stimulatory domain of non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, 0X40, BTLA, CD27, CD30, GITR, and HVEM.
- 4-1BB CD137
- CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, 0X40, BTLA, CD27, CD30, GITR, and HVEM include, but are not limited to, 4-1BB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, 0X40, BTLA, CD27, CD30, GITR, and HVEM.
- a co-stimulatory domain of an aspect of the invention can have at least 80%, 90%, or 95% sequence identity to the co-stimulatory domain of 4-1BB (CD 137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, 0X40, BTLA, CD27, CD30, GITR, or HVEM.
- 4-1BB CD 137
- CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, 0X40, BTLA, CD27, CD30, GITR, or HVEM 4-1BB (CD 137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, 0X40, BTLA, CD27, CD30, GITR, or HVEM.
- a co-stimulatory domain suitable for inclusion in an engineered signaling polypeptide can have a length of from about 30 amino acids to about 70 amino acids (aa), e.g., a co-stimulatory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
- the co-stimulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.
- the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD137 (also known as TNFRSF9; CD137; 4-1BB; CDwl37; ILA; etc.).
- a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:53.
- the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
- the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44).
- a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:54.
- the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
- the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD28 deleted for Lck binding (ICA).
- a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:55.
- the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
- the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1).
- a suitable co stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
- the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
- the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein 0X40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGP1L).
- 0X40 contains a p85 PI3K binding motif at residues 34-57 and a TRAF binding motif at residues 76-102, each of SEQ ID NO: 296 (of Table 1).
- the costimulatory domain can include the p85 PI3K binding motif of 0X40.
- the costimulatory domain can include the TRAF binding motif of 0X40.
- Lysines corresponding to amino acids 17 and 41 of SEQ ID NO: 296 are potentially negative regulatory sites that function as parts of ubiquitin targeting motifs.
- one or both of these Lysines in the costimulatory domain of 0X40 are mutated Arginines or another amino acid.
- a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:57.
- the co-stimulatory domain has a length of from about 20 aa to about 25 aa, about 25 aa to about 30 aa, 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.
- the co-stimulatory domain has a length of from about 20 aa to about 50 aa, for example 20 aa to 45 aa, or 20 aa to 42 aa.
- the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, T 14, TNFRSF7, and Tp55).
- a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:58.
- the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.
- the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272).
- BTLA also known as BTLA1 and CD272.
- a suitable co stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
- the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-l).
- a suitable co stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
- the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D).
- GITR also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D.
- a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:6l.
- the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
- the co-stimulatory domain derived from an intracellular portion of the transmembrane protein HVEM also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, EIGHTR, and TR2.
- a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:62.
- the co-stimulatory domain of both the first and the second polypeptide has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
- the engineered signaling polypeptide includes a linker between any two adjacent domains.
- a linker can be between the transmembrane domain and the first co stimulatory domain.
- the ASTR can be an antibody and a linker can be between the heavy chain and the light chain.
- a linker can be between the ASTR and the transmembrane domain and a co-stimulatory domain.
- a linker can be between the co-stimulatory domain and the intracellular activating domain of the second polypeptide.
- the linker can be between the ASTR and the intracellular signaling domain.
- the linker peptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded.
- a linker can be a peptide of between about 1 and about 100 amino acids in length, or between about 1 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used.
- the linking peptides may have virtually any amino acid sequence, bearing in mind that suitable linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.
- Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
- Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , GSGGS n , GGGS n , and GGGGS n where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components.
- Glycine polymers are of particular interest since glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).
- Exemplary flexible linkers include, but are not limited GGGGSGGGGSGGGGS (SEQ ID NO:63),
- GGGGSGGGGSGGGGSGGGGSGGGGGGSGGGGS SEQ ID NO:64
- GGGGSGGGSGGGGS SEQ ID NO:65
- GGSG SEQ ID NO:66
- GGSGG SEQ ID NO:67
- GSGSG SEQ ID NO:68
- GSGGG SEQ ID NO:69
- GGGSG SEQ ID NO:70
- GSSSG SEQ ID NO:71
- linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.
- a polynucleotide provided by the replication incompetent recombinant retroviral particles has one or more transcriptional units that encode certain combinations of the one or more engineered signaling polypeptides.
- genetically modified T cells include the combinations of the one or more engineered signaling polypeptides after transduction of T cells by the replication incompetent recombinant retroviral particles. It will be understood that the reference of a first polypeptide, a second polypeptide, a third polypeptide, etc. is for convenience and elements on a“first polypeptide” and those on a“second polypeptide” means that the elements are on different polypeptides that are referenced as first or second for reference and convention only, typically in further elements or steps to that specific polypeptide.
- the first engineered signaling polypeptide includes an extracellular antigen binding domain, which is capable of binding an antigen, and an intracellular signaling domain. In other embodiments, the first engineered signaling polypeptide also includes a T cell survival motif and/or a transmembrane domain. In some embodiments, the first engineered signaling polypeptide does not include a co-stimulatory domain, while in other embodiments, the first engineered signaling polypeptide does include a co-stimulatory domain.
- a second engineered signaling polypeptide includes a lymphoproliferative gene product and optionally an extracellular antigen binding domain.
- the second engineered signaling polypeptide also includes one or more of the following: a T cell survival motif, an intracellular signaling domain, and one or more co-stimulatory domains.
- at least one is a CAR.
- the one or more engineered signaling polypeptides are expressed under a T cell specific promoter or a general promoter under the same transcript wherein in the transcript, nucleic acids encoding the engineered signaling polypeptides are separated by nucleic acids that encode one or more internal ribosomal entry sites (IREs) or one or more protease cleavage peptides.
- IREs internal ribosomal entry sites
- the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes a first extracellular antigen binding domain, which is capable of binding to a first antigen, and a first intracellular signaling domain but not a co stimulatory domain, and the second polypeptide includes a second extracellular antigen binding domain, which is capable of binding VEGF, and a second intracellular signaling domain, such as for example, the signaling domain of a co-stimulatory molecule.
- the first antigen is PSCA, PSMA, or BCMA.
- the first extracellular antigen binding domain comprises an antibody or fragment thereof (e.g., scFv), e.g., an antibody or fragment thereof specific to PSCA, PSMA, or BCMA.
- the second extracellular antigen binding domain that binds VEGF is a receptor for VEGF, i.e., VEGFR.
- the VEGFR is VEGFR1, VEGFR2, or VEGFR3. In a certain embodiment, the VEGFR is VEGFR2.
- the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes an extracellular tumor antigen binding domain and a O ⁇ 3z signaling domain, and the second engineered signaling polypeptide includes an antigen binding domain, wherein the antigen is an angiogenic or vasculogenic factor, and one or more co stimulatory molecule signaling domains.
- the angiogenic factor can be, e.g., VEGF.
- the one or more co stimulatory molecule signaling motifs can comprise, e.g., co-stimulatory signaling domains from each of CD27, CD28, 0X40, ICOS, and 4-1BB.
- the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes an extracellular tumor antigen-binding domain and a O ⁇ 3z signaling domain, the second polypeptide comprises an antigen-binding domain, which is capable of binding to VEGF, and co-stimulatory signaling domains from each of CD27, CD28, 0X40, ICOS, and 4-1BB.
- the first signaling polypeptide or second signaling polypeptide also has a T cell survival motif.
- the T cell survival motif is, or is derived from, an intracellular signaling domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL-12 receptor, an intracellular signaling domain of IL-15 receptor, an intracellular signaling domain of IL-21 receptor, or an intracellular signaling domain of transforming growth factor b (TOHb) receptor or the T ⁇ Rb decoy receptor (TGF-b— dominant-negative receptor II (DNRII)).
- IL-7R IL-7 receptor
- TOHb transforming growth factor b
- the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes an extracellular tumor antigen-binding domain and a CD3z signaling domain, and the second engineered signaling polypeptide includes an antigen-binding domain, which is capable of binding to VEGF, an IL-7 receptor intracellular T cell survival motif, and co-stimulatory signaling domains from each of CD27, CD28, 0X40, ICOS, and 4- 1BB.
- more than two signaling polypeptides are encoded by the polynucleotide.
- only one of the engineered signaling polypeptides includes an antigen binding domain that binds to a tumor-associated antigen or a tumor-specific antigen; each of the remainder of the engineered signaling polypeptides comprises an antigen binding domain that binds to an antigen that is not a tumor-associated antigen or a tumor-specific antigen.
- two or more of the engineered signaling polypeptides include antigen binding domains that bind to one or more tumor- associated antigens or tumor-specific antigens, wherein at least one of the engineered signaling polypeptides comprises an antigen binding domain that does not bind to a tumor-associated antigen or a tumor-specific antigen.
- the tumor-associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-l25 (CA-125), CA19-9, calretinin, MUC-l, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-l), myo- Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (
- the first engineered signaling polypeptide includes a first extracellular antigen binding domain that binds a first antigen, and a first intracellular signaling domain; and a second engineered signaling polypeptide includes a second extracellular antigen binding domain that binds a second antigen, or a receptor that binds the second antigen; and a second intracellular signaling domain, wherein the second engineered signaling polypeptide does not comprise a co-stimulatory domain.
- the first antigen-binding domain and the second antigen-binding domain are independently an antigen-binding portion of a receptor o r an antigen-binding portion of an antibody.
- first antigen binding domain or the second antigen binding domain are scFv antibody fragments.
- first engineered signaling polypeptide and/or the second engineered signaling polypeptide additionally comprises a transmembrane domain.
- the first engineered signaling polypeptide or the second engineered signaling polypeptide comprises a T cell survival motif, e.g., any of the T cell survival motifs described herein.
- the first engineered signaling polypeptide includes a first extracellular antigen binding domain that binds HER2 and the second engineered signaling polypeptide includes a second extracellular antigen binding domain that binds MUC-l.
- the second extracellular antigen binding domain of the second engineered signaling polypeptide binds an interleukin.
- the second extracellular antigen binding domain of the second engineered signaling polypeptide binds a damage associated molecular pattern molecule (DAMP; also known as an alarmin).
- DAMP is a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGB1), S100A8 (also known as MRP8, or calgranulin A), S100A9 (also known as MRP14, or calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.
- HMGB1 chromatin-associated protein high mobility group box 1
- S100A8 also known as MRP8, or calgranulin A
- S100A9 also known as MRP14, or calgranulin B
- SAA serum amyloid A
- said second antigen is an antigen on an antibody that binds to an antigen presented by a tumor cell.
- signal transduction activation through the second engineered signaling polypeptide is non-antigenic, but is associated with hypoxia.
- hypoxia is induced by activation of hypoxia-inducible factor-la (HIF-la), HIF-Ib, HIF-2a, HIH-2b, HIF-3a, or HIR-3b.
- expression of the one or more engineered signaling polypeptides is regulated by a control element, which is disclosed in more detail herein.
- the engineered signaling polypeptides can further include one or more additional polypeptide domains, where such domains include, but are not limited to, a signal sequence; an epitope tag; an affinity domain; and a polypeptide whose presence or activity can be detected (detectable marker), for example by an antibody assay or because it is a polypeptide that produces a detectable signal.
- Non limiting examples of additional domains for any of the aspects or embodiments provided herein include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the following sequences as described below: a signal sequence, an epitope tag, an affinity domain, or a polypeptide that produces a detectable signal.
- Signal sequences that are suitable for use in a subject CAR include any eukaryotic signal sequence, including a naturally-occurring signal sequence, a synthetic (e.g., man-made) signal sequence, etc.
- the signal sequence can be the CD8 signal sequence M AFP VT AFFFPFAFFFHAARP (SEQ ID NO:72).
- Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO:73); FLAG (e.g. ,D YKDDDDK; SEQ ID NO:74); c-myc (e.g., EQKLISEEDL; SEQ ID NO:75), and the like.
- Affinity domains include peptide sequences that can interact with a binding partner, e.g., such as one immobilized on a solid support, useful for identification or purification.
- DNA sequences encoding multiple consecutive single amino acids, such as histidine, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high affinity binding to a resin column, such as nickel sepharose.
- affinity domains include His5 (HHHHH; SEQ ID NO:76), HisX6 (HHHHHH; SEQ ID NO:77), c-myc (EQKLISEEDL; SEQ ID NO:75), Flag (D YKDDDDK; SEQ ID NO:74), Strep Tag (WSHPQFEK; SEQ ID NO:78), hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQ ID NO:73), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:79), Phe-His-His-Thr (SEQ ID NO: 80), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag,
- WEAAAREACCRECCARA SEQ ID NO:81
- metal binding domains e.g., zinc binding domains or calcium binding domains such as those from calcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large-subunit, SlOOproteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltose binding protein.
- Suitable detectable signal-producing proteins include, e.g., fluorescent proteins; enzymes that catalyze a reaction that generates a detectable signal as a product; and the like.
- Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP
- Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin.
- Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, m PI urn (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol.
- Suitable enzymes include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N- acetylglucosaminidase, b-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, glucose oxidase (GO), and the like.
- HRP horse radish peroxidase
- AP alkaline phosphatase
- GAL beta-galactosidase
- glucose-6-phosphate dehydrogenase beta-N- acetylglucosaminidase
- b-glucuronidase invertase
- Xanthine Oxidase firefly luciferase
- glucose oxidase GO
- any of the replication incompetent recombinant retroviral particles provided herein can include nucleic acids that encode a recognition or elimination domain as part of, or separate from, nucleic acids encoding any of the engineered signaling polypeptides provided herein.
- any of the engineered signaling polypeptides provided herein can include a recognition or elimination domain.
- any of the CARs disclosed herein can include a recognition or elimination domain.
- a recognition or elimination domain can be expressed together with, or even fused with any of the lymphoproliferative elements disclosed herein. The recognition or elimination domains are expressed on the T cell and/or NK cell but are not expressed on the replication incompetent recombinant retroviral particles.
- the recognition or elimination domain can be derived from herpes simplex virus-derived enzyme thymidine kinase (HSV-tk) or inducible caspase-9.
- the recognition or elimination domain can include a modified endogenous cell-surface molecule, for example as disclosed in U.S. Patent 8,802,374.
- the modified endogenous cell-surface molecule can be any cell- surface related receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin, or cluster of differentiation (CD) that is modified.
- the modified endogenous cell-surface molecule is a truncated tyrosine kinase receptor.
- the truncated tyrosine kinase receptor is a member of the epidermal growth factor receptor (EGFR) family (e.g., ErbBl, ErbB2, ErbB3, and ErbB4).
- the recognition domain can be a polypeptide that is recognized by an antibody that recognizes the extracellular domain of an EGFR member.
- the recognition domain can be at least 20 contiguous amino acids of an EGFR family member, or for example, between 20 and 50 contiguous amino acids of an EGFR family member.
- SEQ ID NO:82 is an exemplary polypeptide that is recognized by, and under the appropriate conditions bound by an antibody that recognizes the extracellular domain of an EGFR member.
- Such extracellular EGFR epitopes are sometimes referred to herein as eTags. In illustrative embodiments, such epitopes are recognized by commercially available anti-EGFR monoclonal antibodies.
- Epidermal growth factor receptor also known as EGFR, ErbBl and F1ER1 is a cell-surface receptor for members of the epidermal growth factor family of extracellular ligands. Alterations in EGFR activity have been implicated in certain cancers.
- a gene encoding an EGFR polypeptide including human epidermal growth factor receptor (EGFR) is constructed by removal of nucleic acid sequences that encode polypeptides including the membrane distal EGF-binding domain and the cytoplasmic signaling tail, but retains the extracellular membrane proximal epitope recognized by an anti-EGFR antibody.
- the antibody is a known, commercially available anti-EGFR monoclonal antibody, such as cetuximab, matuzumab, necitumumab or panitumumab.
- EGFR was demonstrated to have suicide gene potential through Erbitux® mediated antibody dependent cellular cytotoxicity (ADCC) pathways.
- ADCC antibody dependent cellular cytotoxicity
- the inventors of the present disclosure have successfully expressed eTag in PBMCs using lentiviral vectors, and have found that expression of eTag in vitro by PBMCs exposed to Cetuximab, provided an effective elimination mechanism for PBMCs.
- EGFR may be used as a non-immunogenic selection tool, tracking marker, and suicide gene for transduced T cells that have immunotherapeutic potential.
- the EGFR nucleic acid may also be detected by means well known in the art.
- EGFR is expressed as part of a single polypeptide that also includes the CAR or as part of a single polypeptide that includes the lymphoproliferative element.
- the amino acid sequence encoding the EGFR recognition domain can be separated from the amino acid sequence encoding the chimeric antigen receptor by a cleavage signal and/or a ribosomal skip sequence.
- the ribosomal skip and/or cleavage signal can be any ribosomal skip and/or cleavage signal known in the art.
- the ribosomal skip sequence can be, for example T2A (also referred to as 2A-1 herein) with amino acid sequence
- GSGEGRGSEETCGDVEENPGP SEQ ID NO:83.
- other examples of cleavage signals and ribosomal skip sequences include FMDV 2 A (F2A); equine rhinitis A virus 2 A (abbreviated as E2A); porcine teschovirus-l 2A (P2A); and Thoseaasigna virus 2A (T2A).
- the polynucleotide sequence encoding the recognition domain can be on the same transcript as the CAR or lymphoproliferative element but separated from the polynucleotide sequence encoding the CAR or lymphoproliferative element by an internal ribosome entry site.
- a recognition domain can be expressed as part of a fusion polypeptide, fused to a lymphoproliferative element.
- Such constructs provide the advantage, especially in combination with other“space saving” elements provided herein, of taking up less genomic space on an RNA genome compared to separate polypeptides.
- an eTag is expressed as a fusion polypeptide, fused to an IL7Roc mutant, as experimentally demonstrated herein.
- an engineered signaling polypeptide is a chimeric antigen receptor (CAR) or a polynucleotide encoding a CAR, which, for simplicity, is referred to herein as“CAR.”
- a CAR of the present disclosure includes: a) at least one antigen-specific targeting region (ASTR); b) a transmembrane domain; and c) an intracellular activating domain.
- the antigen-specific targeting region of the CAR is an scFv portion of an antibody to the target antigen.
- the intracellular activating domain is from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70, and some further illustrative embodiments, from CD3z.
- the CAR further comprises a co-stimulatory domain, for example any of the co-stimulatory domains provided above in the Modulatory Domains section, and in further illustrative embodiments the co-stimulatory domain is the intracellular co-stimulatory domain of 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and F1VEM.
- the CAR includes any of the transmembrane domains listed in the Transmembrane Domain section above.
- a CAR of the present disclosure can be present in the plasma membrane of a eukaryotic cell, e.g., a mammalian cell, where suitable mammalian cells include, but are not limited to, a cytotoxic cell, a T lymphocyte, a stem cell, a progeny of a stem cell, a progenitor cell, a progeny of a progenitor cell, and an NK cell, an NK-T cell, and a macrophage.
- a CAR of the present disclosure is active in the presence of one or more target antigens that, in certain conditions, binds the ASTR.
- the target antigen is the second member of the specific binding pair.
- the target antigen of the specific binding pair can be a soluble (e.g., not bound to a cell) factor; a factor present on the surface of a cell such as a target cell; a factor presented on a solid surface; a factor present in a lipid bilayer; and the like.
- the antigen can be a soluble (e.g., not bound to a cell) antigen; an antigen present on the surface of a cell such as a target cell; an antigen presented on a solid surface; an antigen present in a lipid bilayer; and the like.
- a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, increases expression of at least one nucleic acid in the cell.
- a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by the one or more target antigens, increases expression of at least one nucleic acid in the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about lO-fold, or more than 10- fold, compared with the level of transcription of the nucleic acid in the absence of the one or more target antigens.
- the CAR of the present disclosure can include an immunoreceptor tyrosine-based activation motif (IT AM) -containing intracellular signaling polypeptide.
- IT AM immunoreceptor tyrosine-based activation motif
- a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can, in some instances, result in increased production of one or more cytokines by the cell.
- a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by the one or more target antigens, can increase production of a cytokine by the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about lO-fold, or more than 10- fold, compared with the amount of cytokine produced by the cell in the absence of the one or more target antigens.
- Cytokines whose production can be increased include, but are not limited to interferon gamma (IFN-g), tumor necrosis factor-alpha (TNF-a), IL-2, IL-15, IL-12, IL-4, IL-5, IL-10; a chemokine; a growth factor; and the like.
- IFN-g interferon gamma
- TNF-a tumor necrosis factor-alpha
- IL-2 tumor necrosis factor-alpha
- IL-15 IL-12
- IL-4 IL-5
- IL-10 a chemokine
- chemokine a growth factor
- a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can result in both an increase in transcription of a nucleic acid in the cell and an increase in production of a cytokine by the cell.
- a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, results in cytotoxic activity by the cell toward a target cell that expresses on its cell surface an antigen to which the antigen-binding domain of the first polypeptide of the CAR binds.
- a CAR of the present disclosure when present in the plasma membrane of the cell, and when activated by the one or more target antigens, increases cytotoxic activity of the cell toward a target cell that expresses on its cell surface the one or more target antigens.
- a CAR of the present disclosure when present in the plasma membrane of the cell, and when activated by the one or more target antigens, increases cytotoxic activity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about lO-fold, or more than lO-fold, compared to the cytotoxic activity of the cell in the absence of the one or more target antigens.
- a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can result in other CAR activation related events such as proliferation and expansion (either due to increased cellular division or anti- apoptotic responses).
- a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can result in other CAR activation related events such as intracellular signaling modulation, cellular differentiation, or cell death.
- CARs of the present disclosure are microenvironment restricted. This property is typically the result of the microenvironment restricted nature of the ASTR domain of the CAR.
- CARs of the present disclosure can have a lower binding affinity or, in illustrative embodiments, can have a higher binding affinity to one or more target antigens under a condition(s) in a microenvironment than under a condition in a normal physiological environment.
- CARs provided herein comprise a co-stimulatory domain in addition to an intracellular activating domain, wherein the co-stimulatory domain is any of the intracellular signaling domains provided herein for lymphoproliferative elements (LEs), such as, for example, intracellular domains of CLEs.
- the co-stimulatory domains of CARs herein are first intracellular domains (P3 domains) identified herein for CLEs or P4 domains that are shown as effective intracellular signaling domains of CLEs herein in the absence of a P3 domain.
- co-stimulatory domains of CARs can comprise both a P3 and a P4 intracellular signaling domain identified herein for CLEs.
- subembodiments include especially effective P3 and P4 partner intracellular signaling domains as identified herein for CLEs.
- the co-stimulatory domain is other than an ITAM-containing intracellular domain of a CAR either as part of the co-stimulatory domain, or in further illustrative embodiments as the only co-stimulatory domain.
- the co-stimulatory domain of a CAR can be any intracellular signaling domain in Table 1 provided herein. Active fragments of any of the intracellular domains in Table 1 can be a co-stimulatory domain of a CAR.
- the ASTR of the CAR comprises an scFV.
- these CARs comprise an intracellular activating domain that in illustrative embodiments is a CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP 12, FCER1G, FCGR2A, FCGR2C.
- DAP10/CD28, or ZAP70 intracellular activating domain or in further illustrative embodiments is a CD3z intracellular activating domain.
- the co-stimulatory domain of a CAR can comprise an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA,
- the co-stimulatory domain of a CAR can include an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL31RA, LEPR, LIFR, LMP1, MPL, MyD88, OSMR, or PRLR.
- the co-stimulatory domain of a CAR can include an intracellular domain or a functional fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL12RB 1, IL12RB2, IL13RA2, IL15RA, IL17RD, IL21R, IL23R, IL27RA, IL31RA, LEPR, MPL, MyD88, or OSMR.
- the co-stimulatory domain of a CAR can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL13RA2, IL17RD, IL31RA, LEPR, MPL, MyD88, or OSMR.
- the co-stimulatory domain of a CAR can include an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNAR1, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL12RB2, IL17RC, IL17RE, IL18R1, IL27RA, IL31RA, MPL, MyD88,
- the co-stimulatory domain of a CAR can include an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL17RE, IL31RA, MPL, or MyD88.
- the co-stimulatory domain of a CAR can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF3R, IL6ST, IL27RA, MPL, and MyD88.
- the intracellular activating domain of the CAR is derived from CD3z. Recombinant T Cell Receptors (TCRs)
- T Cell Receptors recognize specific protein fragments derived from intracellular and well as extracellular proteins. When proteins are broken into peptide fragments, they are presented on the cell surface with another protein called major histocompatibility complex, or MHC, which is called the HLA (human leukocyte antigen) complex in humans.
- MHC major histocompatibility complex
- HLA human leukocyte antigen
- Such combinations are formed by dimerization between members of dimerizing subtypes, such as an a TCR subunit and a b TCR subunit, a g TCR subunit and a d TCR subunit, and for pre-TCRs, a pTa subunit and a b TCR subunit.
- a set of TCR subunits dimerize and recognize a target peptide fragment presented in the context of an MHC.
- the pre-TCR is expressed only on the surface of immature ab T cells while the ab TCR is expressed on the surface of mature ab T cells and NK T cells, and ydTCR is expressed on the surface of gdT cells.
- o ⁇ TCRs on the surface of a T cell recognize the peptide presented by MHCI or MHCII and the ab TCR on the surface of NK T cells recognize lipid antigens presented by CD1.
- ydTCRs can recognize MHC and MHC-like molecules, and can also recognize non-MHC molecules such as viral glycoproteins.
- o ⁇ TCRs and ydTCRs transmit activation signals through the CD3zeta chain that stimulate T cell proliferation and cytokine secretion.
- TCR molecules belong to the immunoglobulin superfamily with its antigen-specific presence in the V region, where CDR3 has more variability than CDR1 and CDR2, directly determining the antigen binding specificity of the TCR.
- CDR3 has more variability than CDR1 and CDR2 directly determining the antigen binding specificity of the TCR.
- the CDR1 and CDR2 recognize and bind the sidewall of the MHC molecule antigen binding channel, and the CDR3 binds directly to the antigenic peptide.
- Recombinant TCRs may thus be engineered that recognize a tumor-specific protein fragment presented on MHC.
- Recombinant TCR such as those derived from human TCRa and TCRb pairs that recognize specific peptides with common HLAs can thus be generated with specificity to a tumor specific protein (Schmitt, TM et al., 2009).
- the target of recombinant TCRs may be peptides derived from any of the antigen targets for CAR ASTRs provided herein, but are more commonly derived from intracellular tumor specific proteins such as oncofetal antigens, or mutated variants of normal intracellular proteins or other cancer specific neoepitopes. Libraries of TCR subunits may be screened for their selectivity to a target antigen.
- TCR subunits Screens of natural and/or recombinant TCR subunits can identify sets of TCR subunits with high avidities and/or reactivities towards a target antigen. Members of such sets of TCR subunits can be selected and cloned to produce one or more polynucleotide encoding the TCR subunit.
- Polynucleotides encoding such a set of TCR subunits can be included in a replication incompetent recombinant retroviral particle to genetically modify a lymphocyte, or in illustrative embodiments, a T cell or an NK cell, such that the lymphocyte expresses the recombinant TCR.
- the engineered signaling polypeptide(s) can include or can be one or more sets of recombinant ydTCR chains, or in illustrative embodiments a TCR chains.
- TCR chains that form a set may be co-expressed using a number of different techniques to co-express the two TCR chains as is disclosed herein for expressing two or more other engineered signaling polypeptides such as CARs and lymphoproliferative elements.
- protease cleavage epitopes such as 2A protease, internal ribosomal entry sites (IRES), and separate promoters may be used.
- this involves modification of the constant (C) domains of the TCRa and TCR chains to promote the preferential pairing of the introduced TCR chains with each other, while rendering them less likely to successfully pair with endogenous TCR chains.
- C constant domains of the TCRa and TCR chains
- One approach that has shown some promise in vitro involves replacement of the C domain of human TCRa and TCR chains with their mouse counterparts.
- Another approach involves mutation of the human TCRa common domain and TCR chain common regions to promote self-pairing, or the expression of an endogenous TCR alpha and TCR beta miRNA within the viral gene construct.
- each member of the set of TCR chains in illustrative embodiments a TCR chains, comprises a modified constant domain that promotes preferential pairing with each other.
- each member of a set of TCR chains in illustrative embodiments a TCR chains, comprises a mouse constant domain from the same TCR chain type, or a constant domain from the same TCR chain subtype with enough sequences derived from a mouse constant domain from the same TCR chain subtype, such that dimerization of the set of TCR chains to each other is preferred over, or occurs to the exclusion of, dimerization with human TCR chains.
- each member of a set of TCR chains in illustrative embodiments a TCR chains, comprises corresponding mutations in its constant domain, such that dimerization of the set of TCR chains to each other is preferred over, or occurs to the exclusion of, dimerization with TCR chains that have human constant domains.
- dimerization in illustrative embodiments, is under physiological conditions.
- Peripheral T lymphocyte numbers are maintained at remarkably stable levels throughout adulthood, despite the continuing addition of cells, due to emigration from the thymus and proliferation in response to antigen encounter, and loss of cells owing to the removal of antigen-specific effectors after antigen clearance (Marrak, P. et al. 2000. Nat Immunol 1:107-111; Freitas, A. A. et al. 2000. A unit Rev Immunol 18:83-111).
- the size of the peripheral T cell compartment is regulated by multiple factors that influence both proliferation and survival. However, in a lymphopenic environment, T lymphocytes divide independently of cognate antigen, due to“acute homeostatic proliferation” mechanisms that maintain the size of the peripheral T cell compartment.
- lymphodepletion of a subject is not desirable because it can cause serious side effects, including immune dysfunction and death.
- lymphodepletion removes endogenous lymphocytes functioning as cellular sinks for homeostatic cytokines, thereby freeing cytokines to induce survival and proliferation of adoptively transferred cells.
- Some cytokines such as for example, IL-7 and IL-15, are known to mediate antigen-independent proliferation of T cells and are thus capable of eliciting homeostatic proliferation in non-lymphopenic environments.
- these cytokines and their receptors have intrinsic control mechanisms that prevent lymphoproliferative disorders at homeostasis.
- an engineered signaling polypeptide is a lymphoproliferative element (LE) such as a chimeric lymphoproliferative element (CLE).
- LE comprises an extracellular domain, a transmembrane domain, and at least one intracellular signaling domain that drives proliferation, and in illustrative embodiments a second intracellular signaling domain.
- the lymphoproliferative element can include a first and/or second intracellular signaling domain.
- the first and/or second intracellular signaling domain can include CD2, CD3D, CD3E, CD3G, CD4, CD8A, CD8B, CD27, mutated Delta Lck CD28, CD28, CD40, CD79A, CD79B, CRLF2, CSF2RB, CSF2RA, CSF3R, EPOR, FCER1G, FCGR2C, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1,
- the first intracellular signaling domain can include MyD88, or a functional mutant and/or fragment thereof.
- the first intracellular signaling domain can include MyD88, or a functional mutant and/or fragment thereof
- the second intracellular signaling domain can include ICOS, TNFRSF4, or TNSFR18, or functional mutants and/or fragments thereof.
- the first intracellular domain is MyD88 and the second intracellular domain is an ITAM-containing intracellular domain, for example, an intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70.
- the second intracellular signaling domain can include TNFRSF18, or a functional mutant and/or fragment thereof.
- the lymphoproliferative element can include a fusion of an extracellular domain and a transmembrane domain.
- the fusion of an extracellular domain and a transmembrane domain can include eTAG IL7RA Ins PPCL (interleukin 7 receptor), Myc LMP1, LMP1, eTAG CRLF2, eTAG CSF2RB, eTAG CSF3R, eTAG EPOR, eTAG GHR, eTAG truncated after Fn F523C IL27RA, or eTAG truncated after Fn S505N MPF, or functional mutants and/or fragments thereof.
- the lymphoproliferative element can include an extracellular domain.
- the extracellular domain can include eTag with 0, 1, 2, 3, or 4 additional alanines at the carboxy terminus.
- the extracellular domain can include Myc with 0, 1, 2, 3, or 4 additional alanines at the carboxy terminus, or functional mutants and/or fragments thereof.
- the lymphoproliferative element can include a transmembrane domain.
- the transmembrane domain can include CD2, CD3D, CD3E, CD3G, CD3Z CD247, CD4, CD8A, CD8B, CD27, CD28, CD40, CD79A, CD79B, CRFF2, CSF2RA, CSF2RB, CSF3R, EPOR, FCER1G, FCGR2C, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNER1, IE1R1,
- CLEs for use in any aspect or embodiment herein can include any CLE disclosed in
- WO2019/055946 (incorporated by reference herein, in its entirety), the vast majority of which were designed to be and are believed to be constitutively active. As illustrated therein, where there is a first and a second intracellular signaling domain of a CLE, the first intracellular signaling domain is positioned between the membrane associating motif and the second intracellular domain.
- the LE provides, is capable of providing and/or possesses the property of (or a cell genetically modified and/or transduced with the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) driving T cell expansion in vivo.
- Methods for performing such an in vivo test are provided in Example 6.
- the in vivo test can utilize a mouse model and measure T cell expansion at 15 to 25 days in vivo, or at 19 to 21 days in vivo, or at approximately 21 days in vivo, after T cells are contacted with lenti viral vectors encoding the LEs, are introduced into the mice.
- the lymphoproliferative element can include any of the sequences listed in Table 1 (SEQ ID NOs: 84-302). Table 1 shows the parts, names (including gene names), and amino acid sequences for domains that were tested in CLEs.
- a CLE includes an extracellular domain (denoted Pl), a transmembrane domain (denoted P2), a first intracellular domain (denoted P3), and a second intracellular domain (denoted P4).
- the lymphoproliferative element includes a first intracellular domain.
- the first intracellular domain can include any of the parts listed as S036 to S0216 or in Table 1, or functional mutants and/or fragments thereof.
- the lymphoproliferative element can include a second intracellular domain.
- the second intracellular domain can include any of the parts listed as S036 to S0216 or in Table 1, or functional mutants and/or fragments thereof.
- the lymphoproliferative element can include an extracellular domain.
- the extracellular domain can include any of the sequences of parts listed as M001 to M049 or E006 to E015 in Table 1, or functional mutants and/or fragments thereof.
- the lymphoproliferative element can include a transmembrane domain.
- the transmembrane domain can include any of the parts listed as M001 to M049 or T001 to T082 in Table 1, or functional mutants and/or fragments thereof.
- the lymphoproliferative element can be of fusion of an
- the lymphoproliferative element can be a fusion of an extracellular domain (E006 to E015 in Table 1), a transmembrane domain (T001 to T082 in Table 1), a first intracellular domain (S036 to S0216 in Table 1), and a second intracellular domain (S036 to S0216 in Table 1).
- the lymphoproliferative element can be a fusion of E006, T001, S036, and S216, also written as E006-T001-S036-S216).
- the lymphoproliferative element can be the fusion E010-T072-S192-S212, E007-T054-S197-S212, E006-T006-S194-S211, E009-T073-S062-S053, E008-T001-S121-S212, E006-T044-S186-S053, or E006-T016-S186-S050.
- the intracellular domain of an LE is other than a functional intracellular activating domain from an ITAM-containing intracellular domain, for example, an intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP 12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70, and in a further illustrative subembodiment, CD3z.
- a second intracellular domain of an LE is other than a co-stimulatory domain of 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.
- the extracellular domain of an LE does not comprise a single-chain variable fragment (scFv).
- the extracellular domain of an LE that upon binding to a binding partner activates an LE does not comprise a single-chain variable fragment (scFv).
- a CLE does not comprise both an ASTR and an activation domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP 12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70.
- the extracellular domain and transmembrane domain are believed to play support roles in LEs, assuring that the intracellular signaling domain(s) is in an effective
- a lymphoproliferative element includes an intracellular domain that is a signaling polypeptide that is capable of driving proliferation of T cells or NK cells that is associated with a membrane through a membrane-associating motif (e.g. a transmembrane domain) and is oriented in, or capable of being oriented into, an active conformation.
- the ASTR of an LE in illustrative embodiments, does not include an scFv.
- a lymphoproliferative element does not include an extracellular domain.
- the extracellular domains, transmembrane domains, and intracellular domains of LEs can vary in their respective amino acid lengths.
- the overall length of the LE can be between 3 and 4000 amino acids, for example between 10 and 3000, 10 and 2000, 50 and 2000, 250 and 2000 amino acids, and, in illustrative embodiments between 50 and 1000, 100 and 1000 or 250 and 1000 amino acids.
- the extracellular domain when present to form an extracellular and transmembrane domain, can be between 1 and 1000 amino acids, and is typically between 4 and 400, between 4 and 200, between 4 and 100, between 4 and 50, between 4 and 25, or between 4 and 20 amino acids.
- the extracellular region is GGGS for an extracellular and transmembrane domain of this aspect of the invention.
- the transmembrane domains, or transmembrane regions of extracellular and transmembrane domains, can be between 10 and 250 amino acids, and are more typically at least 15 amino acids in length, and can be, for example, between 15 and 100, 15 and 75, 15 and 50, 15 and 40, or 15 and 30 amino acids in length.
- the intracellular signaling domains can be, for example, between 10 and 1000, 10 and 750, 10 and 500, 10 and 250, or 10 and 100 amino acids.
- the intracellular signaling domain can be at least 30, or between 30 and 500, 30 and 250, 30 and 150, 30 and 100, 50 and 500, 50 and 250, 50 and 150, or 50 and 100 amino acids.
- an intracellular signaling domain for a particular gene is at least 90%, 95%, 98%, 99% or 100% identical to at least 10, 25, 30, 40, or 50 amino acids from a sequence of that intracellular signaling domain, such as a sequence provided herein for that intracellular domain, up to the size of the entire intracellular domain sequence, and can include for example, up to an additional 1, 2, 3, 4, 5, 10, 20, or 25 amino acids, provided that such sequence still is capable of providing any of the properties of LEs disclosed herein.
- the lymphoproliferative element is a chimeric cytokine receptor such as but not limited to a cytokine tethered to its receptor that typically constitutively activates the same STAT pathway as a corresponding activated wild-type cytokine receptor such as STAT3, STAT4, and in illustrative embodiments, STAT5.
- the chimeric cytokine receptor is an interleukin, or a fragment thereof, tethered to or covalently attached to its cognate receptor, or a fragment thereof, via a linker.
- the chimeric cytokine receptor is IL7 tethered to IL7Ra (also known as IL7RA).
- the chimeric cytokine receptor is IL-7 tethered to a domain of IL7Ra, such as for example, the extracellular domain of IL-7Ra and/or the transmembrane domain of IL-7Ra.
- the lymphoproliferative element is a cytokine receptor that is not tethered to a cytokine, and in fact in illustrative embodiments, provided herein a lymphoproliferative element is a constitutively active cytokine receptor that is not tethered to a cytokine.
- These chimeric IL-7 receptors typically constitutively activate STAT5 when expressed.
- the lymphoproliferative element can comprise an interleukin polypeptide covalently attached to a portion of its cognate interleukin receptor polypeptide via a linker.
- this portion of the cognate interleukin receptor includes a functional portion of the extracellular domain capable of binding the interleukin cytokine and the transmembrane domain.
- the intracellular domain is an intracellular portion of the cognate interleukin receptor.
- the intracellular domain is an intracellular portion of a different cytokine receptor that is capable of promoting lymphocyte proliferation.
- the lymphoproliferative element is an interleukin polypeptide covalently attached to its full length cognate interleukin receptor polypeptide via a linker.
- the intracellular domain can be derived from a portion of the protein IL7RA.
- the domains, motifs, and point mutations of IL7RA that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IL7RA polypeptides, some of which are discussed in this paragraph.
- the IL7RA protein has an S region rich in serine residues (359-394 of full-length IL7RA, corresponding to residues 96-133 of SEQ ID NO:248), a T region with three tyrosine residues (residues Y401, Y449, and Y456 of full-length IL7RA, corresponding to residues Y138, Y18, and Y193 of SEQ ID NO:248), and a Boxl motif that can bind the signaling kinase Jakl (residues 272-280 of full-length IL7RA corresponding to residues 9-17 of SEQ ID NO:248 and 249) (Jiang, Qiong et al. Mol. and Cell.
- a lymphoproliferative element herein can include one or more, for example all of the domains and motifs of IL7RA disclosed herein or otherwise known to induce proliferation and/or survival of T cells and/or NK cells.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NOs:248 or 249.
- the intracellular domain derived from IL7RA has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, or from about 175 aa to about 200 aa.
- the intracellular domain derived from IL7RA has a length of from about 30 aa to about 200 aa.
- the second intracellular domain can be derived from TNFRSF8.
- the intracellular domain can be derived from a portion of the protein IL12RB.
- the domains, motifs, and point mutations of IL12RB that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IL12RB polypeptides, some of which are discussed in this paragraph.
- IL12RB contains at least one Boxl motif PXXP (SEQ ID NO:306) where each X can be any amino acid (residues 10-12 of SEQ ID NOs:254 and 255; and residues 107-110 and 139-142 of SEQ ID NO:256) (Presky DH et al. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24)).
- a lymphoproliferative element that includes an IL12RB intracellular domain can include one or more of the above Boxl motifs or other motifs, domains, or mutations of IL12RB known to induce proliferation and/or survival of T cells and/or NK cells.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NOs:254-256.
- the intracellular domain derived from IL12RB has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, or from about 200 aa to about 219 aa.
- the intracellular domain derived from IL12RB has a length of from about 30 aa to about 219 aa, for example, 30 aa to 92 aa, or 30 aa to 90 aa.
- the intracellular domain can be derived from a portion of the protein IL31RA.
- the domains, motifs, and point mutations of IL31RA that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IL31RA polypeptides, some of which are discussed in this paragraph.
- Full- length IL31RA contains the Boxl motif PXXP (SEQ ID NO:306) where each X can be any amino acid (corresponding to residues 12-15 of SEQ ID NOs:275 and 276) (Cornelissen C et al. Eur J Cell Biol. 2012 Jun-Jul;9l(6-7):552-66).
- a lymphoproliferative element that includes an IL31RA intracellular domain can include the Boxl motif.
- Full-length IL31RA also contains three
- phosphorylatable tyrosine residues that are important for downstream signaling, Y652, Y683, and Y721 (corresponding to residues Y96, Y237, and Y165 of SEQ ID NO:275; these tyrosine residues are not present in SEQ ID NO:276) (Cornelissen C et al. Eur J Cell Biol. 2012 Jun-Jul;9l(6-7):552-66). All three tyrosine residues contribute to the activation of STAT1, while Y652 is required for STAT5 activation and Y721 recruits STAT3.
- a lymphoproliferative element with an IL31RA intracellular domain includes the Boxl motif and/or the known phosphorylation sites disclosed herein.
- the Boxl motif and phosphorylatable tyrosines of IL31RA are known in the art and a skilled artisan will be able to identify corresponding motifs and phosphorylatable tyrosines in similar IL31RA polypeptides.
- a lymphoproliferative element with an IL31RA intracellular domain does not include the known phosphorylation sites disclosed herein.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NOs:275 or 276.
- the intracellular domain derived from IL31RA has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, or from about 175 aa to about 189 aa.
- the intracellular domain derived from IL31RA has a length of from about 30 aa to about 200 aa, for example, 30 aa to 189 aa, 30 aa to 106 aa.
- the intracellular domain can be derived from an intracellular portion of the transmembrane protein CD40.
- the domains, motifs, and point mutations of CD40 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in CD40 polypeptides, some of which are discussed in this paragraph.
- the CD40 protein contains several binding sites for TRAF proteins.
- binding sites for TRAF1, TRAF2, and TRAF3 are located at the membrane distal domain of the intracellular portion of CD40 and include the amino acid sequence PXQXT (SEQ ID NO:303) where each X can be any amino acid, (corresponding to amino acids 35-39 of SEQ ID NO:208) (Elgueta et al. Immunol Rev. 2009 May; 229(1): 152-72).
- TRAF2 has also been shown to bind to the consensus sequence SXXE (SEQ ID NO:304) where each X can be any amino acid, (corresponding to amino acids 57-60 of SEQ ID NO:208) (Elgueta et al. Immunol Rev. 2009 May; 229(1): 152-72).
- a distinct binding site for TRAF6 is situated at the membrane proximal domain of intracellular portion of CD40 and includes the consensus sequence QXPXEX (SEQ ID NO:305) where each X can be any amino acid (corresponding to amino acids 16-21 of SEQ ID NO:208) (Lu et al. J Biol Chem. 2003 Nov 14; 278(46):454l4-8).
- the intracellular portion of the transmembrane protein CD40 can include all the binding sites for the TRAF proteins.
- the TRAF binding sites are known in the art and a skilled artisan will be able to identify corresponding TRAF binding sites in similar CD40 polypeptides.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:208 or SEQ ID NO:209.
- the intracellular domain derived from CD40 has a length of from about 30 amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to about 65 aa.
- the intracellular domain derived from CD40 has a length of from about 30 aa to about 66 aa, for example, 30 aa to 65 aa, or 50 aa to 66 aa.
- the second intracellular domain can be other than an intracellular domain derived from MyD88, a CD28 family member (e.g. CD28, ICOS), Pattern Recognition Receptor, a C-reactive protein receptor (i.e., Nodi ,
- Pattern Recognition Receptors include, but are not limited to endocytic pattern- recognition receptors (i.e., mannose receptors, scavenger receptors (i.e., Mac-l , LRP, peptidoglycan, techoic acids, toxins, CD1 1 c/CR4)); external signal pattern-recognition receptors (Toll-like receptors (TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), peptidoglycan recognition protein, (PGRPs bind bacterial peptidoglycan, and CD 14); internal signal pattern-recognition receptors (i.e., NOD-receptors 1 & 2), and RIG1
- endocytic pattern- recognition receptors i.e., mannose receptors, scavenger receptors (i.e., Mac-l , LRP, peptidoglycan, techoic acids, toxins, CD1 1
- the intracellular domain can be derived from an intracellular portion of CD27.
- the domains, motifs, and point mutations of CD27 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in CD27 polypeptides, some of which are discussed in this paragraph.
- the serine at amino acid 219 of full-length CD27 (corresponding to the serine at amino acid 6 of SEQ ID NO:205) has been shown to be phosphorylated.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:205.
- the intracellular domain derived from CD27 has a length of from about 30 amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.
- the intracellular domain can be derived from an intracellular portion of CSF2RB.
- the domains, motifs, and point mutations of CSF2RB that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in CSF2RB polypeptides, some of which are discussed in this paragraph.
- Full-length CSF2RB contains a Boxl motif at amino acids 474-482 (corresponding to amino acids 14-22 of SEQ ID NO:213).
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or ah of the amino acids in SEQ ID NO: 213.
- the intracellular domain derived from CSF2RB has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, from about 300 aa to 350 aa, from about 350 aa to about 400 aa, or from about 400 aa to about 450 aa.
- the intracellular domain can be derived from an intracellular portion of IL2RB.
- the domains, motifs, and point mutations of IL2RB that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IL2RB polypeptides, some of which are discussed in this paragraph.
- Full- length IL2RB contains a Boxl motif at amino acids 278-286 (corresponding to amino acids 13-21 of SEQ ID NO:240).
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:240.
- the intracellular domain derived from IL2RB has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, or from about 250 aa to 300 aa.
- the intracellular domain can be derived from an intracellular portion of IL6ST.
- the domains, motifs, and point mutations of IL6ST that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IL6ST polypeptides, some of which are discussed in this paragraph.
- Full- length IL6ST contains a Boxl motif at amino acids 651-659 (corresponding to amino acids 10-18 of SEQ ID NO:247).
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or ah of the amino acids in SEQ ID NO:246 or SEQ ID NO:247.
- the intracellular domain derived from IL6ST has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, or from about 250 aa to 300 aa.
- the intracellular domain can be derived from an intracellular portion of IL17RE.
- the domains, motifs, and point mutations of IL17RE that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IL17RE polypeptides, some of which are discussed in this paragraph.
- Full- length IL17RE contains a TIR domain at amino acids 372-495 (corresponding to amino acids 13-136 of SEQ ID NO:265).
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:265.
- the intracellular domain derived from IL17RE has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, or from about 175 aa to about 200 aa.
- the intracellular domain can be derived from an intracellular portion of IL2RG.
- the domains, motifs, and point mutations of IL2RG that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IL2RG polypeptides, some of which are discussed in this paragraph.
- Full- length IL2RG contains a Boxl motif at amino acids 286-294 (corresponding to amino acids 3-11 of SEQ ID NO:241).
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:241.
- the intracellular domain derived from IL2RG has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, or from about 70 aa to about 100 aa.
- the intracellular domain can be derived from an intracellular portion of IL18R1.
- the domains, motifs, and point mutations of IL18R1 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IL18R1 polypeptides, some of which are discussed in this paragraph.
- Full- length IL18R1 contains a TIR domain at amino acids 222-364 (corresponding to amino acids 28-170 of SEQ ID NO:266).
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:266.
- the intracellular domain derived from IL18R1 has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, or from about 70 aa to about 100 aa.
- the intracellular domain can be derived from an intracellular portion of IL27RA.
- the domains, motifs, and point mutations of IL27RA that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IL27RA polypeptides, some of which are discussed in this paragraph.
- Full- length IL27RA contains a Boxl motif at amino acids 554-562 (corresponding to amino acids 17-25 of SEQ ID NO:273).
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:273 or SEQ ID NO:274.
- the intracellular domain derived from IL27RA has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, or from about 70 aa to about 100 aa.
- the intracellular domain can be derived from an intracellular portion of IFNGR2.
- the domains, motifs, and point mutations of IFNGR2 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IFNGR2 polypeptides, some of which are discussed in this paragraph.
- Full- length IFNGR2 contains a dileucine internalization motif at amino acids 276-277 (corresponding to amino acids 8-9 of SEQ ID NO:230).
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:230.
- the intracellular domain derived from IFNGR2 has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
- the intracellular domain can be derived from a portion of the protein MyD88.
- the domains, motifs, and point mutations of MyD88 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in MyD88 polypeptides, some of which are discussed in this paragraph.
- the MyD88 protein has an N-terminal death domain that mediates interactions with other death domain- containing proteins (corresponding to amino acids 29-106 of SEQ ID NO:284), an intermediate domain that interacts with IL-1R associated kinase (corresponding to amino acids 107-156 of SEQ ID NO:284), and a C-terminal TIR domain (corresponding to amino acids 160-304 of SEQ ID NO:284) that associates with the TLR-TIR domain (Biol Res. 2007; 40(2): 97- 112).
- MyD88 also has canonical nuclear localization and export motifs.
- Point mutations have been identified in MyD88 and include the loss-of-function mutations L93P and R193C (corresponding to L93P and R196C in SEQ ID NO:284), and the gain-of- function mutation L265P (corresponding to L260P in SEQ ID NO:284) (Deguine and Barton.
- a lymophoproliferative element herein can include one or more, for example all of the domains and motifs of MyD88 disclosed herein.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:284-293, and in illustrative embodiments includes one or more, in illustrative embodiments all, of the following MyD88 domains/motifs: the death domain, the intermediate domain, the TIR domain, the nuclear localization and export motifs, an amino acid corresponding to position L93, R193, and L265 or P265.
- the intracellular domain derived from MyD88 has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, or from about 300 aa to 350 aa.
- the intracellular domain derived from MyD88 has a length of from about 30 aa to about 350 aa, for example, 50 aa to 350 aa, or 100 aa to 350 aa, 100 aa to 304 aa, 100 aa to 296 aa, 100 aa to 251 aa, 100 aa to 191 aa, 100 aa to 172 aa, 100 aa to 146 aa, or 100 aa to 127 aa.
- the second intracellular domain can be derived from the first intracellular domain derived from MyD88.
- the second intracellular domain can be other than an intracellular domain derived from a CD28 family member (e.g. CD28, ICOS), Pattern Recognition Receptor, a C-reactive protein receptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor (i.e., CD40,
- CD28 family member e.g. CD28, ICOS
- Pattern Recognition Receptor e.g. CD28, ICOS
- C-reactive protein receptor i.e., Nodi, Nod2, PtX3-R
- TNF receptor i.e., CD40
- RANK/TRANCE-R 0X40, 4-1BB
- HSP receptor Lix-l and CD91
- CD28 CD28
- the intracellular domain can be derived from a portion of the transmembrane protein MPL.
- the domains, motifs, and point mutations of MPL that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in MPL polypeptides, some of which are discussed in this paragraph.
- the transmembrane MPL protein contains the Boxl motif PXXP (SEQ ID NO:306) where each X can be any amino acid (corresponding to amino acids 17-20 in SEQ ID NO:283) and the Box2 motif, a region with increased serine and glutamic acid content (corresponding to amino acids 46-64 in SEQ ID NO:283) (Drachman and Kaushansky. Proc Natl Acad Sci U S A. 1997 Mar 18; 94(6):2350-5).
- the Boxl and Box2 motifs are involved in binding to JAKs and signal transduction, although the Box2 motif presence is not always required for a proliferative signal (Murakami et al. Proc Natl Acad Sci U S A.
- cytokine receptors have hydrophobic residues at positions -1, -2, and -6 relative to the Boxl motif (corresponding to amino acids 16, 15, and 11, respectively, of SEQ ID NO:283), that form a“switch motif,” which is required for cytokine-induced JAK2 activation but not for JAK2 binding (Constantinescu et al. Mol Cell. 2001 Feb; 7(2):377-85; and Huang et al. Mol Cell.
- a MPL intracellular signaling domain does not comprise the region comprising amino acids 70-95 in SEQ ID NO:283.
- the lysines K553 corresponding to K40 of SEQ ID NO: 283
- K573 corresponding to K60 of SEQ ID NO: 283
- a MPL intracellular signaling domain does not comprise these ubiquitination targeting motif residues.
- the tyrosines Y521 (corresponding to Y8 of SEQ ID NO: 283), Y542 (corresponding to Y29 of SEQ ID NO:283), Y591 (corresponding to Y78 of SEQ ID NO: 283), Y626 (corresponding to Y113 of SEQ ID NO: 283), and Y631 (corresponding to Y118 of SEQ ID NO: 283) have been shown to be phosphorylated (Varghese et al. Front Endocrinol (Lausanne). 2017 Mar 31; 8:59).
- Y521 and Y591 of full-length MPL are negative regulatory sites that function either as part of a lysosomal targeting motif (Y521) or via an interaction with adaptor protein AP2 (Y591) (Drachman and Kaushansky. Proc Natl Acad Sci U S A. 1997 Mar 18; 94(6):2350-5; and Hitchcock et al. Blood. 2008 Sep 15; 112(6):2222-31).
- Y626 and Y631 of full-length MPL are positive regulatory sites (Drachman and Kaushansky. Proc Natl Acad Sci U S A.
- MPL contains the She phosphotyrosine-binding binding motif NXXY (SEQ ID NO:307) where each X can be any amino acid (corresponding to amino acids 110-113 of SEQ ID NO: 283), and this tyrosine is phosphorylated and important for the TPO-dependent
- MPL also contains the STAT3 consensus binding sequence YXXQ (SEQ ID NO:308) where each X can be any amino acid
- MPL also contains the sequence YLPL (SEQ ID NO: 309) (corresponding to amino acid 113-116 of SEQ ID NO: 283), which is similar to the consensus binding site for STAT5 recruitment pYLXL (SEQ ID NOG 10) where pY is phosphotyrosine and X can be any amino acid (March et al. FEBS Lett. 1996 Sep 30; 394(2):221-6).
- YLPL SEQ ID NO: 309
- pYLXL SEQ ID NOG 10
- pY is phosphotyrosine
- X can be any amino acid
- the intracellular portion of MPL can include one or more, or all the domains and motifs described herein that are present in SEQ ID NO: 283.
- a transmembrane portion of MPL can include one or more, or all the domains and motifs described herein that are present in SEQ ID NO: 187.
- the domains, motifs, and point mutations of MPL provided herein are known in the art and a skilled artisan would recognize that MPL intracellular signaling domains herein in illustrative embodiments would include one or more corresponding domains, motifs, and point mutations in that have been shown to promote proliferative activity and would not include that that have been shown to inhibit MPLs proliferative activity.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 283.
- the intracellular domain derived from MPL has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, from about 300 aa to 350 aa, from about 350 aa to about 400 aa, from about 400 aa to about 450 aa, from about
- the intracellular domain derived from MPL has a length of from about 30 aa to about 200 aa, for example, 30 aa to 150 aa, 30 aa to 119 aa, 30 aa to 121 aa, 30 aa to 122 aa, or 50 aa to 125 aa.
- the second intracellular domain can be derived from CD79B.
- the intracellular domain can be derived from a portion of the intracellular domain
- CD79B transmembrane protein CD79B, also known as B29; IGB; AGM6.
- the domains, motifs, and point mutations of CD79B that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in CD79B polypeptides, some of which are discussed in this paragraph.
- CD79B contains an IT AM motif at residues 193-212 (corresponding to amino acids 16-30 of SEQ ID NO:2l l).
- CD79B has two tyrosines that are known to be phosphorylated, Y196 and Y207 (corresponding to Y16 and Y27 of SEQ ID NO: 211).
- the intracellular portion of the transmembrane protein CD79B includes the IT AM motif and/or the known phosphorylation sites disclosed herein.
- the motif and phosphorylatable tyrosines of CD79B are known in the art and a skilled artisan will be able to identify corresponding motifs and phosphorylatable tyrosines in similar CD79B polypeptides.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 211.
- the intracellular domain derived from CD79B has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.). In illustrative embodiments, the intracellular domain derived from CD79B has a length of from about 30 aa to about 50 aa.
- a suitable CD79B intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids of the following sequence: LDKDDSKAGMEEDHT[YEGLDIDQTATYEDI]VTLRTGEVKWSVGEHPGQE (SEQ ID NO: 211), where the IT AM motif is set out in brackets.
- the first intracellular domain can be derived from CSF3R.
- the intracellular domain can be derived from a portion of the intracellular domain
- OSMR transmembrane protein transmembrane protein transmembrane protein.
- the domains, motifs, and point mutations of OSMR that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in OSMR polypeptides, some of which are discussed in this paragraph.
- OSMR contains a Boxl motif at amino acids 771-779 of isoform 3
- OSMR has two serines at amino acids 829 and 890 of isoform 3 that are known to be phosphorylated (serines at amino acids 65 and 128 of SEQ ID NO:294).
- the intracellular portion of the protein OSMR can include the Boxl motif and the known phosphorylation sites disclosed herein.
- the motif and phosphorylatable serines of OSMR are known in the art and a skilled artisan will be able to identify corresponding motifs and phosphorylatable serines in similar OSMR polypeptides.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:294.
- the intracellular domain derived from OSMR has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa., from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, or from about 200 aa to about 250 aa.
- the intracellular domain can be derived from a portion of the intracellular domain
- PRLR transmembrane protein PRLR.
- the domains, motifs, and point mutations of PRLR that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in PRLR polypeptides, some of which are discussed in this paragraph.
- PRLR contains a growth hormone receptor binding domain at amino acids 185-261 of isoform 6 (corresponding to amino acids 28-104 of SEQ ID NO:295).
- the growth hormone receptor binding domain of PRLR is known in the art and a skilled artisan will be able to identify corresponding domain in similar PRLR polypeptides.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:295.
- the intracellular domain derived from PRLR has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, from about 300 aa to 350 aa, or from about 350 aa to about 400 aa.
- an intracellular domain of a lymphoproliferative element is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki- 1).
- the intracellular domain can be derived from a portion of the protein CD28.
- the domains, motifs, and point mutations of CD28 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in CD28 polypeptides, some of which are discussed in this paragraph.
- Full- length CD28 contains a PI3-K- and Grb2-binding motif that corresponds to residues 12-15 of SEQ ID NOs:206 and 207 (Flarada et al. J Exp Med. 2003 Jan 20;l97(2):257-62).
- a lymphoproliferative element that includes a CD28 intracellular domain can include the PI3-K- and Grb2- binding motif.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or ah of the amino acids in SEQ ID NOs:206 or 207.
- the intracellular domain derived from CD28 has a length of from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, or from about 35 aa to about 42 aa.
- the intracellular domain can be derived from a portion of the protein ICOS.
- the domains, motifs, and point mutations of ICOS that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in ICOS polypeptides, some of which are discussed in this paragraph.
- ICOS binds PI3-K and not Grb2.
- the PI3-K-binding motif of full-length ICOS corresponds to residues 19-22 of SEQ ID NO:225.
- a single amino acid substitution in this motif can lead to Grb2 binding by ICOS and increased IL-2 production (Harada et al. J Exp Med. 2003 Jan 20;l97(2):257-62).
- This mutation corresponds to mutating phenylalanine 21 of SEQ ID NO:225 to an asparagine.
- a skilled artisan will understand how to mutate this residue in SEQ ID NO:225 and generate an ICOS intracellular domain that binds Grb2 in addition to PI3-K.
- a lymphoproliferative element that includes an ICOS intracellular domain can include the PI3-K-binding motif.
- a lymphoproliferative element that includes an ICOS intracellular domain can include the PI3-K-binding motif that has been mutated to additionally bind Grb2.
- ICOS also contains a membrane proximal motif in the cytoplasmic tail that is essential for ICOS-assisted calcium signaling (Leconte et al. Mol Immunol. 2016 Nov;79:38-46). This calcium signaling-motif corresponds to residues 5-8 of SEQ ID NO:225.
- a lymphoproliferative element that includes an ICOS intracellular domain can include the calcium-signaling motif. Two other conserved motifs have been identified in full-length ICOS.
- a lymphoproliferative element that includes an ICOS intracellular domain can include at least one of the first or second conserved motifs.
- a lymphoproliferative element that includes an ICOS intracellular domain does not include the first conserved motif, does not include the second conserved motif, or does not include the first and second conserved motifs.
- a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 225.
- the intracellular domain derived from ICOS has a length of from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, or from about 35 aa to about 38 aa.
- an intracellular domain of a chimeric lymphoproliferative element is derived from an intracellular portion of the transmembrane protein 0X40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGP1L).
- 0X40 also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGP1L.
- the domains, motifs, and point mutations of 0X40 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in 0X40 polypeptides, some of which are discussed in this paragraph.
- 0X40 contains a TRAF binding motif at residues 256-263 of full- length 0X40 (corresponding to residues 20-27 of SEQ ID NO:296) that are important for binding TRAF1, TRAF2, TRAF3, and TRAF5 (Kawamata, S, et al. J Biol Chem. 1998 Mar 6;273(l0):5808-l4; Hori, T. Int J Hematol. 2006 Jan;83(l): 17-22).
- Full-length 0X40 also contains a p85 PI3K binding motif at residues 34-57.
- 0X40 when 0X40 is present as an intracellular domain of a lymphoproliferative element, it includes the p85 PI3K binding motif of 0X40.
- an intracellular domain of 0X40 can include the TRAF binding motif of 0X40. In some embodiments, an intracellular domain of 0X40 can bind TRAF1, TRAF2, TRAF3, and TRAF5. Lysines corresponding to amino acids 17 and 41 of SEQ ID NO: 296 are potentially negative regulatory sites that function as parts of ubiquitin targeting motifs. In some embodiments, one or both of these lysines in the intracellular domain of 0X40 are mutated arginines or another amino acid.
- a suitable intracellular domain of a lymphoproliferative element can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:57.
- the intracellular domain of 0X40 has a length of from about 20 aa to about 25 aa, about 25 aa to about 30 aa, 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.
- the intracellular domain of 0X40 has a length of from about 20 aa to about 50 aa, for example 20 aa to 45 aa, or 20 aa to 42 aa.
- an intracellular domain of a chimeric lymphoproliferative element is derived from an intracellular portion of the transmembrane protein IFNAR2.
- the domains, motifs, and point mutations of IFNAR2 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in IFNAR2 polypeptides, some of which are discussed in this paragraph.
- Full-length IFNAR2 contains a Boxl motif and two Box2 motifs (known as Box2A and Box2B). (Usacheva A et al. J Biol Chem. 2002 Dec l3;277(50):48220-6).
- a lymphoproliferative element that includes a IFNAR2 intracellular domain can include one or more of the Boxl or Box2 motifs.
- the IFNAR2 intracellular domain can include one or more of the Boxl, Box2A, or Box2B motifs.
- IFNAR2 contains a JAKl-binding site (Gauzzi MC et al. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):l l839-44; Schindler et al. J Biol Chem. 2007 Jul l3;282(28):20059-63).
- a JAKl-binding site Gauzzi MC et al. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):l l839-44; Schindler et al. J Biol Chem. 2007 Jul l3;282(28):20059-63).
- a JAKl-binding site Gauzzi MC et al
- lymphoproliferative element that includes a IFNAR2 intracellular domain can include the JAKl-binding site.
- a suitable intracellular domain of a lymphoproliferative element can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NOs:227 or 228.
- the intracellular domain of IFNAR2 has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, or from about 200 aa to about 251 aa.
- the intracellular domain of 0X40 has a length of from about 30 aa to about 251 aa, for example 30 aa to 67 aa.
- an intracellular domain of a chimeric lymphoproliferative element is derived from an intracellular portion of the transmembrane protein CSF3R.
- the domains, motifs, and point mutations of CSF3R that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in CSF3R polypeptides, some of which are discussed in this paragraph.
- Full-length CSF3R contains a Boxl and Box2 motif as well as a Box3 motif (Nguyen-Jackson F1T et al. G-CSF Receptor Structure, Function, and Intracellular Signal Transduction.
- a lymphoproliferative element that includes a CSF3R intracellular domain can include one or more of the Boxl, Box2, or Box3 motifs.
- CSF3R contains four tyrosine residues, Y704, Y729, Y744, and Y764 in full-length CSF3R, that are important for binding STAT3 (Y704 and Y744), SOCS3 (Y729), and Grb2 and p2lRas (Y764).
- a lymphoproliferative element that includes a CSF3R intracellular domain can include one, two, three, or all of the tyrosine residues corresponding to Y704, Y729, Y744, and Y764 of full-length CSF3R.
- CSF3R contains two threonine residues, T615 and T618 in full-length CSF3R, that can increase receptor dimerization and activity when mutated to alanine and isoleucine, respectively (T615A and T618I) (Maxson et al. J Biol Chem. 2014 Feb 28;289(9):5820-7).
- a lymphoproliferative element that includes a CSF3R intracellular domain can include one or more of the mutations corresponding to T615A and T618I.
- a suitable intracellular domain of a lymphoproliferative element can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NOs:2l6, 217, or 218.
- the intracellular domain of CSF3R has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, or from about 200 aa to about 213 aa.
- the intracellular domain of CSF3R has a length of from about 30 aa to about 213 aa, for example from about 30 aa to about 186 or from about 30 aa to about 133 aa.
- an intracellular domain of a chimeric lymphoproliferative element is derived from an intracellular portion of the transmembrane protein EPOR.
- the domains, motifs, and point mutations of EPOR that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in EPOR polypeptides, some of which are discussed in this paragraph.
- EPOR contains a Boxl (residues 257-264 of full-length EPOR) and Box2 (residues 303-313 of full-length EPOR) motif (Constantinescu SN. Trends Endocrinol Metab.
- a lymphoproliferative element that includes an EPOR intracellular domain can include one or more of the Boxl, Box2, or extended Box2 motifs.
- EPOR also contains a short segment important for EPOR internalization (residues 267-276 of full-length EPOR).
- a lymphoproliferative element that includes an EPOR intracellular domain does not include the internalization segment.
- a suitable intracellular domain of a lymphoproliferative element can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NOs:2l9 or 220.
- the intracellular domain of EPOR has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, or from about 200 aa to about 235 aa.
- the intracellular domain of EPOR has a length of from about 30 aa to about 235 aa.
- an intracellular domain of a chimeric lymphoproliferative element is derived from an intracellular portion of the transmembrane protein CD3G.
- the domains, motifs, and point mutations of CD3G that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in CD3G polypeptides, some of which are discussed in this paragraph.
- Two serine residues, S123 and S126 of full- length CD3G have been shown to be phosphorylated in T cells in response to ionomycin (Davies et al. J Biol Chem. 1987 Aug 15;262(23):10918-21).
- a lymphoproliferative element that includes a CD3G intracellular domain can include one or more of the serine residues corresponding to full-length S123 and S126. Furthermore, phosphorylation at S 126 but not S 123 was shown to be required for PKC-mediated down-regulation (Dietrich J et al. EMBO J. 1994 May 1;13(9):2156- 66). In some embodiments, a lymphoproliferative element that includes a CD3G intracellular domain can include the serine residue corresponding to full-length S123 and not include serine residue corresponding to full-length S126.
- a lymphoproliferative element that includes a CD3G intracellular domain can include a non-phosphorylatable amino acid substitution at the serine residue corresponding to full-length S126.
- the amino acid substitution can be a serine to alanine mutation.
- leucine to alanine mutations of either leucine of a di-leucine motif can include a non-phosphorylatable amino acid substitution at the serine residue corresponding to full-length S126.
- lymphoproliferative element that includes a CD3G intracellular domain can include at least one amino acid substitution at the leucine residues corresponding to L131 or L 132 of full-length CD3G.
- the amino acid substitution can be a leucine to alanine mutation.
- a suitable intracellular domain of a lymphoproliferative element can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 199. In some of these
- the intracellular domain of CD3G has a length of from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, or from about 40 aa to about 45 aa.
- the intracellular domain of CD3D has a length of from about 30 aa to about 45 aa.
- TNFRs TNF receptors
- TNFRSF4 TNF receptor-associated factors
- DD “death domain”
- the domains, motifs, and point mutations of TNFRs that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in TNFR polypeptides, some of which are discussed in this paragraph.
- TRAFs TNF receptor-associated factors
- DD “death domain”
- the domains, motifs, and point mutations of TNFRs that induce proliferation and/or survival of T cells and/or NK cells are known in the art and a skilled artisan can identify corresponding domains, motifs, and point mutations in TNFR polypeptides, some of which are discussed in this paragraph.
- mammals there are at least six TRAF molecules and a number of nonreceptor DD molecules.
- TRAF-binding motif is a roughly 60 amino acid globular bundle of 6 conserved a-helices that is also known in the art (Focksley RM et al. Cell. 2001 Feb 23;l04(4):487-50l).
- a skilled artisan will be able to identify the TRAF- and/or DD-binding motif in the different TNFR families using, for example, sequence alignments to known binding motifs.
- TNFRs can recruit TRADD and TRAF2, resulting in the activation of NF-kB, MAPK, and JNK (Sedger and McDermott. Cytokine Growth Factor Rev. 2014 Aug;25(4):453-72).
- a lymphoproliferative element that includes a TNFR intracellular domain can include one or more TRAF-binding motifs.
- a lymphoproliferative element that includes a TNFR intracellular domain does not include a DD-binding motif, or has one or more DD-binding motifs deleted or mutated within the intracellular domain.
- a lymphoproliferative element that includes a TNFR intracellular domain can recruit TRADD and/or TRAF2.
- TNFRs also include cysteine -rich domains (CRDs) that are important for ligand binding (Focksley RM et al. Cell. 2001 Feb 23;l04(4):487-50l).
- CRDs cysteine -rich domains
- a lymphoproliferative element that includes a TNFR intracellular domain does not include a TNFR CRD.
- Fymphoproliferative elements and CFEs that can be included in any of the aspects disclosed herein can be any of the FEs or CFEs disclosed in WO2019/055946.
- CLEs were disclosed therein that promoted proliferation in cell culture of PBMCs that were transduced with lentiviral particles encoding the CFEs between day 7 and day 21, 28, 35 and/or 42 after transduction.
- CFEs were identified therein, that promoted proliferation in vivo in mice in the presence or absence of an antigen recognized by a CAR, wherein T cells expressing one of the CLEs and the CAR were introduced into the mice.
- tests and/or criteria can be used to identify whether any test polypeptide, including LEs, or test domains of an LE, such as a first intracellular domain, or a second intracellular domain, or both a first and second intracellular domain, are indeed LEs or effective intracellular domains of LEs, or especially effective LEs or intracellular domains of LEs.
- any aspect or other embodiment provided herein that includes an LE or a polynucleotide or nucleic acid encoding an LE can recite that the LE meets, or provides the property of, or is capable of providing and/or possesses the property of, any one or more of the identified tests or criteria for identifying an LE provided herein, or that a cell genetically modified and/or transduced with a retroviral particle, such as a lentiviral particle encoding the LE, is capable of providing, is adapted for, possesses the property of, and/or is modified for achieving the results of one or more of the recited tests.
- the LE provides, is capable of providing and/or possesses the property of, (or a cell genetically modified and/or transduced with a retroviral particle encoding the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) improved expansion to pre-activated PBMCs transduced with a lenti virus comprising a nucleic acid encoding the LE and an anti-CD 19 CAR comprising a CD3 zeta intracellular activating domain but no co-stimulatory domain, between day 7 and day 21, 28, 35, and/or 42 of in vitro culturing post-transduction in the absence of exogenously added cytokines, compared to a control retroviral particle, e.g.
- a lymphoproliferative element test for improved or enhanced survival, expansion, and/or proliferation of cells transduced with a retroviral particle e.g. lentiviral particle
- a retroviral particle e.g. lentiviral particle having a genome encoding a test construct encoding a putative LE (test cells)
- test cells can be performed based on a comparison to control cells, which can be, for example, either untransduced cells or cells transduced with a control retroviral (e.g. lentiviral) particle identical to the lentiviral particle comprising the nucleic acid encoding the
- control cells are transduced with a retroviral particle (e.g. lentiviral particle) having a genome encoding a lymphoproliferative element or intracellular domain(s) thereof, identified herein as exemplifying a lymphoproliferative element.
- a retroviral particle e.g. lentiviral particle
- the test criteria can include that there is at least as much enrichment, survival and/or expansion, or no statistical difference of enrichment, survival, and/or expansion when the test is performed using a retroviral particle (e.g. lentiviral particle) having a genome encoding a test construct versus encoding the control lymphoproliferative element, typically by analyzing cells transcribed therewith.
- a retroviral particle e.g. lentiviral particle
- Exemplary or illustrative embodiments of lymphoproliferative elements herein, in some embodiments, are illustrative embodiments of control lymphoproliferative elements for such a test.
- this test for an improved property of a putative or test lymphoproliferative element is performed by performing replicates and/or performing a statistical test.
- a skilled artisan will recognize that many statistical tests can be used for such a lymphoproliferative element test.
- the statistical test can be a T-test or a Mann-Whitney-Wilcoxon test.
- the normalized enrichment level of a test construct is significant at a p-value of less than 0.1 , or less than 0.05, or less than 0.01.
- the LE provides, is capable of providing and/or possesses the property of (or a cell genetically modified and/or transduced with the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold expansion, or between 1.5 fold and 25-fold expansion, or between 2-fold and 20-fold expansion, or between 2-fold and 15-fold expansion, or between 5-fold and 25-fold expansion, or between 5-fold and 20-fold expansion, or between 5-fold and 15-fold expansion, of pre activated PBMCs transduced with a nucleic acid encoding the LE when transduced along with an anti- CD 19 CAR comprising a CD3 zeta intracellular activating domain but no co-stimulatory domain, between day 7 and day 21, 28, 35, and/or 42 of in vitro culturing in the absence of exogenously added cyto
- the test is performed in the presence of PBMCs, for example at a 1:1 ratio of transduced cells to PBMCs, which can be for example, from a matched donor, and in some embodiments, the test is performed in the absence of PBMCs.
- the analysis of expansion for any of these tests is performed as illustrated in WO2019/055946.
- the test can include a further statistical test and a cut-off such as a P value below 0.1, 0.05, or 0.01, wherein a test polypeptide or nucleic acid encoding the same, needs to meet one or both thresholds (i.e. fold expansion and statistical cutoff).
- the number of test cells and the number of control cells can be compared between day 7 and day 14, 21, 28, 35, 42 or 60 post
- the numbers of test and control cells can be determined by sequencing DNA and counting the occurrences of unique identifiers present in each construct. In some embodiments, the numbers of test and control cells can be counted directly, for example with a hemocytometer or a cell counter. In some embodiments, ah the test cells and control cells can be grown within the same vessel, well or flask. In some embodiments, the test cells can be seeded in one or more wells, flasks or vessels, and the control cells can be seeded in one or more flasks or vessels. In some embodiments, test and control cells can be seeded individually into wells or flasks, e.g., one cell per well.
- the numbers of test cells and control cells can be compared using enrichment levels.
- the enrichment level for a test or control construct can be calculated by dividing the number of cells at a later time point (day 14, 21, 28, 35, or day 45) by the number of cells at day 7 for each construct.
- the enrichment level for a test or control construct can be calculated by dividing the number of cells at a time point (day 14, 21, 28, 35, or day 45) by the number of cells at that time point for untransduced cells.
- the enrichment level of each test construct can be normalized to the enrichment level of the respective control construct to generate a normalized enrichment level.
- a LE encoded in the test construct provides (or a cell genetically modified and/or transduced with a retroviral particle (e.g. lentiviral particle) having a genome encoding the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or lO-fold normalized enrichment level, or between 1.5 fold and 25-fold normalized enrichment level, or between 3-fold and 20- fold normalized enrichment level, or between 5-fold and 25-fold normalized enrichment level, or between 5-fold and 20-fold normalized enrichment level, or between 5-fold and 15-fold normalized enrichment level.
- a retroviral particle e.g. lentiviral particle
- Enrichment can be measured, for example, by direct cell counting. Cutoff values can be based on a single test, or two, three, four, or five repeats, or based on many repeats. The cutoff can be met when a lymphoproliferative element meets one or more repeat tests, or meets or exceeds a cutoff for all repeats.
- the enrichment is measured as log2((normalized count data on the test day + l)/(normalized count data on day 7 + 1)).
- CLEs were identified from libraries of constructs that included constructs that encoded test chimeric polypeptides that were designed to comprise an intracellular domain believed to induce proliferation and/or survival of lymphoid or myeloid cells, and an anti-CD 19 CAR that comprised an intracellular activating domain but not a co-stimulatory domain.
- Preactivation which was performed overnight at 37 °C, was performed in a preactivation reaction mixture comprising PBMCs, a commercial media for lymphocytes (Complete OpTmizerTM CTSTM T-Cell Expansion SFM), recombinant human interleukin-2 (lOOIU/ml) and anti-CD3 Ab (OKT3) (50ng/ml).
- transduction was performed overnight at 37 °C after addition of test and control lentiviral particles to the preactivation reaction mixtures at a multiplicity of infection (MOI) of 5.
- MOI multiplicity of infection
- Some control lentiviral particles contained constructs encoding polypeptides with extracellular and transmembrane domains but no intracellular domains.
- the test lentiviral particles contained constructs encoding polypeptides with extracellular and transmembrane domains and either one or two intracellular domains.
- Complete OpTmizerTM CTSTM T-Cell Expansion SFM was added to dilute the reaction mixture 5- to 20-fold and the cells were cultured for up to 45 days at 37 °C.
- test constructs were identified as CLEs because the CLEs induced proliferation/expansion in these fed or unfed cultures without added cytokines such as IL-2 between days 7 and day 21, 28, 35, and/or 42.
- effective CLEs were identified by identifying test CLEs that provided increased expansion of these in vitro cultures, whether fed or unfed with untransduced PBMCs, between day 7 and day 21, 28, 35, and/or 42 post-transduction, compared to control constructs that did not include any intracellular domains.
- WO2019/055946 discloses that at least one and typically more than one test CLE that included an intracellular domain from a test gene provided more expansion than every control construct that was present at day 7 post-transduction, that did not include an intracellular domain.
- WO2019/055946 further provides a statistical method that was used to identify exceptionally effective genes with respect to a first intracellular domain, and one or more exemplary intracellular domain(s) from these genes. The method used a Mann-Whitney-Wilcoxon test and a false discovery cutoff rate of less than 0.1 or less than 0.05.
- WO2019/055946 identified especially effective genes for the first intracellular domain or the second intracellular domain, for example, by analyzing scores for genes calculated as combined score for all constructs with that gene. Such analysis can use a cutoff of greater than 1 , or greater than negative control constructs without any intracellular domains, or greater than 2, as shown for some of the tests disclosed in WO2019/055946.
- the LE provides, is capable of providing and/or possesses the property of (or a cell genetically modified and/or transduced with the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) driving T cell expansion in vivo.
- the in vivo test can utilize a mouse model and measure T cell expansion at 15 to 25 days in vivo, or at 19 to 21 days in vivo, or at approximately 21 days in vivo, after T cells are contacted with lenti viral vectors encoding the LEs, are introduced into the mice, as disclosed in WO2019/055946,
- the genetically modified cell is modified so as to possess new properties not previously possessed by the cell before genetic modification and/or transduction.
- a property can be provided by genetic modification with a nucleic acid encoding a CAR or a LE, and in illustrative embodiments both a CAR and a LE.
- the genetically modified and/or transduced cell is capable of, is adapted for, possesses the property of, and/or is modified for survival and/or proliferation in ex vivo culture for at least 7, 14, 21, 28, 35, 42, or 60 days or from between day 7 and day 14, 21, 28, 35, 42 or 60 post-transduction, in the absence of added IL-2 or in the absence of added cytokines such as IL-2, IL-15, or IL-7, and in certain illustrative embodiments, in the presence of the antigen recognized by the CAR where the method comprises genetically modifying using a retroviral particle having a pseudotyping element and optionally a separate or fused activation domain on its surface and typically does not require pre-activation.
- the genetically modified and/or transduced cell exhibits, is capable of, is adapted for, possesses the property of, and/or is modified for improved survival or expansion in ex vivo or in vitro culture in culture media in the absence of one or more added cytokines such as IL-2, IL-15, or IL-7, or added lymphocyte mitogenic agent, compared to a control cell(s) identical to the genetically modified and/or transduced cell(s) before it was genetically modified and/or transduced or to a control cell that was transduced with a retroviral particle identical to an on-test retroviral particle that comprises an LE or a putative LE, but without the LE or the intracellular domains of the LE, wherein said survival or proliferation of said control cell(s) is promoted by adding said one or more cytokines, such as IL-2, IL-15, or IL-7, or said lymphocyte mitogenic agent to the culture media.
- cytokines such as IL-2, IL-15, or IL-7
- cytokine or lymphocyte mitogenic agent By added cytokine or lymphocyte mitogenic agent, it is meant that cytokine or lymphocyte mitogenic agent is added from an exogenous source to a culture media such that the concentration of said cytokine or lymphocyte mitogenic agent is increased in the culture media during culturing of the cell(s) compared to the initial culture media, and in some embodiments can be absent from the initial culture media before said adding.
- cytokine or lymphocyte mitogenic agent is added from an exogenous source to a culture media such that the concentration of said cytokine or lymphocyte mitogenic agent is increased in the culture media during culturing of the cell(s) compared to the initial culture media, and in some embodiments can be absent from the initial culture media before said adding.
- cytokine or lymphocyte mitogenic agent By“added” or“exogenously added”, it is meant that such cytokine or lymphocyte mitogenic agent is added to a lymphocyte media used to culture the genetically modified and/or transduced cell after the
- All or a portion of the media that includes a mixture of multiple media components is typically stored and in illustrative embodiments has been shipped to a site where the culturing takes place, without the exogenously added cytokine(s) or lymphocyte mitogenic agent(s).
- the lymphocyte media in some embodiments is purchased from a supplier, and a user such as a technician not employed by the supplier and not located within a supplier facility, adds the exogenously added cytokine or lymphocyte mitogenic agent to the lymphocyte media and then the genetically modified and/or transduced cells are cultured in the presence or absence of such exogenously added cytokine or lymphocyte mitogenic agent.
- improved or enhanced survival, expansion, and/or proliferation can be shown as an increase in the number of cells determined by sequencing DNA from cells transduced with retroviral particle (e.g. lentiviral particle) having a genome encoding CLEs and counting the occurrences of sequences present in unique identifiers from each CLE.
- retroviral particle e.g. lentiviral particle
- improved survival and/or improved expansion can be determined by counting the cells directly, for example with a hemocytometer or a cell counter, at each time point.
- improved survival and/or improved expansion and/or enrichment can be calculated by dividing the number of cells at the later time point (day 21, 28, 35, and/or day 45) by the number of cells at day 7 for each construct.
- the cells can be counted by hemocytometer or cell counters.
- the enrichment level determined using the nucleic acid counts or the cell counts of each specific test construct can be normalized to the enrichment level of the respective control construct, i.e., the construct with the same extracellular domain and transmembrane domain but lacking the intracellular domains present in the test construct.
- the LE encoded in the construct provides (or a cell genetically modified and/or transduced with a retroviral particle (e.g.
- lentiviral particle) having a genome encoding the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold normalized enrichment level, or between 1.5 fold and 25-fold normalized enrichment level, or between 3-fold and 20-fold normalized enrichment level, or between 5-fold and 25-fold normalized enrichment level, or between 5- fold and 20-fold normalized enrichment level, or between 5-fold and 15-fold normalized enrichment level.
- the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes an intracellular signaling domain from any of the genes having a P3 signaling domain with or without a P4 domain, or from any of the genes having a P4 domain wherein the P3 domain was a linker, in the CLEs identified in Tables 4 to 8 herein, which promote T cell, e.g. CAR-T cell, expansion in vivo.
- the lymphoproliferative element can include at the P4 position, an intracellular domain or a fragment thereof that includes a signaling domain from any of the genes having a P4 signaling domain in constructs having a P3 and a P4 signaling domain in the CLEs identified in Tables 4 to 8 herein, which promote T cell, e.g. CAR-T cell, expansion in vivo.
- the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from any of the genes having a P3 signaling domain and a signaling domain from any of the genes having a P4 domain in the same CLE, in illustrative embodiments in the P3 and P4 positions respectively, in any of the CLEs identified in Tables 4 to 8 herein, which promote T cell, e.g. CAR-T cell, expansion in vivo.
- T cell e.g. CAR-T cell
- the P2 domain can be from any of the genes identified as having a P2 part in CLEs found in Tables 4 to 8 herein.
- the CLEs can include in some illustrative embodiments a Pl domain from Tables 4 to 8.
- the lymphoproliferative element can include a P3 signaling domain from any of the CLEs identified in Tables 4 to 8 herein, which promote T cell, e.g. CAR-T cell, expansion in vivo, or a P4 signaling domain in a construct having no P3 signaling domain, from any of the CLEs identified in Tables 4 to 8 herein, which promote T cell, e.g. CAR-T cell, expansion in vivo.
- the lymphoproliferative element can include at the P4 position, a P4 signaling domain in constructs having a P3 and a P4 signaling domain in the CLEs identified in Tables 4 to 8 herein, which promote T cell, e.g. CAR-T cell, expansion in vivo.
- the lymphoproliferative element can include a P3 signaling domain and a P4 signaling domain in the P3 and P4 positions respectively, from any one of the CLEs identified in Tables 4 to 8 herein, which promote T cell, e.g. CAR-T cell, expansion in vivo.
- the CLEs can include in some illustrative embodiments, a Pl domain from Tables 4 to 8.
- the P2 domain can comprise or be any P2 domain from a CLE found in Tables 4 to 8 herein, or in illustrative embodiments, a lymphoproliferative element can include a P2 domain, P3 domain and P4 domain, and optionally Pl domain, all from the same CLE identified in Tables 4 to 8 herein.
- a lymphoproliferative element can include a P2 domain, P3 domain and P4 domain, and optionally Pl domain, all from the same CLE identified in Tables 4 to 8 herein.
- lymphoproliferative element can have P3 and P4 domains S121-S212 or S186-S053, or P2, P3, and P4 domains T001-S121-S212 or T044-S186-S053 optionally with a Pl domain E008 or E006.
- the lymphoproliferative element can include a cytokine receptor or a fragment that includes a signaling domain thereof.
- the cytokine receptor can be CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7R, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL13R, IL13RA1, IL13RA2, IL15R, IL15RA, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1,
- the cytokine receptor can be CD27, CD40, CRFF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNER1, IE1R1, IE 1 RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL27RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFR
- the lymphoproliferative element can comprise an intracellular domain from the cytokine receptors CD27, CD40, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR2, IL1R1, IL1RL1, IL2RA, IL2RG, IL3RA, IL5RA, IL6R, IL7R, IL9R, IL10RB, IL11RA, IL12RB1, IL13RA1, IL13RA2, IL15RA, IL17RB, IL18R1, IL18RAP, IL20RB, IL22RA1, IL27RA, IL31RA, LEPR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSFl8In illustrative embodiments, the intracellular domain in a lymphoproliferative element comprises a domain from CD40
- the lymphoproliferative element can comprise a costimulatory domain from CD27, CD28, 0X40 (also referred to as TNFRSF4), GITR (also referred to as TNFRSF18), or HVEM (also referred to as TNFRSF14).
- a lymphoproliferative element comprising a costimulatory domain from 0X40 does not comprise an intracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, GITR, or HVEM.
- a lymphoproliferative element comprising a costimulatory domain from 0X40 does not comprise an intracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, GITR, or HVEM.
- a lymphoproliferative element comprising a costimulatory domain from 0X40 does not comprise an intracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD
- lymphoproliferative element comprising a costimulatory domain from GITR does not comprise an intracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, or HVEM.
- a lymphoproliferative element comprising a costimulatory domain from CD28 does not comprise an intracellular domain from CD3Z, 4-1BB, ICOS, CD27, BTLA, CD30, or HVEM.
- a lymphoproliferative element comprising a costimulatory domain from 0X40, CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, GITR, or HVEM does not comprise a coiled-coil spacer domain N-terminal of the transmembrane domain.
- a lymphoproliferative element comprising a costimulatory domain from GITR does not comprise an intracellular domain from CD3Z that is N-terminal of the costimulatory domain of GITR.
- the lymphoproliferative element comprises an intracellular domain of CD40, MPL and IL2Rb.
- the lymphoproliferative element can be other than a cytokine receptor.
- the lymphoproliferative element other than a cytokine receptor can include an intracellular signaling domain from CD2, CD3D, CD3G, CD3Z, CD4, CD8RA, CD8RB, CD28, CD79A, CD79B, FCER1G, FCGR2A, FCGR2C, or ICOS.
- a lymphoproliferative element including a CLE, comprises an intracellular activating domain as disclosed hereinabove.
- a lymphoproliferative element including a CLE, comprises an intracellular activating domain as disclosed hereinabove.
- lymphoproliferative element is a CLE comprising an intracellular activating domain comprising an ITAM-containing domain
- the CLE can comprise an intracellular activating domain having at least 80%, 90%, 95%, 98%, or 100% sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70 domains provided herein wherein the CLE does not comprise an ASTR.
- the intracellular activating domain is an ITAM-containing domain from CD3D, CD3G, CD3Z, CD79A, CD79B,
- FCER1G FCGR2A, or FCGR2C.
- CLEs comprising these intracellular activating domains are illustrated in WO2019/055946, as being effective at promoting proliferation of PBMCs ex vivo in cultures in the absence of exogenous cytokines such as exogenous IL-2.
- one or more domains of a lymphoproliferative element is fused to a modulatory domain, such as a co-stimulatory domain, and/or an intracellular activating domain of a CAR.
- a modulatory domain such as a co-stimulatory domain
- an intracellular activating domain of a CAR can be part of the same polypeptide as a CAR or can be fused and optionally functionally connected to some components of CARs.
- an engineered signaling polypeptide can include an ASTR, an intracellular activation domain (such as a CD3 zeta signaling domain), a co-stimulatory domain, and a lymphoproliferative domain. Further details regarding co-stimulatory domains, intracellular activating domains, ASTRs and other CAR domains, are disclosed elsewhere herein.
- the lymphoproliferative element is not a polypeptide, but rather comprises an inhibitory RNA.
- methods, uses, compositions, and products of processes according to any aspect herein include both a lymphoproliferative element comprising an inhibitory RNA and a lymphoproliferative element that is an engineered signaling polypeptide.
- a lymphoproliferative element is or includes an inhibitory RNA, or multiple inhibitory RNAs
- the inhibitory RNA or multiple inhibitory RNAs can have any of the structures identified elsewhere herein, for example in the Inhibitory RNA Molecules section herein.
- the inhibitory RNA can be a miRNA that stimulates the STAT5 pathway typically by potentiating activation of STAT5 by degrading or causing down-regulation of a negative regulator in the SOCS pathway.
- Inhibitory RNA can be a miRNA that stimulates the STAT5 pathway typically by potentiating activation of STAT5 by degrading or causing down-regulation of a negative
- lymphoproliferative elements can target any of the mRNAs identified in the Inhibitory RNA Molecules section herein or elsewhere herein.
- inhibitory RNA e.g. miRNAs
- inclusion of introns in transcription units are believed to result in higher expression and/or stability of transcripts.
- 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNAs in illustrative embodiments between 2 and 5, for example 4 miRNAs, one or more of which each bind nucleic acids encoding one or more of the targets disclosed herein., can be included in the recombinant retroviral genome and delivered to a target cell, for example T cells and/or NK cells, using methods provided herein.
- a target cell for example T cells and/or NK cells, using methods provided herein.
- 1, 2, 3, or 4 miRNAs can be delivered in a single intron such as the EFl-a intron.
- the lymphoproliferative element comprises MPL, or is MPL, or a variant and/or fragment thereof, including a variant and/or fragment that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, with or without a transmembrane and/or extracellular domain of MPL, and/or has at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to the intracellular domain of MPL, with or without a transmembrane and/or extracellular domain of MPL, wherein the variant and/or fragment retains the ability to promote cell proliferation of PBMCs, and in some embodiments T cells.
- the lymphoproliferative element comprises an intracellular domain of MPL, or a variant or fragment thereof that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, and the lymphoproliferative element does not comprise a transmembrane domain of MPL.
- the lymphoproliferative element comprises an intracellular domain of MPL, or a variant or fragment thereof that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, and the lymphoproliferative element does not comprise a transmembrane domain of MPL.
- the lymphoproliferative element comprises an intracellular domain of MPL, or a variant or fragment thereof that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, and the lymphoproliferative element does not comprise a transmembrane domain of M
- lymphoproliferative element comprises an intracellular domain of MPL, or a variant or fragment thereof that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, and the lymphoproliferative element comprises a transmembrane domain of MPL.
- a cell expressing the lymphoproliferative element comprising an intracellular and transmembrane domain of MPL can be contacted with, exposed to, or treated with eltrombopag.
- eltrombopag binds to the transmembrane domain of MPL and induces the activation of the intracellular domain of MPL.
- an MPL fragment included in the compositions and methods herein has and/or retains a JAK-2 binding domain. In some embodiments, an MPL fragment included herein has or retains the ability to activate a STAT.
- the full intracellular domain of MPL is SEQ ID NO: 1
- MPL is the receptor for thrombopoietin.
- cytokines such as thrombopoietin and EPO fire referred to in the literature and herein as either a hormone or a cytokine.
- chimeric polypeptides that are chimeric lymphoproliferative elements (CLEs), as well as isolated polynucleotides and nucleic acid sequences that encode the same.
- CLEs can include any of the domains and/or domains derived from specific genes discussed in the section.
- isolated polynucleotides and nucleic acid sequences encoding CLEs can encode as part of the CLE any of the domains and/or domains derived from specific genes discussed in this section.
- Lymphoproliferative elements typically include a transmembrane domain.
- the transmembrane domain can have 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to any one of the transmembrane domains from the following genes and representative sequences disclosed in WO2019/055946: CD8 beta, CD4, CD3 zeta, CD28, CD134, CD7, CD2, CD3D, CD3E, CD3G, CD3Z, CD4, CD8A CD8B, CD27, CD28, CD40, CD79A, CD79B, CRLF2, CRLF2, CSF2RA, CSF2RB, CSF2RB, CSF3R, EPOR, FCER1G, FCGR2C, FCGRA2, GHR, GHR, ICOS, IFNAR,
- Transmembrane (TM) domains suitable for use in any engineered signaling polypeptide include, but are not limited to, constitutively active cytokine receptors, the TM domain from LMP1, and TM domains from type 1 TM proteins comprising a dimerizing motif, as discussed in more detail herein.
- the transmembrane domain can be a Type I growth factor receptor, a hormone receptor, a T cell receptor, or a TNF-family receptor.
- Eltrombopag is a small molecule activator of the thrombopoietin receptor MPL (also known as TPOR).
- a cell expressing an LE comprising a MPL transmembrane domain can be exposed to or contacted with eltrombopag, or a patient or subject to which such a cell has been infused, can be treated with eltrombopag.
- the proliferative and/or survival properties of the LE are activated and provided to the cell, thereby increasing survival and/or proliferation of the cell compared to the absence of the eltrombopag.
- binding of eltrombopag occurs in the transmembrane domain and can activate one or more intracellular domains that are part of the same polypeptide.
- a skilled artisan will understand the amount of eltrombopag to be used to activate a CLE comprising a MPL transmembrane domain.
- CLEs include both an extracellular portion and a transmembrane portion that is from the same protein, in illustrative embodiments the same receptor, either of which in illustrative embodiments is a mutant, thus forming an extracellular and transmembrane domain.
- These domains can be from a cytokine receptor, or a mutant thereof, or a hormone receptor, or a mutant thereof in some embodiments that have been reported to be constitutively active when expressed at least in some cell types.
- extracellular and transmembrane domains do not include a ligand binding region. It is believed that such domains do not bind a ligand when present in CLEs and expressed in B cells, T cells, and/or NK cells. Mutations in such receptor mutants can occur in the transmembrane region or in the extracellular juxtamembrane region.
- a mutation in at least some extracellular - transmembrane domains of CLEs are responsible for signaling of the CLE in the absence of ligand, by bringing activating chains together that are not normally together, or by changing the confirmation of a linked transmembrane and/or intracellular domain.
- Exemplary extracellular and transmembrane domains for CLEs of embodiments that include such domains are extracellular regions, typically less than 30 amino acids of the membrane -proximal extracellular domains along with transmembrane domains from mutant receptors that have been reported to be constitutive, that is not require ligand binding for activation of an associated intracellular domain.
- extracellular and transmembrane domains include IL7RA Ins PPCL, CRLF2 F232C, CSF2RB V449E, CSF3R T640N, EPOR L251C I252C, GHR E260C I270C, IL27RA F523C, and MPL S505N.
- the extracellular and transmembrane domain does not comprise more than 10, 20, 25 30 or 50 consecutive amino acids that are identical in sequence to a portion of the extracellular and/or transmembrane domain of IL7RA, or a mutant thereof.
- the extracellular and transmembrane domain is other than IL7RA Ins PPCL.
- the extracellular and transmembrane does not comprise more than 10, 20, 25, 30, or 50 consecutive amino acids that are identical in sequence to a portion of the extracellular and/or
- an LE provided herein comprises an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerizing motif.
- the extracellular domain comprises a leucine zipper.
- the leucine zipper is from a jun polypeptide, for example c-jun.
- the c-jun polypeptide is the c-jun polypeptide region of ECD-l 1.
- the transmembrane domain is a type I transmembrane protein
- the transmembrane domain can be a Type I growth factor receptor, a hormone receptor, a T cell receptor, or a TNF-family receptor.
- the chimeric polypeptide comprises an extracellular domain and wherein the extracellular domain comprises a dimerizing motif
- the transmembrane domain can be a Type I cytokine receptor, a hormone receptor, a T cell receptor, or a TNF-family receptor.
- transmembrane domains include any transmembrane domain that was illustrated in WO2019/055946.
- the transmembrane domain is from CD4, CD8RB, CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2C, GHR, ICOS, IFNAR1, IFNGR1, IFNGR2, IL1R1,
- the transmembrane domain is from CD40, ICOS, FCGR2C, PRLR, IL3RA, or IL6ST.
- the extracellular and transmembrane domain is the viral protein LMP1, or a mutant and/or fragment thereof.
- LMP1 is a multispan transmembrane protein that is known to activate cell signaling independent of ligand when targeted to lipid rafts or when fused to CD40 (Kaykas et al. EMBO J. 20: 2641 (2001)).
- a fragment of LMP1 is typically long enough to span a plasma membrane and to activate a linked intracellular domain(s).
- the LMP1 can be between 15 and 386, 15 and 200, 15 and 150, 15 and 100, 18 and 50, 18 and 30, 20 and 200, 20 and 150, 20 and 50, 20 and 30, 20 and 100, 20 and 40, or 20 and 25 amino acids.
- the extracellular domain includes at least 1 , but typically at least 4 amino acids and is typically linked to another functional polypeptide, such as a clearance domain, for example, an eTag.
- the lymphoproliferative element comprises an LMP1 transmembrane domain.
- the lymphoproliferative element comprises an LMP1 transmembrane domain and the one or more intracellular domains do not comprise an intracellular domain from TNFRSF proteins (i.e. CD40, 4- IBB, RANK, TACI, 0X40, CD27, GITR, LTR, and BAFFR), TLR1 to TLR13, integrins, FcyRIII, Dectinl, Dectin2, NOD1, NOD2, CD 16, IL-2R, Type I II interferon receptor, chemokine receptors such as CCR5 and CCR7, G-protein coupled receptors, TREM1, CD79A, CD79B, Ig-alpha, IPS-l, MyD88, RIG- 1, MDA5, CD3Z, MyD88ATIR, TRIF, TRAM, TIRAP, MAL, BTK, RTK, RAC1, SYK, NALP3 (NLRP3), NALP3ALRR, NALP1, CARD9, DAI, IPAG, STING
- the extracellular domain includes a dimerizing moiety.
- dimerizing moieties are capable of homodimerizing.
- dimerizing moieties can provide an activating function on intracellular domains connected thereto via transmembrane domains. Such activation can be provided, for example, upon dimerization of a dimerizing moiety, which can cause a change in orientation of intracellular domains connected thereto via a transmembrane domain, or which can cause intracellular domains to come into proximity.
- An extracellular domain with a dimerizing moiety can also serve a function of connecting a recognition tag to a cell expressing a CLE.
- the dimerizing agent can be located intracellularly rather than extracellularly. In some embodiments, more than one or multiples of dimerizing domains can be used.
- Extracellular domains for embodiments where extracellular domains have a dimerizing motif are long enough to form dimers, such as leucine zipper dimers.
- extracellular domains that include a dimerizing moiety can be from 15 to 100, 20 to 50, 30 to 45, or 35 to 40 amino acids, of in illustrative embodiments is a c-Jun portion of a c-Jun extracellular domain.
- Extracellular domains of polypeptides that include a dimerizing moiety may not retain other functionalities.
- leucine zippers embodiments such leucine zippers are capable of forming dimers because they retain a motif of leucines spaced 7 residues apart along an alpha helix.
- leucine zipper moieties of certain embodiments of CLEs provided herein may or may not retain their DNA binding function.
- a spacer of between 1 and 4 alanine residues can be included in CLEs between the extracellular domain that has a dimerizing moiety, and the transmembrane domain. Not to be limited by theory, it is believed that the alanine spacer affects signaling of intracellular domains connected to the leucine zipper extracellular region via the transmembrane domain, by changing the orientation of the intracellular domains.
- the first and optional second intracellular domains of CLEs provided herein are intracellular signaling domains of genes that are known in at least some cell types, to promote proliferation, survival (anti-apoptotic), and/or provide a co-stimulatory signal that enhances proliferative potential or resistance to cell death.
- these intracellular domains can be intracellular domains from lymphoproliferative elements and co-stimulatory domains provided herein.
- Some of the intracellular domains of candidate chimeric polypeptides are known to activate JAK1/JAK2, JAK3, STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6 signaling.
- a lymphoproliferative element herein is a transgenic B OX1 -containing cytokine receptor that includes an intracellular domain of a cytokine receptor comprising a Boxl Janus kinase (JAK)-binding motif, optionally a Box2 JAK-binding motif, and a Signal Transducer and Activator of Transcription (STAT) binding motif comprising a tyrosine residue.
- JAK Boxl Janus kinase
- STAT Signal Transducer and Activator of Transcription
- transgenic B OX1 -containing cytokine receptor lymphoproliferative element has a switch motif, which in illustrative embodiments has one or more, and preferably all hydrophobic residues at positions -1, -2, and -6 relative to the Boxl motif.
- the Boxl motif an ICD of a lymphoproliferative element is located proximal to the transmembrane (TM) domain (for example between 5 and 15 or about 10 residues downstream from the TM domain) relative to the Box2 motif, which is located proximal to the transmembrane domain (for example between 10 and 50 residues downstream from the TM domain) relative to the STAT binding motif.
- the STAT binding motif typically comprising a tyrosine residue, the phosphorylation of which affects binding of a STAT to the STAT binding motif of the lymphoproliferative element.
- the ICDs comprising multiple STAT binding motifs where multiple STAT binding motifs are present in a native ICD (e.g. EPO receptor and IL-6 receptor signaling chain (gpl30).
- Intracellular domains from IFNAR1, IFNGR1, IFNLR1, IL2RB, IL4R, IL5RB, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL21R, IL27R, IL31RA, LIFR, and OSMR are known in the art to activate JAK1 signaling.
- Intracellular domains from CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNGR2, IL3RA, IL5RA, IL6ST, IL20RA, IL20RB, IL23R, IL27R, LEPR, MPL, and PRLR are known in the art to activate JAK2.
- Intracellular domains from IL2RG are known in the art to activate JAK3.
- Intracellular domains from GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL2RB, IL2RG, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL21R, IL22RA1, IL31RA, LIFR, MPL, and OSMR are known in the art to activate STAT1.
- Intracellular domains from IFNAR1 and IFNAR2 are known in the art to activate STAT2.
- Intracellular domains from GHR, IL2RB, IL2RG, IL6R, IL7RA, IL9R, IL10RA, IL10RB, IL21R, IL22RA1, IL23R, IL27R, IL31RA, LEPR, LIFR, MPL, and OSMR are known in the art to activate STAT3.
- Intracellular domains from ILl2RBl are known in the art to activate STAT4.
- Intracellular domains from CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL15RA, IL20RA, IL20RB, IL21R, IL22RA1, IL31RA, LIFR, MPL, OSMR, and PRLR are known in the art to activate STAT5.
- Intracellular domains from IL4R and OSMR are known in the art to activate STAT6.
- genes and intracellular domains thereof that are found in a first intracellular domain are the same as the optional second intracellular domain, except that if the first and second intracellular domain are identical, then at least one, and typically both the transmembrane domain and the extracellular domain are not from the same gene.
- all domains of a CLE are other than an IL-7 receptor, or a mutant thereof, and/or a fragment thereof that has at least 10, 15, 20, or 25 contiguous amino acids of IL-7 receptor, or other than an IL-15 receptor, or a mutant thereof, and/or a fragment thereof that has at least 10, 15, 20, or 25 contiguous amino acids of IL-15 receptor.
- a CLE does not comprise a combination of first intracellular domain and second intracellular domain of CD40 and MyD88.
- CLEs include a recognition and/or elimination domain. Details regarding recognition and/or elimination domains are provided in other sections herein. Any of the recognition and/or elimination domains provided herein can be part of a CLE. Typically the recognition domain is linked to the N terminus of the extracellular domain. Not to be limited by theory, in some embodiments, the extracellular domain includes the function of providing a linker, in illustrative embodiments a flexible linker, linking a recognition domain to a cell that expresses the CLE.
- polynucleotides that include a nucleic acid sequence encoding a CLE provided herein also typically comprise a signal sequence to direct expression to the plasma membrane.
- Exemplary signal sequences are provided herein in other sections. Elements can be provided on the transcript such that both a CAR and CLE are expressed from the same transcript in certain embodiments.
- the dimerizing motif can be selected from the group consisting of: a leucine zipper motif-containing polypeptide, CD69, CD71, CD72, CD96, Cdl05, Cdl61, Cdl62, Cd249, CD271, and Cd324, as well as mutants and/or active fragments thereof that retain the ability to dimerize.
- the dimerizing motif can require a dimerizing agent, and the dimerizing motif and associated dimerizing agent can be selected from the group consisting of: FKBP and rapamycin or analogs thereof, GyrB and coumermycin or analogs thereof, DHFR and methotrexate or analogs thereof, or DmrB and AP20187 or analogs thereof, as well as mutants and/or active fragments of the recited dimerizing proteins that retain the ability to dimerize.
- a lymphoproliferative element is constitutively active, and is other than a lymphoproliferative element that requires a dimerizing agent for activation.
- the extracellular domain of a CLE comprises a dimerizing motif
- the extracellular domain can comprise a leucine zipper motif.
- the leucine zipper motif is from a jun polypeptide, for example c-jun.
- the c-jun polypeptide is the c-jun polypeptide region of ECD-11.
- Internally dimerizing and/or multimerizing lymphoproliferative elements in one embodiment are an integral part of a system that uses a dimeric analog of the lipid permeable immunosuppressant drug, FK506, which loses its normal bioactivity while gaining the ability to crosslink molecules genetically fused to the FK506-binding protein, FKBP 12.
- FKBP lipid permeable immunosuppressant drug
- FKBP12 permits specific activation of the recombinant receptor in vivo without the induction of non-specific side effects through endogenous FKBP12.
- FKBP12 variants having amino acid substitutions and deletions, such as FKBP12V36, that bind to a dimerizer drug, may also be used.
- the synthetic ligands are resistant to protease degradation, making them more efficient at activating receptors in vivo than most delivered protein agents.
- pseudotyping elements Many of the methods and compositions provided herein include pseudotyping elements.
- a pseudotyping element as used herein can include a "binding polypeptide" that includes one or more polypeptides, typically glycoproteins, that identify and bind the target host cell, and one or more
- pseudotyping elements are provided as polypeptide(s)/protein(s), or as nucleic acid sequences encoding the polypeptide(s)/protein(s).
- the pseudotyping element is the feline endogenous virus (RD114) envelope protein, an oncoretroviral amphotropic envelope protein, an oncoretroviral ecotropic envelope protein, the vesicular stomatitis virus envelope protein (VSV-G) (SEQ ID NO: 336), the baboon retroviral envelope glycoprotein (BaEV) (SEQ ID NO: 337), the murine leukemia envelope protein (MuLV) (SEQ ID NO: 338), the influenza glycoprotein F1A surface glycoprotein (F1A), the influenza glycoprotein neurominidase (NA), the paramyxovirus Measles envelope protein H, the paramyxovirus Measles envelope protein F, and/or functional variants or fragments of any of these envelope proteins.
- RD114 feline endogenous virus envelope protein
- VSV-G SEQ ID NO: 336
- BaEV baboon retroviral envelope glycoprotein
- MuLV murine leukemia envelope protein
- F1A surface glycoprotein
- NA
- the pseudotyping element can be wild-type BaEV.
- BaEV contains an R peptide that has been shown to inhibit transduction.
- the BaEV can contain a deletion of the R peptide.
- the BaEV can contain a deletion of the inhibitory R peptide after the nucleotides encoding the amino acid sequence HA, referred to herein as BaEVAR (HA) (SEQ ID NO: 339).
- the BaEV can contain a deletion of the inhibitory R peptide after the nucleotides encoding the amino acid sequence HAM, referred to herein as BaEVAR (HAM) (SEQ ID NO: 340).
- the pseudotyping element can be wild-type MuLV.
- the MuLV can contain one or more mutations to remove the furin-mediated cleavage site located between the transmembrane (TM) and surface (SU) subunits of the envelope glycoprotein.
- the MuLV contains the SUx mutation (MuLVSUx) (SEQ ID NO: 453) which inhibits furin -mediated cleavage of MuLV envelope protein in packaging cells.
- the C- terminus of the cytoplasmic tail of the MuLV or MuLVSUx protein is truncated by 4 to 31 amino acids.
- the C-terminus of the cytoplasmic tail of the MuLV or MuLVSUx protein is truncated by 4, 8, 12, 16, 20, 24, 28, or 31 amino acids.
- the pseudotyping elements include a binding polypeptide and a fusogenic polypeptide derived from different proteins.
- the replication incompetent recombinant retroviral particles of the methods and compositions disclosed herein can be pseudotyped with the fusion (F) and/or hemagglutinin (H) polypeptides of the measles virus (MV), as non-limiting examples, clinical wildtype strains of MV, and vaccine strains including the Edmonston strain (MV-Edm) (GenBank; AF266288.2) or fragments thereof.
- both hemagglutinin (H) and fusion (F) polypeptides are believed to play a role in entry into host cells wherein the H protein binds MV to receptors CD46, SLAM, and Nectin-4 on target cells and F mediates fusion of the retroviral and host cell membranes.
- the binding polypeptide is a Measles Virus H polypeptide and the fusogenic polypeptide is a Measles Virus F polypeptide.
- optimal truncation occurred when 18 or 19 residues were deleted (MV(Ed)-HAl8 (SEQ ID NOG 14) or MV(Ed)-HAl9), although variants with a truncation of 24 residues with and without replacement of deleted residues with alanine (MV(Ed)-HA24 (SEQ ID NO:3l5) and MV(Ed)-HA24+A) also resulted in optimal titers.
- the replication incompetent recombinant retroviral particles of the methods and compositions disclosed herein are pseudotyped with mutated or variant versions of the measles virus fusion (F) and hemagglutinin (H) polypeptides, in illustrative examples, cytoplasmic domain deletion variants of measles virus F and H polypeptides.
- the mutated F and H polypeptides are "truncated H" or "truncated F" polypeptides, whose cytoplasmic portion has been truncated, i.e.
- HAY and F polypeptide designate such truncated H and F polypeptide, respectively, wherein “Y” refers to 1-34 residues that have been deleted from the amino termini and “X” refers to 1-35 residues that have been deleted from the carboxy termini of the cytoplasmic domains.
- the "truncated F polypeptide" is HD24 or HD30 and/or the “truncated H protein” is selected from the group consisting of HD14, HD15, HD16, HD17, HD18, HD19, HD20, HD21+A, HD24 and HD24+4A, more preferably HA18 or HD24.
- the truncated F polypeptide is MV(Ed)-FA30 and the truncated Fl polypeptide is MV(Ed)-HAl8.
- the pseudotyping element includes polypeptides derived from different proteins.
- the pseudotyping element can comprise an influenza protein hemagglutinin HA and/or a neuraminidase (NA).
- NA neuraminidase
- the HA is from influenza A virus subtype H1N1.
- the HA is from H1N1 PR8 1934 in which the monobasic trypsin-dependent cleavage site has been mutated to a more promiscuous multibasic sequence (SEQ ID NO:3l 1).
- the NA is from influenza A virus subtype H10N7.
- the NA is from H 10N7-HKWF446C-07 (SEQ ID NO:3l2).
- the viral particles are copseudotyped with envelope glycoproteins from 2 or more heterologous viruses.
- the viral particles are copseudotyped with VSV-G, or a functional variant or fragment thereof, and an envelope protein from RD114, BaEV, MuLV, influenza virus, measles virus, and/or a functional variant or fragment thereof.
- the viral particles are copseudotyped with VSV-G and the MV(Ed)-H glycoprotein or the MV(Ed)-H glycoprotein with a truncated cytoplasmic domain.
- the viral particles are copseudotyped with VSV-G and MV(Ed)-HA24.
- VSV-G is copseudotyped with MuLV or MuLV with a truncated cytoplasmic domain.
- VSV-G is copseudotyped with MuLVSUx or MuLVSUx with a truncated cytoplasmic domain.
- VSV-G is copseudotyped with a fusion of an antiCD3scFv to MuLV.
- the fusogenic polypeptide includes multiple elements expressed as one polypeptide.
- the binding polypeptide and fusogenic polypeptide are translated from the same transcript but from separate ribosome binding sites; in other embodiments, the binding polypeptide and fusogenic polypeptide are separated by a cleavage peptide site, which not to be bound by theory, is cleaved after translation, as is common in the literature, or a ribosomal skip sequence.
- the translation of the binding polypeptide and fusogenic polypeptide from separate ribosome binding sites results in a higher amount of the fusogenic polypeptide as compared to the binding polypeptide.
- the ratio of the fusogenic polypeptide to the binding polypeptide is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, or at least 8:1.
- the ratio of the fusogenic polypeptide to the binding polypeptide is between 1.5:1, 2:1, or 3:1, on the low end of the range, and 3:1, 4:1, 5:1, 6:1, 7:1, 8:1. 9:1 or 10:1 on the high end of the range.
- an activation element also referred to herein as a T cell activation element, or a nucleic acid encoding an activation element.
- lentiviral (LV) transduction into resting T cells are attributed to a series of pre-entry and post-entry barriers as well as cellular restrictive factors (Strebel et al 2009. BMC Medicine 7:48).
- One restriction is the inability for the envelope pseudotyped-LV particles to recognize potential receptors and mediate fusion with the cellular membrane.
- TCR T cell receptor
- T lymphocytes recognize and interact with specific antigens through receptors or receptor complexes which, upon recognition or an interaction with such antigens, cause activation of the cell and expansion in the body.
- An example of such a receptor is the antigen- specific T lymphocyte receptor complex (TCR/CD3).
- TCR T cell receptor
- CD3 One component, CD3, is responsible for intracellular signaling following occupancy of the TCR by ligand.
- T lymphocyte receptor for antigen-CD3 complex recognizes antigenic peptides that are presented to it by the proteins of the major histocompatibility complex (MHC).
- MHC major histocompatibility complex
- TCR/CD3 complexes of MHC and peptide are expressed on the surface of antigen presenting cells and other T lymphocyte targets. Stimulation of the TCR/CD3 complex results in activation of the T lymphocyte and a consequent antigen-specific immune response.
- the TCR/CD3 complex plays a central role in the effector function and regulation of the immune system.
- activation elements provided herein, activate T cells by binding to one or more components of the T cell receptor associated complex, for example by binding to CD3.
- the activation element can activate alone. In other cases, the activation requires activation through the TCR receptor complex in order to further activate cells.
- T lymphocytes also require a second, co-stimulatory signal to become fully active in vivo.
- T lymphocytes are either non-responsive to antigen binding to the TCR, or become anergic.
- the second, co-stimulatory signal is not required for the transduction and expansion of T cells.
- a co-stimulatory signal for example, is provided by CD28, a T lymphocyte protein, which interacts with CD80 and CD86 on antigen-producing cells.
- CD80 a T lymphocyte protein
- CD86 a functional extracellular fragment of CD80 retains its ability to interact with CD28.
- 0X40, 4-1BB, and ICOS Inducible
- T cell receptor (TCR) CD3 complex and co-stimulation with CD28 can occur by ex vivo exposure to solid surfaces (e.g. beads) coated with anti-CD3 and anti-CD28.
- resting T cells are activated by exposure to solid surfaces coated with anti-CD3 and anti-CD28 ex vivo.
- resting T cells or NK cells are activated by exposure to soluble anti-CD3 antibodies (e.g. at 50-150, or 75-125, or 100 ng/ml).
- soluble anti-CD3 antibodies e.g. at 50-150, or 75-125, or 100 ng/ml.
- activation and/or contacting can be carried out by including anti-CD3 in a transduction reaction mixture and contacting with optional incubating for any of the times provided herein.
- such activation with soluble anti-CD3 can occur by incubating lymphocytes, such as PBMCs, and in illustrative embodiments NK cells and in more illustrative embodiments, T cells, after they are contacted with retroviral particles in a media containing an anti-CD3.
- lymphocytes such as PBMCs
- NK cells and in more illustrative embodiments, T cells
- retroviral particles in a media containing an anti-CD3.
- Such incubation can be for example, for between 5, 10, 15, 30, 45, 60, or 120 minutes on the low end of the range, and 15, 30, 45, 60, 120, 180, or 240 minutes on the high end of the range, for example, between 15 and 1 hours or 2 hours.
- polypeptides that are capable of binding to an activating T cell surface protein are presented as "activation elements" on the surface of replication incompetent recombinant retroviral particles of the methods and compositions disclosed herein, which are also aspects of the invention.
- the activation elements on the surfaces of the replication incompetent recombinant retroviral particles can include one or more polypeptides capable of binding CD3.
- the activation elements on the surfaces of the replication incompetent recombinant retroviral particles can include one or more polypeptides capable of binding the epsilon chain of CD3 (CD3 epsilon).
- the activation element on the surfaces of the replication incompetent recombinant retroviral particles can include one or more polypeptides capable of binding CD28, 0X40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, and/or CD82 and optionally one or more polypeptides capable of binding CD3.
- the activation element can be a T cell surface protein agonist.
- the activation element can include a polypeptide that acts as a ligand for a T cell surface protein.
- the polypeptide that acts as a ligand for a T cell surface protein is, or includes, one or more of OX40L, 4- 1BBL, or ICOSLG.
- one or typically more copies of one or more of these activation elements can be expressed on the surfaces of the replication incompetent recombinant retroviral particles as polypeptides separate and distinct from the pseudotyping elements.
- the activation elements can be expressed on the surfaces of the replication incompetent recombinant retroviral particles as fusion polypeptides.
- the fusion polypeptides include one or more activation elements and one or more pseudotyping elements.
- the fusion polypeptide includes anti-CD3, for example an anti-CD3scFv, or an anti-CD3scFvFc, and a viral envelope protein.
- the fusion polypeptide is the OKT-3scFv fused to the amino terminal end of a viral envelope protein such as the MuLV envelope protein, as shown in Maurice et al. (2002).
- the fusion polypeptide is UCFlTlscFv fused to a viral envelope protein, for example the MuLV envelope protein (SEQ ID NO:34l), the MuLVSUx envelope protein (SEQ ID NO:454), VSV-G (SEQ ID NO:455 or SEQ ID NO:456), or functional variants or fragments thereof, including any of the membrane protein truncations provided herein.
- illustrative embodiments especially for compositions and methods herein for transducing lymphocytes in whole blood do not include any blood protein (e.g. blood Factor (e.g. Factor X)) cleavage sites in the portion of the fusion protein that resides outside the retroviral particle.
- the fusion constructs do not include any furin cleavage sites.
- Furin is a membrane bound protease expressed in all mammalian cells examined, some of which is secreted and active in blood plasma (See e.g. C. Fernandez et al. J. Internal. Medicine (2016) 284; 377-387). Mutations can be made to fusion constructs using known methods to remove such protease cleavage sites.
- polypeptides that bind CD3, CD28, 0X40, 4-1BB, or ICOS are referred to as activation elements because of their ability to activate resting T cells.
- nucleic acids encoding such an activating element are found in the genome of a replication incompetent recombinant retroviral particle that contains the activating element on its surface. In other embodiments, nucleic acids encoding an activating element are not found in the replication incompetent recombinant retroviral particle genome.
- the nucleic acids encoding an activating element are found in the genome of a virus packaging cell.
- the activation element is a polypeptide capable of binding to CD3.
- the polypeptide capable of binding to CD3, binds to CD3D, CD3E, CD3G, or CD3Z.
- the activation element is a polypeptide capable of binding to CD3E.
- the polypeptide capable of binding to CD3 is an anti-CD3 antibody, or a fragment thereof that retains the ability to bind to CD3.
- the anti-CD3 antibody or fragment thereof is a single chain anti-CD3 antibody, such as but not limited to, an anti-CD3 scFv.
- the polypeptide capable of binding to CD3 is anti-CD3scFvFc.
- a number of anti-human CD3 monoclonal antibodies and antibody fragments thereof are available, and can be used in the present invention, including but not limited to UCF1T1, OKT-3, F1IT3A, TRX4, X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, Fl 11409, CLB-T3.4.2, TR- 66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2 and F101.01.
- the activation element is a polypeptide capable of binding to CD28.
- the polypeptide capable of binding to CD28 is an anti-CD28 antibody, or a fragment thereof that retains the ability to bind to CD28.
- the polypeptide capable of binding to CD28 is CD80, CD86, or a functional fragment thereof that is capable of binding CD28 and inducing CD28-mediated activation of Akt, such as an external fragment of CD80.
- an external fragment of CD80 means a fragment that is typically present on the outside of a cell in the normal cellular location of CD80, that retains the ability to bind to CD28.
- the anti-CD28 antibody or fragment thereof is a single chain anti-CD28 antibody, such as, but not limited to, an anti-CD28 scFv.
- the polypeptide capable of binding to CD28 is CD80, or a fragment of CD80 such as an external fragment of CD80.
- Anti-CD28 antibodies are known in the art and can include, as non-limiting examples, monoclonal antibody 9.3, an IgG2a antibody (Dr. Jeffery Ledbetter, Bristol Myers Squibb Corporation, Seattle, Wash.), monoclonal antibody KOLT-2, an IgGl antibody, 15E8, an IgGl antibody, 248.23.2, an IgM antibody and EX5.3D10, an IgG2a antibody.
- an activation element includes two polypeptides, a polypeptide capable of binding to CD3 and a polypeptide capable of binding to CD28.
- the polypeptide capable of binding to CD3 or CD28 is an antibody, a single chain monoclonal antibody or an antibody fragment, for example a single chain antibody fragment.
- the antibody fragment can be, for example, a single chain fragment variable region (scFv), an antibody binding (Fab) fragment of an antibody, a single chain antigen-binding fragment (scFab), a single chain antigen-binding fragment without cysteines (scFabAC), a fragment variable region (Fv), a construct specific to adjacent epitopes of an antigen (CRAb), or a single domain antibody (VH or VL).
- an activation element can include a dimerizing or higher order multimerizing motif. Dimerizing and multimerizing motifs are well-known in the art and a skilled artisan will understand how to incorporate them into the polypeptides for effective dimerization or multimerization.
- the activation element that includes a dimerizing motif can be one or more polypeptides capable of binding to CD3 and/or CD28.
- the polypeptide capable of binding to CD3 is an anti-CD3 antibody, or a fragment thereof that retains the ability to bind to CD3.
- the anti-CD3 antibody or fragment thereof is a single chain anti-CD3 antibody, such as but not limited to, an anti-CD3 scFv.
- the polypeptide capable of binding to CD3 is anti- CD3scFvFc, which in some embodiments is considered an anti-CD3 with a dimerizing motif without any additional dimerizing motif, since anti-CD3scFvFc constructs are known to be capable of dimerizing without the need for a separate dimerizing motif.
- the dimerizing or multimerizing motif, or a nucleic acid sequence encoding the same can be an amino acid sequence from transmembrane polypeptides that naturally exist as homodimers or multimers.
- the dimerizing or multimerizing motif, or a nucleic acid sequence encoding the same can be an amino acid sequence from a fragment of a natural protein or an engineered protein.
- the homodimeric polypeptide is a leucine zipper motif- containing polypeptide (leucine zipper polypeptide).
- leucine zipper polypeptide derived from c-JUN, non-limiting examples of which are disclosed related to chimeric lymphoproliferative elements (CLEs) herein.
- these transmembrane homodimeric polypeptides can include early activation antigen CD69 (CD69), Transferrin receptor protein 1 (CD71), B-cell differentiation antigen (CD72), T-cell surface protein tactile (CD96), Endoglin (Cdl05), Killer cell lectin-like receptor subfamily B member 1 (Cdl6l), P-selectin glycoprotein ligand 1 (Cdl62), Glutamyl aminopeptidase (Cd249), Tumor necrosis factor receptor superfamily member 16 (CD271), Cadherin-l (E-Cadherin) (Cd324), or active fragments thereof.
- CD69 early activation antigen CD69
- CD71 Transferrin receptor protein 1
- CD72 B-cell differentiation antigen
- T-cell surface protein tactile CD96
- Endoglin Cdl05
- Killer cell lectin-like receptor subfamily B member 1 Cdl6l
- P-selectin glycoprotein ligand 1 Cdl62
- the dimerizing motif, and nucleic acid encoding the same can include an amino acid sequence from transmembrane proteins that dimerize upon ligand (also referred to herein as a dimerizer or dimerizing agent) binding.
- the dimerizing motif and dimerizer can include (where the dimerizer is in parentheses following the dimerizer-binding pair): FKBP and FKBP (rapamycin); GyrB and GyrB (coumermycin); DF1FR and DF1FR (methotrexate); or DmrB and DmrB (AP20187).
- rapamycin can serve as a dimerizer.
- rapamycin derivative or analog can be used (see, e.g., W096/41865; WO 99/36553; WO 01/14387; and Ye et al (1999) Science 283:88-91).
- analogs, homologs, derivatives, and other compounds related structurally to rapamycin include, among others, variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxy at C13,
- coumermycin can serve as a dimerizing agent.
- a coumermycin analog can be used (see, e.g., Farrar et al. (1996) Nature 383:178-181; and U.S. Pat. No. 6,916,846).
- the dimerizing agent is methotrexate, e.g., a non-cytotoxic, homo-bifunctional methotrexate dimer (see, e.g., U.S. Pat. No.
- lymphoproliferative elements include a dimerizing agent
- a lymphoproliferative element is constitutively active, and is other than a lymphoproliferative element that requires a dimerizing agent for activation.
- an activation element including a dimerizing motif when present on the surface of replication incompetent recombinant retroviral particles, can be active in the absence of a dimerizing agent.
- activation elements including a dimerizing motif from transmembrane homodimeric polypeptides including CD69, CD71, CD72, CD96, Cdl05, Cdl6l, Cdl62, Cd249, CD271, Cd324, active mutants thereof, and/or active fragments thereof can be active in the absence a dimerizing agent.
- the activation element can be an anti-CD3 single chain fragment and include a dimerizing motif selected from the group consisting of CD69, CD71, CD72, CD96, Cdl05, Cdl6l, Cdl62, Cd249, CD271, Cd324, active mutants thereof, and/or active fragments thereof.
- an activation element including a dimerizing motif when present on the surface of replication incompetent recombinant retroviral particles, can be active in the presence of a dimerizing agent.
- activation elements including a dimerizing motif from FKBP, GyrB, DF1FR, or DmrB can be active in the presence of the respective dimerizing agents or analogs thereof, e.g.
- the activation element can be a single chain antibody fragment against anti-CD3 or anti-CD28, or another molecule that binds CD3 or CD28
- the dimerizing motif and dimerizing agent can be selected from the group consisting of FKBP and rapamycin or analogs thereof, GyrB and coumermycin or analogs thereof, DF1FR and methotrexate or analogs thereof, or DmrB and AP20187 or analogs thereof.
- an activation element is fused to a heterologous signal sequence and/or a heterologous membrane attachment sequence or a membrane bound protein, all of which help direct the activation element to the membrane.
- the heterologous signal sequence targets the activation element to the endoplasmic reticulum, where the heterologous membrane attachment sequence covalently attaches to one or several fatty acids (also known as posttranslational lipid modification) such that the activation elements that are fused to the heterologous membrane attachment sequence are anchored in the lipid rafts of the plasma membrane.
- posttranslational lipid modification can occur via myristoylation, palmitoylation, or GPI anchorage.
- Myristoylation is a post-translational protein modification which corresponds to the covalent linkage of a 14-carbon saturated fatty acid, the myristic acid, to the N-terminal glycine of a eukaryotic or viral protein. Palmitoylation is a post-translational protein modification which corresponds to the covalent linkage of a C16 acyl chain to cysteines, and less frequently to serine and threonine residues, of proteins. GPI anchorage refers to the attachment of glycosylphosphatidylinositol, or GPI, to the C-terminus of a protein during posttranslational modification.
- the heterologous membrane attachment sequence is a GPI anchor attachment sequence.
- the heterologous GPI anchor attachment sequence can be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Gly cobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11).
- GPI-anchored protein Reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Gly cobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11).
- the heterologous GPI anchor attachment sequence is the GPI anchor attachment sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, and CD87.
- the heterologous GPI anchor attachment sequence is derived from CD 16.
- the heterologous GPI anchor attachment sequence is derived from Fc receptor FcyRIIIb (CDl6b) or decay accelerating factor (DAF), otherwise known as complement decay-accelerating factor or CD55.
- one or both of the activation elements include a heterologous signal sequence to help direct expression of the activation element to the cell membrane.
- Any signal sequence that is active in the packaging cell line can be used.
- the signal sequence is a DAF signal sequence.
- an activation element is fused to a DAF signal sequence at its N terminus and a GPI anchor attachment sequence at its C terminus.
- the activation element includes anti-CD3 scFvFc fused to a GPI anchor attachment sequence derived from CD14 and CD80 fused to a GPI anchor attachment sequence derived from CDl6b; and both are expressed on the surface of a replication incompetent recombinant retroviral particle provided herein.
- the anti-CD3 scFvFc is fused to a DAF signal sequence at its N terminus and a GPI anchor attachment sequence derived from CD 14 at its C terminus and the CD80 is fused to a DAF signal sequence at its N terminus and a GPI anchor attachment sequence derived from CDl6b at its C terminus; and both are expressed on the surface of a replication incompetent recombinant retroviral particle provided herein.
- the DAF signal sequence includes amino acid residues 1-30 of the DAF protein.
- Some embodiments of the method and composition aspects provided herein include a membrane- bound cytokine, or polynucleotides encoding a membrane-bound cytokine.
- Cytokines are typically, but not always, secreted proteins. Cytokines that are naturally secreted can be engineered as fusion proteins to be membrane-bound.
- Membrane-bound cytokine fusion polypeptides are included in methods and compositions disclosed herein, and are also an aspect of the invention.
- replication incompetent recombinant retroviral particles have a membrane-bound cytokine fusion polypeptide on their surface that is capable of binding a T cell and/or NK cell and promoting proliferation and/or survival thereof.
- membrane-bound polypeptides are incorporated into the membranes of replication incompetent recombinant retroviral particles, and when a cell is transduced by the replication incompetent recombinant retroviral particles, the fusion of the retroviral and host cell membranes results in the polypeptide being bound to the membrane of the transduced cell.
- the cytokine fusion polypeptide includes IL-2, IL-7, IL-15, or an active fragment thereof.
- the membrane-bound cytokine fusion polypeptides are typically a cytokine fused to heterologous signal sequence and/or a heterologous membrane attachment sequence.
- the heterologous membrane attachment sequence is a GPI anchor attachment sequence.
- the heterologous GPI anchor attachment sequence can be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Gly cobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11).
- the heterologous GPI anchor attachment sequence is the GPI anchor attachment sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, and CD87.
- the heterologous GPI anchor attachment sequence is derived from CD 16.
- the heterologous GPI anchor attachment sequence is derived from Fc receptor FcyRIIIb (CDl6b).
- the GPI anchor is the GPI anchor of DAF.
- the membrane-bound cytokine is a fusion polypeptide of a cytokine fused to DAF.
- DAF is known to accumulate in lipid rafts that are incorporated into the membranes of replication incompetent recombinant retroviral particles budding from packaging cells. Accordingly, not to be limited by theory, it is believed that DAF fusion proteins are preferentially targeted to portions of membranes of packaging cells that will become part of a recombinant retroviral membrane.
- the cytokine fusion polypeptide is an IL-7, or an active fragment thereof, fused to DAF.
- the fusion cytokine polypeptide includes in order: the DAF signal sequence (residues 1-31 of DAF), IL-7 without its signal sequence, and residues 36-525 of DAF.
- the present disclosure provides mammalian packaging cells and packaging cell lines that produce replication incompetent recombinant retroviral particles.
- the cell lines that produce replication incompetent recombinant retroviral particles are also referred to herein as packaging cell lines.
- a non limiting example of such method is illustrated in WO2019/055946. Further exemplary methods for making retroviral particles are provided herein, for example in the Examples section herein.
- Such methods include, for example, a 4 plasmid system or a 5 plasmid system when a nucleic acid encoding an additional membrane bound protein, such as a T cell activation element that is not a fusion with the viral envelope, such as a GPI-linked anti-CD3, is included (See WO2019/05546).
- a 4 plasmid system in which a T cell activation element, such as a GPI- linked anti-CD3, is encoded on one of the packaging plasmids such as the plasmid encoding the viral envelope or the plasmid encoding REV, and optionally a second viral membrane-associated transgene such as a membrane bound cytokine can be encoded on the other packaging plasmid.
- a T cell activation element such as a GPI- linked anti-CD3
- a second viral membrane-associated transgene such as a membrane bound cytokine
- the nucleic acid encoding the viral protein is separated from the transgene by an IRES or a ribosomal skip sequence such as P2A or T2A.
- the present disclosure provides packaging cells and mammalian cell lines that are packaging cell lines that produce replication incompetent recombinant retroviral particles that genetically modify target mammalian cells and the target mammalian cells themselves.
- the packaging cell comprises nucleic acid sequences encoding a packageable RNA genome of the replication incompetent retroviral particle, a REV protein, a gag polypeptide, a pol polypeptide, and a pseudotyping element.
- the cells of the packaging cell line can be adherent or suspension cells. Exemplary cell types are provided hereinbelow.
- the packaging cell line can be a suspension cell line, i.e. a cell line that does not adhere to a surface during growth.
- the cells can be grown in a chemically- defined media and/or a serum-free media.
- the packaging cell line can be a suspension cell line derived from an adherent cell line, for example, the HEK293 cell line can be grown in conditions to generate a suspension-adapted HEK293 cell line according to methods known in the art.
- the packaging cell line is typically grown in a chemically defined media.
- the packaging cell line media can include serum.
- the packaging cell line media can include a serum replacement, as known in the art.
- the packaging cell line media can be serum-free media.
- Such media can be a chemically defined, serum-free formulation manufactured in compliance with Current Good Manufacturing Practice (CGMP) regulations of the US Food and Drug Administration (FDA).
- CGMP Current Good Manufacturing Practice
- FDA US Food and Drug Administration
- the packaging cell line media can be xeno-free and complete.
- the packaging cell line media has been cleared by regulatory agencies for use in ex vivo cell processing, such as an FDA 510(k) cleared device.
- a method of making a replication incompetent recombinant retroviral particle including: A. culturing a packaging cell in suspension in serum-free media, wherein the packaging cell comprises nucleic acid sequences encoding a packageable RNA genome of the replication incompetent retroviral particle, a REV protein, a gag polypeptide, a pol polypeptide, and a pseudotyping element; and B. harvesting the replication incompetent recombinant retroviral particle from the serum-free media.
- a method of transducing a lymphocyte with a replication incompetent recombinant retroviral particle comprising: A.
- the packaging cell comprises nucleic acid sequences encoding a packageable RNA genome of the replication incompetent retroviral particle, a REV protein, a gag polypeptide, a pol polypeptide, and a pseudotyping element; B. harvesting the replication incompetent recombinant retroviral particle from the serum-free media; and C.
- the contacting is performed for less than 24 hours, 20 hours, 18 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 30 minutes, or 15 minutes (or between contacting and no incubation, or 15 minutes, 30 minutes, 1, 2, 3, or 4 hours on the low end of the range and 1, 2, 3, 4, 6, 8, 12, 18, 20, or 24 hours on the high end of the range), thereby transducing the lymphocyte.
- the packageable RNA genome in certain illustrative embodiments, is designed to express one or more target polypeptides, including as a non-limiting example, any of the engineered signaling polypeptides disclosed herein and/or one or more (e.g. two or more) inhibitory RNA molecules in opposite orientation (e.g., encoding on the opposite strand and in the opposite orientation), from retroviral components such as gag and pol.
- target polypeptides including as a non-limiting example, any of the engineered signaling polypeptides disclosed herein and/or one or more (e.g. two or more) inhibitory RNA molecules in opposite orientation (e.g., encoding on the opposite strand and in the opposite orientation), from retroviral components such as gag and pol.
- the packageable RNA genome can include from 5' to 3': a 5' long terminal repeat, or active truncated fragment thereof; a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element; a nucleic acid sequence encoding a first and optionally second target polypeptide, such as, but not limited to, an engineered signaling polypeptide(s) in opposite orientation, which can be driven off a promoter in this opposite orientation with respect to the 5’ long terminal repeat and the cis-acting RNA packaging element, which in some embodiments is called a“fourth” promoter for convenience only (and sometimes referred to herein as the promoter active in T cells and/or NK cells), which is active in a target cell such as a T cell and/or an NK cell but in illustrative examples is not active in the packaging cell or is only inducibly or minimally active in the packaging cell; and a 3' long terminal repeat, or active truncated fragment thereof.
- the packageable RNA genome can include a central polypurine tract (cPPT)/central termination sequence (CTS) element.
- the retroviral cis-acting RNA packaging element can be HIV Psi.
- the retroviral cis-acting RNA packaging element can be the Rev Response Element.
- the engineered signaling polypeptide driven by the promoter in the opposite orientation from the 5’ long terminal repeat in illustrative embodiments, is one or more of the engineered signaling polypeptides disclosed herein and can optionally express one or more inhibitory RNA molecules as disclosed in more detail herein and in WO2017/165245A2, WO2018/009923 Al, and W02018/161064A1.
- promoter number such as a first, second, third, fourth, etc. promoter is for convenience only.
- a promoter that is called a“fourth” promoter should not be taken to imply that there are any additional promoters, such as first, second or third promoters, unless such other promoters are explicitly recited.
- each of the promoters are capable of driving expression of a transcript in an appropriate cell type and such transcript forms a transcription unit.
- the engineered signaling polypeptide can include a first
- the lymphoproliferative element can be expressed as a fusion with a recognition domain, such as an eTag, as disclosed herein.
- the packageable RNA genome can further include a nucleic acid sequence encoding a second engineered polypeptide including a chimeric antigen receptor, encoding any CAR embodiment provided herein.
- the second engineered polypeptide can include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activating domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activating domains are disclosed elsewhere herein.
- the promoter that is active in a target cell is active in a T cell, as disclosed elsewhere herein.
- the engineered signaling polypeptide can include a CAR, and the nucleic acid sequence can encode any CAR embodiment provided herein.
- the engineered polypeptide can include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activating domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activating domains are disclosed elsewhere herein.
- the packageable RNA genome can further include a nucleic acid sequence encoding a second engineered polypeptide.
- the second engineered polypeptide can be a lymphoproliferative element.
- the promoter that is active in a target cell is active in a T cell or NK cell, as disclosed elsewhere herein.
- the packageable RNA genome included in any of the aspects provided herein can further include a riboswitch, as discussed in WO2017/165245A2, W02018/009923A1, and W02018/161064A1.
- the nucleic acid sequence encoding the engineered signaling polypeptide can be in a reverse orientation with respect to the 5’ to 3’ orientation established by the 5’ LTR and the 3’ LTR.
- the packageable RNA genome can further include a riboswitch and, optionally, the riboswitch can be in reverse orientation.
- a polynucleotide including any of the elements can include a primer binding site.
- insulators and/or polyadenylation sequences can be placed before, after, between, or near genes to prevent or reduce unregulated transcription.
- the insulator can be chicken HS4 insulator, Kaiso insulator, SAR/MAR elements, chimeric chicken insulator-SAR elements, CTCF insulator, the gypsy insulator, or the b-globin insulator or fragments thereof known in the art.
- the insulator and/or polyadenylation sequence can be hGH polyA (SEQ ID NO:3l6), SPA1 (SEQ ID NO:3l7), SPA2 (SEQ ID NO:3l8), b-globin polyA spacer B (SEQ ID NO:3l6), hGH polyA (SEQ ID NO:3l6), SPA1 (SEQ ID NO:3l7), SPA2 (SEQ ID NO:3l8), b-globin polyA spacer B (SEQ ID NO:3l6), SPA1 (SEQ ID NO:3l7), SPA2 (SEQ ID NO:3l8), b-globin polyA spacer B (SEQ ID NO:3l6), hGH polyA (SEQ ID NO:3l6), SPA1 (SEQ ID NO:3l7), SPA2 (SEQ ID NO:3l8), b-globin polyA spacer B (SEQ ID NO:3l6), b-globin polyA spacer B
- a nucleic acid sequence encoding Vpx can be on the second or an optional third transcriptional unit, or on an additional transcriptional unit that is operably linked to the first inducible promoter.
- Some aspects of the present disclosure include or are cells, in illustrative examples, mammalian cells, that are used as packaging cells to make replication incompetent recombinant retroviral particles, such as lentiviruses, for transduction of T cells and/or NK cells.
- mammalian cells that are used as packaging cells to make replication incompetent recombinant retroviral particles, such as lentiviruses, for transduction of T cells and/or NK cells.
- any of a wide variety of cells can be selected for in vitro production of a virus or virus particle, such as a redirected recombinant retroviral particle, according to the invention.
- Eukaryotic cells are typically used, particularly mammalian cells including human, simian, canine, feline, equine and rodent cells.
- the cells are human cells.
- the cells reproduce indefinitely, and are therefore immortal.
- Examples of cells that can be advantageously used in the present invention include NIH 3T3 cells, COS cells, Madin-Darby canine kidney cells, human embryonic 293T cells and any cells derived from such cells, such as gpnlslacZ fNC cells, which are derived from 293T cells.
- Highly transfectable cells such as human embryonic kidney 293T cells, can be used.
- “highly transfectable” it is meant that at least about 50%, more preferably at least about 70% and most preferably at least about 80% of the cells can express the genes of the introduced DNA.
- Suitable mammalian cells include primary cells and immortalized cell lines.
- Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.
- Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No.
- the recombinant retroviral genomes in non limiting illustrative examples, lentiviral genomes, have a limitation to the number of polynucleotides that can be packaged into the viral particle.
- the polypeptides encoded by the polynucleotide encoding region can be truncations or other deletions that retain a functional activity such that the polynucleotide encoding region is encoded by less nucleotides than the
- polypeptides encoded by the polynucleotide encoding region can be fusion polypeptides that can be expressed from one promoter.
- the fusion polypeptide can have a cleavage signal to generate two or more functional polypeptides from one fusion polypeptide and one promoter.
- some functions that are not required after initial ex vivo transduction are not included in the retroviral genome, but rather are present on the surface of the replication incompetent recombinant retroviral particles via the packaging cell membrane. These various strategies are used herein to maximize the functional elements that are packaged within the replication incompetent recombinant retroviral particles.
- the recombinant retroviral genome to be packaged can be between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, and 8,000 nucleotides on the low end of the range and 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, and 11,000 nucleotides on the high end of the range.
- the retroviral genome to be packaged includes one or more polynucleotide regions encoding a first and second engineering signaling polypeptide as disclosed in detail herein.
- the recombinant retroviral genome to be packaged can be less than 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 11,000 nucleotides.
- Functions discussed elsewhere herein that can be packaged include required retroviral sequences for retroviral assembly and packaging, such as a retroviral rev, gag, and pol coding regions, as well as a 5' LTR and a 3' LTR, or an active truncated fragment thereof, a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element, and a cPPT/CTS element.
- required retroviral sequences for retroviral assembly and packaging such as a retroviral rev, gag, and pol coding regions, as well as a 5' LTR and a 3' LTR, or an active truncated fragment thereof, a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element, and a cPPT/CTS element.
- a replication incompetent recombinant retroviral particle herein can include any one or more or all of the following, in some embodiments in reverse orientation with respect to a 5’ to 3’ orientation established by the retroviral 5’ LTR and 3’ LTR (as illustrated in
- WO2019/055946 as a non-limiting example: one or more polynucleotide regions encoding a first and second engineering signaling polypeptide, at least one of which includes at least one lymphoproliferative element; a second engineered signaling polypeptide that can include a chimeric antigen receptor; an miRNA, a control element, such as a riboswitch, which typically regulates expression of the first and/or the second engineering signaling polypeptide; a recognition domain, an intron, a promoter that is active in a target cell, such as a T cell, a 2A cleavage signal and/or an IRES.
- RECOMBINANT RETROVIRAL PARTICLES RECOMBINANT RETROVIRAL PARTICLES
- Recombinant retroviral particles are disclosed in methods and compositions provided herein, for example, to transduce T cells and/or NK cells to make genetically modified T cells and/or NK cells.
- the recombinant retroviral particles are themselves aspects of the present invention.
- the recombinant retroviral particles included in aspects provided herein are replication incompetent, meaning that a recombinant retroviral particle cannot replicate once it leaves the packaging cell.
- the recombinant retroviral particles are lentiviral particles.
- replication incompetent recombinant retroviral particles for use in transducing cells, typically lymphocytes and illustrative embodiments T cells and/or NK cells.
- the replication incompetent recombinant retroviral particles can include any of the pseudotyping elements discussed elsewhere herein.
- the replication incompetent recombinant retroviral particles can include any of the activation elements discussed elsewhere herein.
- a replication incompetent recombinant retroviral particle including a polynucleotide including:
- the T cell activation element can be any of the activation elements discussed elsewhere herein.
- the T cell activation element can be anti-CD3 scFvFc.
- a replication incompetent recombinant retroviral particle including a polynucleotide including one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide including a chimeric antigen receptor (CAR) and a second polypeptide including a lymphoproliferative element.
- the lymphoproliferative element can be a chimeric lymphoproliferative element.
- the lymphoproliferative element does not comprise IL-7 tethered to the IL-7 receptor alpha chain or a fragment thereof.
- the lymphoproliferative element does not comprise IL-15 tethered to the IL-2/IL-15 receptor beta chain.
- a replication incompetent recombinant retroviral particle comprising a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a chimeric lymphoproliferative element, for example a constitutively active chimeric lymphoproliferative element.
- CAR chimeric antigen receptor
- the chimeric lymphoproliferative element does not comprise a cytokine tethered to its cognate receptor or tethered to a fragment of its cognate receptor.
- a recombinant retroviral particle that includes (i) a pseudotyping element capable of binding to a T cell and/or NK cell and facilitating membrane fusion of the recombinant reto viral particle thereto; (ii) a polynucleotide having one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first engineered signaling polypeptide having a chimeric antigen receptor that includes an antigen-specific targeting region, a transmembrane domain, and an intracellular activating domain, and a second engineered signaling polypeptide that includes at least one lymphoproliferative element; wherein expression of the first engineered signaling
- the promoter active in T cells and/or NK cells is not active in the packaging cell line or is only active in the packaging cell line in an inducible manner.
- either of the first and second engineered signaling polypeptides can have a chimeric antigen receptor and the other engineered signaling polypeptide can have at least one lymphoproliferative element.
- RNA molecules include, for example, pseudotyping elements, activation elements, and membrane bound cytokines, as well as nucleic acid sequences that are included in a genome of a replication incompetent, recombinant retroviral particle such as, but not limited to, a nucleic acid encoding a CAR; a nucleic acid encoding a lymphoproliferative element; a nucleic acid encoding a control element, such as a riboswitch; a promoter, especially a promoter that is constitutively active or inducible in a T cell; and a nucleic acid encoding an inhibitory RNA molecule.
- various aspects provided herein such as methods of making recombinant retroviral particles, methods for performing adoptive cell therapy, and methods for transducing T cells, produce and/or include replication incompetent, recombinant retroviral particles.
- Replication incompetent recombinant retroviruses that are produced and/or included in such methods themselves form separate aspects of the present invention as replication incompetent, recombinant retroviral particle compositions, which can be in an isolated form.
- Such compositions can be in dried down (e.g. lyophilized) form or can be in a suitable solution or medium known in the art for storage and use of retroviral particles.
- a replication incompetent recombinant retroviral particle having in its genome a polynucleotide having one or more nucleic acid sequences operatively linked to a promoter active in T cells and/or NK cells that in some instances, includes a first nucleic acid sequence that encodes one or more (e.g. two or more) inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence that encodes a chimeric antigen receptor, or CAR, as described herein.
- a first nucleic acid sequence that encodes one or more (e.g. two or more) inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence that encodes a chimeric antigen receptor, or CAR, as described herein.
- a third nucleic acid sequence is present that encodes at least one lymphoproliferative element described previously herein that is not an inhibitory RNA molecule.
- the polynucleotide incudes one or more riboswitches as presented herein, operably linked to the first nucleic acid sequence, the second nucleic acid sequence, and/or the third nucleic acid sequence, if present.
- expression of one or more inhibitory RNAs, the CAR, and/or one or more lymphoproliferative elements that are not inhibitory RNAs is controlled by the riboswitch.
- two to 10 inhibitory RNA molecules are encoded by the first nucleic acid sequence.
- two to six inhibitory RNA molecules are encoded by the first nucleic acid sequence.
- 4 inhibitory RNA molecules are encoded by the first nucleic acid sequence.
- the first nucleic acid sequence encodes one or more inhibitory RNA molecules and is located within an intron.
- the intron includes all or a portion of a promoter.
- the promoter can be a Pol I, Pol II, or Pol III promoter.
- the promoter is a Pol II promoter.
- the intron is adjacent to and downstream of the promoter active in a T cell and/or NK cell.
- the intron is EFl-a intron A.
- Recombinant retroviral particle embodiments herein include those wherein the retroviral particle comprises a genome that includes one or more nucleic acids encoding one or more inhibitory RNA molecules.
- the retroviral particle comprises a genome that includes one or more nucleic acids encoding one or more inhibitory RNA molecules.
- nucleic acids that encode inhibitory RNA molecules that can be included in a genome of a retroviral particle including combinations of such nucleic acids with other nucleic acids that encode a CAR or a lymphoproliferative element other than an inhibitory RNA molecule, are included for example, in the inhibitory RNA section provided herein, as well as in various other paragraphs that combine these embodiments.
- various alternatives of such replication incompetent recombinant retroviruses can be identified by exemplary nucleic acids that are disclosed within packaging cell line aspects disclosed herein.
- disclosure in this section of a recombinant retroviral particle that includes a genome that encodes one or more (e.g. two or more) inhibitory RNA molecules can be combined with various alternatives for such nucleic acids encoding inhibitory RNA molecules provided in other sections herein.
- nucleic acids encoding one or more inhibitory RNA molecules can be combined with various other functional nucleic acid elements provided herein, as for example, disclosed in the section herein that focuses on inhibitory RNA molecules and nucleic acid encoding these molecules.
- the various embodiments of specific inhibitory RNA molecules provided herein in other sections can be used in recombinant retroviral particle aspects of the present disclosure.
- recombinant retroviral vectors such as lentiviral vectors
- These elements are included in the packaging cell line section and in details for making replication incompetent, recombinant retroviral particles provided in the Examples section and as illustrated in WO2019/055946.
- lentiviral particles typically include packaging elements REV, GAG and POL, which can be delivered to packaging cell lines via one or more packaging plasmids, a pseudotyping element, various examples which are provided herein, which can be delivered to a packaging cell line via a pseudotyping plasmid, and a genome, which is produced by a polynucleotide that is delivered to a host cell via a transfer plasmid.
- This polynucleotide typically includes the viral LTRs and a psi packaging signal.
- the 5’ LTR can be a chimeric 5’ LTR fused to a heterologous promoter, which includes 5’ LTRs that are not dependent on Tat transactivation.
- Vpu such as a polypeptide comprising Vpu (sometimes called a“Vpu polypeptide” herein) including but not limited to, Src-FLAG-Vpu
- Vpx such as Src-FLAG-Vpx, is packaged within the retroviral particle.
- Vpu and Vpx is packaged within the retroviral particle for any composition or method aspect and embodiment that includes a retroviral particle provided herein.
- Retroviral particles included in various aspects of the present invention are in illustrative embodiments, replication incompetent, especially for safety reasons for embodiments that include introducing cells transduced with such retroviral particles into a subject.
- retroviral particles are not produced from the transduced cell. Modifications to the retroviral genome are known in the art to assure that retroviral particles that include the genome are replication incompetent.
- replication competent recombinant retroviral particles can be used.
- expression vectors can be delivered to packaging cells and/or to T cells using different types of vectors, such as expression vectors.
- Illustrative aspects of the invention utilize retroviral vectors, and in some particularly illustrative embodiments lentiviral vectors.
- Other suitable expression vectors can be used to achieve certain embodiments herein.
- Such expression vectors include, but are not limited to, viral vectors (e.g.
- viral vectors based on vaccinia virus; poliovirus; adenovirus see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther
- SV40 herpes simplex virus
- a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
- retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
- a gamma retrovirus e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816
- replication incompetent recombinant retroviral particles are a common tool for gene delivery (Miller, Nature (1992) 357:455-460).
- the ability of replication incompetent recombinant retroviral particles to deliver an unrearranged nucleic acid sequence into a broad range of rodent, primate and human somatic cells makes replication incompetent recombinant retroviral particles well suited for transferring genes to a cell.
- the replication incompetent recombinant retroviral particles can be derived from the Alpharetrovirus genus, the Betaretrovirus genus, the Gammaretrovirus genus, the Deltaretrovirus genus, the Epsilonretrovirus genus, the Lentivirus genus, or the Spumavirus genus.
- retroviruses suitable for use in the methods disclosed herein.
- murine leukemia virus MMV
- human immunodeficiency virus HIV
- equine infectious anaemia virus EIAV
- mouse mammary tumor virus MMTV
- Rous sarcoma virus RSV
- Fujinami sarcoma virus FuSV
- Moloney murine leukemia virus Mo-MLV
- FBR MSV FBR murine osteosarcoma virus
- Mo-MSV Abelson murine leukemia virus
- A-MLV Avian myelocytomatosis virus-29
- AEV Avian erythroblastosis virus
- retroviruses A detailed list of retroviruses may be found in Coffin et al (“Retroviruses” 1997 Cold Spring Harbor Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763). Details on the genomic structure of some retroviruses may be found in the art. By way of example, details on HIV may be found from the NCBI Genbank (i.e. Genome Accession No. AF033819).
- the replication incompetent recombinant retroviral particles can be derived from the Lentivirus genus. In some embodiments, the replication incompetent recombinant retroviral particles can be derived from HIV, SIV, or FIV. In further illustrative embodiments, the replication incompetent recombinant retroviral particles can be derived from the human
- HIV immunodeficiency virus
- Lentiviruses are complex retroviruses which, in addition to the common retroviral genes gag, pol and env, contain other genes with regulatory or structural function. The higher complexity enables the lentivirus to modulate the life cycle thereof, as in the course of latent infection.
- a typical lentivirus is the human immunodeficiency virus (HIV), the etiologic agent of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages.
- replication incompetent recombinant retroviral particles provided herein contain Vpx polypeptide.
- replication incompetent recombinant retroviral particles provided herein comprise and/or contain Vpu polypeptide.
- a retroviral particle is a lentiviral particle.
- Such retroviral particle typically includes a retroviral genome within a capsid which is located within a viral envelope.
- DNA-containing viral particles are utilized instead of recombinant retroviral particles.
- viral particles can be adenoviruses, adeno-associated viruses, herpesviruses, cytomegaloviruses, poxviruses, avipox viruses, influenza viruses, vesicular stomatitis virus (VSV), or Sindbis virus.
- VSV vesicular stomatitis virus
- a skilled artisan will appreciate how to modify the methods disclosed herein for use with different viruses and retroviruses, or retroviral particles.
- functional units can be included in such genomes to induce integration of all or a portion of the DNA genome of the viral particle into the genome of a T cell transduced with such virus.
- the HIV RREs and the polynucleotide region encoding HIV Rev can be replaced with N-terminal RGG box RNA binding motifs and a polynucleotide region encoding ICP27.
- the polynucleotide region encoding HIV Rev can be replaced with one or more polynucleotide regions encoding adenovirus E1B 55-kDa and E4 Orf6.
- a container such as a commercial container or package, or a kit comprising the same, comprising isolated replication incompetent recombinant retroviral particles according to any of the replication incompetent recombinant retroviral particle aspects provided herein.
- a container such as a commercial container or package, or a kit comprising the same, comprising isolated packaging cells, in illustrative embodiments isolated packaging cells from a packaging cell line, according to any of the packaging cell and/or packaging cell line aspects provided herein.
- the kit includes additional containers that include additional reagents such as buffers or reagents used in methods provided herein.
- any replication incompetent recombinant retroviral particle provided herein in any aspect, in the manufacture of a kit for genetically modifying a T cell or NK cell according to any aspect provided herein.
- any packaging cell or packaging cell line provided herein in any aspect, in the manufacture of a kit for producing the replication incompetent recombinant retroviral particles according to any aspect provided herein.
- a commercial container containing a replication incompetent recombinant retroviral particle and instructions for the use thereof to treat tumor growth in a subject, wherein the replication incompetent recombinant retroviral particle comprises in its genome a polynucleotide comprising one or more nucleic acid sequences operatively linked to a promoter active in T cells and/or NK cells.
- a nucleic acid sequence of the one or more nucleic acid sequences can encode a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
- a nucleic acid sequence of the one or more nucleic acid sequences can encode two or more inhibitory RNA molecules directed against one or more RNA targets.
- the container that contains the recombinant retroviral particles can be a tube, vial, well of a plate, or other vessel for storage of a recombinant retroviral particle.
- the kit can include two or more containers wherein a second or other container can include, for example, a solution or media for transduction of T cells and/or NK cells, and/or the second or other container can include a pH-modulating pharmacologic agent. Any of these containers can be of industrial strength and grade.
- the replication incompetent recombinant retroviral particle in such aspects that include a kit and a nucleic acid encoding an inhibitory RNA molecule, can be any of the embodiments for such replication incompetent recombinant retroviral particles provided herein, which include any of the embodiments for inhibitory RNA provided herein.
- kits for genetically modifying a T cell or NK cell wherein the use of the kit includes: contacting the T cell or NK cell ex vivo with the replication incompetent recombinant retroviral particle, wherein the replication incompetent recombinant retroviral particle includes a pseudotyping element on a surface and a T cell activation element on the surface, wherein said contacting facilitates transduction of the T cell or NK cell by the replication incompetent recombinant retroviral particle, thereby producing a genetically modified T cell or NK cell.
- the T cell or NK cell can be from a subject.
- the T cell activation element can be membrane- bound.
- the contacting can be performed for between 1, 2, 3, 4, 5, 6, 7, or 8 hours on the low end of the range and 4, 5, 6, 7, 8, 10, 12, 15, 18, 21, and 24 hours on the high end of the range, for example, between 1 and 12 hours.
- the replication incompetent recombinant retroviral particle for use in the manufacture of a kit can include any of the aspects, embodiments, or subembodiments discussed elsewhere herein.
- a pharmaceutical composition for treating or preventing cancer or tumor growth comprising a replication incompetent recombinant retroviral particle as an active ingredient.
- an infusion composition or other delivery solution for treating or preventing cancer or tumor growth comprising a replication incompetent recombinant retroviral particle.
- the replication incompetent recombinant retroviral particle of the pharmaceutical composition or infusion composition can include any of the aspects, embodiments, or subembodiments discussed above or elsewhere herein.
- genetically modified lymphocytes are produced, which themselves are a separate aspect of the invention.
- Such genetically modified lymphocytes are produced, which themselves are a separate aspect of the invention.
- lymphocytes can be genetically modified and/or transduced lymphocytes.
- a genetically modified T cell or NK cell is made using a method according to any aspect for genetically modifying T cells and/or NK cells in blood or a component thereof, provided herein.
- the T cell or NK cell has been genetically modified to express a first engineered signaling polypeptide.
- the first engineered signaling polypeptide can be a lymphoproliferative element or a CAR that includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
- the T cell or NK cell can further include a second engineered signaling polypeptide that can be a CAR or a
- the lymphoproliferative element can be a chimeric lymphoproliferative element.
- the T cell or NK cell can further include a pseudotyping element on a surface.
- the T cell or NK cell can further include an activation element on a surface.
- the CAR, lymphoproliferative element, pseudotyping element, and activation element of the genetically modified T cell or NK cell can include any of the aspects, embodiments, or subembodiments disclosed herein.
- the activation element can be anti-CD3 antibody, such as an anti-CD3 scFvFc.
- genetically modified lymphocytes are lymphocytes such as T cells or NK cells that have been genetically modified to express a first engineered signaling polypeptide comprising at least one lymphoproliferative element and/or a second engineered signaling polypeptide comprising a chimeric antigen receptor, which includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
- the NK cells are NKT cells. NKT cells are a subset of T cells that express CD3 and typically coexpress an ab T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells (such as NK1.1 or CD56).
- heterologous nucleic acid sequence that has been introduced into the lymphocyte by a recombinant DNA method.
- the heterologous sequence in illustrative embodiments is inserted into the lymphocyte during a method for transducing the lymphocyte provided herein.
- the heterologous nucleic acid is found within the lymphocyte and in some embodiments is or is not integrated into the genome of the genetically modified lymphocyte.
- the heterologous nucleic acid is integrated into the genome of the genetically modified lymphocyte.
- lymphocytes are produced, in illustrative embodiments, using a method for transducing lymphocytes provided herein, that utilizes a recombinant retroviral particle.
- recombinant retroviral particle can include a polynucleotide that encodes a chimeric antigen receptor that typically includes at least an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
- ASTR antigen-specific targeting region
- replication incompetent recombinant retroviral particles and polynucleotides encoded in a genome of the replication incompetent retroviral particle that can be used to produce genetically modified lymphocytes that themselves form another aspect of the present disclosure.
- lymphocytes of the present disclosure can be isolated outside the body.
- lymphocytes can be found in media and other solutions that are used for ex vivo transduction as provided herein.
- the lymphocytes can be present in a genetically unmodified form in blood that is collected from a subject in methods provided herein, and then genetically modified during method of transduction.
- the genetically modified lymphocytes can be found inside a subject after they are introduced or reintroduced into the subject after they have been genetically modified.
- the genetically modified lymphocytes can be a resting T cell or a resting NK cell, or the genetically modified T cell or NK cell can be actively dividing, especially after it expresses some of the functional elements provided in nucleic acids that are inserted into the T cell or NK cell after transduction as disclosed herein.
- transduced and/or genetically modified T cell or NK cell comprising a recombinant polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, in its genome.
- T cells and/or NK cells that relate to either aspects for transduction of T cells and/or NK cells in blood or a component thereof, that include transcription units that encode one, two, or more (e.g. 1-10, 2-10, 4-10, 1-6, 2-6, 3-6, 4-6, 1-4, 2-4, 3-4) inhibitory RNA molecules.
- such inhibitory RNA molecules are lymphoproliferative elements and therefore, can be included in any aspect or embodiment disclosed herein as the lymphoproliferative element as long as they induce proliferation of a T cell and/or an NK cell, or otherwise meet a test for a lymphoproliferative element provided herein.
- RNA target RNAs can be used in embodiments of any of the aspects provided herein.
- one, most or all of the one (e.g. two) or more inhibitory RNA molecules decrease expression of an endogenous TCR.
- the RNA target is mRNA transcribed from a gene selected from the group consisting of: PD-l, CTLA4, TCR alpha, TCR beta, CD3 zeta, SOCS, SMAD2, a miR-155 target, IFN gamma, cCBL, TRAIL2, PP2A, and ABCG1.
- at least one of the one (e.g. two) or more inhibitory RNA molecules is miR-1 5.
- the ASTR of the CAR is an MRB ASTR and/or the ASTR of the CAR binds to a tumor associated antigen.
- the first nucleic acid sequence is operably linked to a riboswitch, which for example is capable of binding a nucleoside analog, and in illustrative embodiments is an antiviral drug such as acyclovir.
- control element is a polynucleotide comprising a riboswitch.
- the riboswitch is capable of binding a nucleoside analog and when the nucleoside analog is present, one or both of the engineered signaling polypeptides are expressed.
- a nucleic acid will in some embodiments be DNA, including, e.g., a recombinant expression vector.
- a nucleic acid will in some embodiments be RNA, e.g., in vitro synthesized RNA.
- a nucleic acid provides for production of a polypeptide of the present disclosure, e.g., in a mammalian cell.
- a subject nucleic acid provides for amplification of the nucleic acid encoding a polypeptide of the present disclosure.
- a nucleotide sequence encoding a polypeptide of the present disclosure can be operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc.
- Suitable promoter and enhancer elements are known in the art.
- suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc.
- suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters.
- Suitable reversible promoters including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art.
- Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (ale A) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters
- the locus or construct or trans gene containing the suitable promoter is irreversibly switched through the induction of an inducible system.
- Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al., PNAS (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific
- the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter.
- a CD4 gene promoter can be used; see, e.g., Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; and Marodon et al. (2003) Blood 101 :3416.
- a CD8 gene promoter can be used.
- NK cell-specific expression can be achieved by use of an Neri (p46) promoter; see, e.g., Eckelhart et al. (2011) Blood 117: 1565.
- a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter such as a GALI promoter, a GALIO promoter, an ADH2 promoter, a PH05 promoter, a CUP1 promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well
- Suitable promoters for use in prokaryotic host cells include, but are not limited to, a
- bacteriophage T7 RNA polymerase promoter a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J.
- a hybrid promoter e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like
- a sigma70 promoter e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPI-2 (see, e.g., W096/17951); an actA promoter (see, e.g., Shetron-Rama et al. (2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g., Valdivia and Falkow (1996). Mai. Microbial.
- a sigma70 promoter e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183
- Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and PLambda.
- Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (Laci repressor protein changes conformation when contacted with lactose, thereby preventing the Laci repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25).
- a nucleotide sequence encoding a polypeptide of the disclosure can be present in an expression vector and/or a cloning vector. Nucleotide sequences encoding two separate polypeptides can be cloned in the same or separate vectors.
- An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like.
- Suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating subject recombinant constructs.
- the following bacterial vectors are provided by way of example: pBs, phagescript, PsiXl74, pBluescript SK, pBs KS, pNH8a, pNHl6a, pNHl8a, pNH46a (Stratagene, La Jolla, CA, USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
- eukaryotic vectors are provided by way of example: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
- Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins.
- a selectable marker operative in the expression host may be present.
- a nucleic acid encoding a polypeptide of the present disclosure will in some embodiments be RNA, e.g., in vitro synthesized RNA.
- Methods for in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA including a nucleotide sequence encoding a polypeptide of the present disclosure.
- Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. (2010) Cancer Res. 15:9053.
- RNA including a nucleotide sequence encoding a polypeptide of the present disclosure into a host cell can be carried out in vitro or ex vivo or in vivo.
- a host cell e.g., an NK cell, a cytotoxic T lymphocyte, etc.
- RNA comprising a nucleotide sequence encoding a polypeptide of the present disclosure.
- Various aspects and embodiments that include a polynucleotide, a nucleic acid sequence, and/or a transcriptional unit, and/or a vector including the same further include one or more of a Kozak-type sequence (also called a Kozak-related sequence herein), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a double stop codon or a triple stop codon, wherein one or more stop codons of the double stop codon or the triple stop codon define a termination of a reading from of at least one of the one or more transcriptional units.
- a Kozak-type sequence also called a Kozak-related sequence herein
- WPRE woodchuck hepatitis virus post-transcriptional regulatory element
- a double stop codon or a triple stop codon wherein one or more stop codons of the double stop codon or the triple stop codon define a termination of a reading from of at least one of the one or more transcriptional units.
- a polynucleotide a nucleic acid sequence, and/or a transcriptional unit, and/or a vector including the same further includes a Kozak-type sequence having a 5’ nucleotide within 10 nucleotides upstream of a start codon of at least one of the one or more transcriptional units.
- Kozak determined the Kozak consensus sequence, (GCC)GCCRCCATG (SEQ ID NO:327), for 699 vertebrate mRNAs, where R is a purine (A or G) (Kozak. Nucleic Acids Res. 1987 Oct 26;15(20):8125-48).
- the Kozak-type sequence is or includes CCACCAT/UG(G) (SEQ ID NO:328), CCGCCAT/UG(G) (SEQ ID NO:329), GCCGCCGCCAT/UG(G) (SEQ ID NO:330), or GCCGCCACCAT/UG(G) (SEQ ID NO:33l) (with nucleotides in parenthesis representing optional nucleotides and nucleotides separated by a slash indicated different possible nucleotides at that position, for example depending on whether the nucleic acid is DNA or RNA.
- the A can be considered position 0.
- the nucleotides at -3 and +4 are identical, for example the -3 and +4 nucleotides can be G.
- the Kozak-type sequence includes an A or G in the 3rd position upstream of ATG where ATG is the start codon.
- the Kozak-type sequence includes an A or G in the 3rd position upstream of AUG where AUG is the start codon.
- the Kozak sequence is (GCC)GCCRCCATG (SEQ ID NO:327), where R is a purine (A or G).
- the Kozak-type sequence is GCCGCCACCAUG (SEQ ID NO:332).
- the polynucleotide includes a WPRE element.
- WPREs have been characterized in the art (See e.g., (Higashimoto et al., Gene Ther. 2007; 14: 1298)) and as illustrated in WO2019/055946.
- the WPRE element is located 3’ of a stop codon of the one or more transcriptional units and 5’ to a 3’ LTR of the polynucleotide.
- the preceding embodiments i.e.
- the one or more transcriptional units terminates with one or more stop codons of a double stop codon or a triple stop codon, wherein the double stop codon includes a first stop codon in a first reading frame and a second stop codon in a second reading frame, or a first stop codon in frame with a second stop codon, and wherein the triple stop codon includes a first stop codon in a first reading frame, a second stop codon in a second reading frame, and a third stop codon in a third reading frame, or a first stop codon in frame with a second stop codon and a third stop codon.
- a triple stop codon herein includes three stop codons, one in each reading frame, within 10 nucleotides of each other, and preferably having overlapping sequence, or three stop codons in the same reading frame, preferably at consecutive codons.
- a double stop codon means two stop codons, each in a different reading frame, within 10 nucleotides of each other, and preferably having overlapping sequences, or two stop codons in the same reading frame, preferably at consecutive codons.
- the introduction of DNA into PBMCs, B cells, T cells and/or NK cells and optionally the incorporation of the DNA into the host cell genome is performed using methods that do not utilize replication incompetent recombinant retroviral particles.
- retroviral particles such as those derived from adenovirus, adeno-associated virus, or herpes simplex virus- 1, as non-limiting examples.
- methods provided herein can include transfecting target cells with non- viral vectors.
- the non-viral vectors including naked DNA, can be introduced into the target cells, such as for example, PBMCs, B cells, T cells and/or NK cells using methods that include electroporation, nucleofection, liposomal formulations, lipids, dendrimers, cationic polymers such as poly(ethylenimine) (PEI) and poly(l-lysine) (PLL), nanoparticles, cell-penetrating peptides, microinjection, and/or non integrating lentiviral vectors.
- PEI poly(ethylenimine)
- PLL poly(l-lysine)
- DNA can be introduced into target cells, such as PBMCs, B cells, T cells and/or NK cells in a complex with liposomes and protamine.
- target cells such as PBMCs, B cells, T cells and/or NK cells in a complex with liposomes and protamine.
- Other methods for transfecting T cells and/or NK cells ex vivo that can be used in embodiments of methods provided herein, are known in the art (see e.g., Morgan and Boyerinas, Biomedicines. 2016 Apr 20;4(2). pii: E9, incorporated by reference herein in its entirety).
- DNA can be integrated into the genome using transposon-based carrier systems by co-transfection, co-nucleofection or co-electroporation of target DNA as plasmid containing the transposon ITR fragments in 5’ and 3’ ends of the gene of interest and transposase carrier system as DNA or mRNA or protein or site specific serine recombinases such as phiC3l that integrates the gene of interest in pseudo attP sites in the human genome, in this instance the DNA vector contains a 34 to 40 bp attB site that is the recognition sequence for the recombinase enzyme (Bhaskar Thyagarajan et al.
- transposon-based systems that can be used in certain methods provided herein utilize the Sleeping Beauty DNA carrier system (see e.g., U.S. Pat. No. 6,489,458 and U.S. Pat. Appl. No. 15/434,595, incorporated by reference herein in their entireties), the PiggyBac DNA carrier system (see e.g., Manuri et al., Hum Gene Ther.
- the transposon and/or transposase of the transposon-based vector systems can be produced as a minicircle DNA vector before introduction into T cells and/or NK cells (see e.g., Hudecek et al., Recent Results Cancer Res. 2016;209:37-50 and Monjezi et al., Feukemia.
- the CAR or lymphoproliferative element can also be integrated into the defined and specific sites in the genome using CRISPR or TAFEN mediated integration, by adding 50-1000 bp homology arms homologous to the integration 5’ and 3’ of the target site (Jae Seong Fee et al. Scientific Reports 5, Article number: 8572 (2015), Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway).
- CRISPR or TAFEN provide specificity and genomic-targeted cleavage and the construct will be integrated via homology- mediated end joining (Yao X at al. Cell Res. 2017 Jun;27(6):801-814. doi: 10.1038/cr.2017.76. Epub 2017 May 19).
- the CRISPR or TAFEN can be co-transfected with target plasmid as DNA, mRNA, or protein.
- Embodiments of any of the aspects provided herein can include recombinant retroviral particles whose genomes are constructed to induce expression of one or more, and in illustrative embodiments two or more, inhibitory RNA molecules, such as for example, a miRNA or shRNA, after integration into a host cell, such as a lymphocyte (e.g. a T cell and/or an NK cell).
- inhibitory RNA molecules can be encoded within introns, including for example, an EFl-a intron. This takes advantage of the present teachings of methods to maximize the functional elements that can be included in a packageable retroviral genome to overcome shortcomings of prior teachings and maximize the effectiveness of such
- the inhibitory RNA molecule includes a 5’ strand and a 3’ strand (in some examples, sense strand and antisense strand) that are partially or fully complementary to one another such that the two strands are capable of forming a 18-25 nucleotide RNA duplex within a cellular environment.
- the 5' strand can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length
- the 3' strand can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
- the 5' strand and the 3' strand can be the same or different lengths, and the RNA duplex can include one or more mismatches. Alternatively, the RNA duplex has no mismatches.
- the inhibitory RNA molecule is a miRNA or an shRNA.
- a nucleic acid encoding an siRNA especially in a context where the nucleic acid is part of a genome, it will be understood that such nucleic acid is capable of forming an siRNA precursor such as miRNA or shRNA in a cell that is processed by DICER to form a double stranded RNA that typically interacts with, or becomes part of a RISK complex.
- an inhibitory molecule of an embodiment of the present disclosure is a precursor of a miRNA, such as for example, a Pri-miRNA or a Pre-miRNA, or a precursor of an shRNA.
- the miRNA or shRNA are artificially derived (i.e. artificial miRNAs or siRNAs).
- the inhibitory RNA molecule is a dsRNA (either transcribed or artificially introduced) that is processed into an siRNA or the siRNA itself.
- the miRNA or shRNA has a sequence that is not found in nature, or has at least one functional segment that is not found in nature, or has a combination of functional segments that are not found in nature.
- inhibitory RNA molecules are positioned in the first nucleic acid molecule in a series or multiplex arrangement such that multiple miRNA sequences are simultaneously expressed from a single polycistronic miRNA transcript.
- the inhibitory RNA molecules can be adjoined to one another either directly or indirectly by non-functional linker sequence(s).
- the linker sequence in some embodiments, is between 5 and 120 nucleotides in length, and in some embodiments can be between 10 and 40 nucleotides in length, as non-limiting examples.
- the first nucleic acid sequence encoding one or more (e.g. two or more) inhibitory RNAs and the second nucleic acid sequence encoding a CAR e.g.
- an MRB-CAR are operably linked to a promoter that is active constitutively or that can be induced in a T cell or NK cell.
- the inhibitory RNA molecule(s) e.g. miRNAs
- the CAR are expressed in a polycistronic manner.
- functional sequences can be expressed from the same transcript. For example, any of the
- lymphoproliferative elements provided herein that are not inhibitory RNA molecules can be expressed from the same transcript as the CAR and the one or more (e.g. two or more) inhibitory RNA molecules.
- the inhibitory RNA molecule is a naturally occurring miRNA such as but not limited to miR-l55.
- artificial miRNAs can be produced in which sequences capable of forming a hybridizing/complementary stem structure and directed against a target RNA, are placed in a miRNA framework that includes microRNA flanking sequences for microRNA processing and a loop, which can optionally be derived from the same naturally occurring miRNA as the flanking sequences, between the stem sequences.
- an inhibitory RNA molecule includes from 5' to 3' orientation: a 5’ microRNA flanking sequence, a 5’ stem, a loop, a 3’ stem that is partially or fully complementary to said 5' stem, and a 3’ microRNA flanking sequence.
- the 5' stem also called a 5’ arm herein
- the 5' stem is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
- the 3' stem (also called a 3’ arm herein) is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
- the loop is 3 to 40, 10 to 40, 20 to 40, or 20 to 30 nucleotides in length, and in illustrative embodiments the loop can be 18, 19, 20, 21, or 22 nucleotides in length.
- one stem is two nucleotides longer than the other stem. The longer stem can be the 5' or the 3' stem.
- the 5’ microRNA flanking sequence, 3’ microRNA flanking sequence, or both are derived from a naturally occurring miRNA, such as but not limited to miR-l55, miR-30, miR- 17-92, miR-l22, and miR-21.
- the 5’ microRNA flanking sequence, 3’ microRNA flanking sequence, or both are derived from a miR-155, such as, e.g., the miR-155 from Mus musculus or Homo sapiens. Inserting a synthetic miRNA stem-loop into a miR-155 framework (i.e.
- RNA flanking sequence consisting of nucleotides 134-161 (SEQ ID NO:333) of the Mus musculus BIC noncoding mRNA (Genbank ID AY096003.1) and a 3' microRNA flanking sequence consisting of nucleotides 223-283 of the Mus musculus BIC noncoding mRNA (Genbank ID AY096003.1).
- SIBR sequence was modified (eSIBR) to enhance expression of miRNAs (Fowler, D.K. et al. 2015. Nucleic acids Research 44(5):e48).
- miRNAs can be placed in the SIBR or eSIBR miR-155 framework.
- miRNAs are placed in a miR-155 framework that includes the 5' microRNA flanking sequence of miR-1 5 represented by SEQ ID NO:333, the 3' microRNA flanking sequence represented by SEQ ID NO:334 (nucleotides 221-265 of the Mus musculus BIC noncoding mRNA); and a modified miR-155 loop (SEQ ID NO:335).
- the 5’ microRNA flanking sequence of miR-155 is SEQ ID NO:333 or a functional variant thereof, such as, for example, a sequence that is the same length as SEQ ID NO:333, or 95%, 90%, 85%, 80%, 75%, or 50% as long as SEQ ID NO: 333 or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
- the 3’ microRNA flanking sequence of miR-155 is SEQ ID NO:334 or a functional variant thereof, such as, for example, the same length as SEQ ID NO:334, or 95%, 90%, 85%, 80%, 75%, or 50% as long as SEQ ID NO:334 or is a sequence that is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%
- At least one, at least two, at least three, or at least four of the inhibitory RNA molecules encoded by a nucleic acid sequence in a polynucleotide of a replication incompetent recombinant retroviral particle has the following arrangement in the 5' to 3' orientation: a 5’ microRNA flanking sequence, a 5’ stem, a loop, a 3’ stem that is partially or fully complementary to said 5' stem, and a 3’ microRNA flanking sequence.
- all of the inhibitory RNA molecules have the following arrangement in the 5' to 3' orientation: a 5’ microRNA flanking sequence, a 5’ stem, a loop, a 3’ stem that is partially or fully complementary to said 5' stem, and a 3’ microRNA flanking sequence.
- the inhibitory RNA molecules can be separated by one or more linker sequences, which in some embodiments have no function except to act as spacers between inhibitory RNA molecules.
- inhibitory RNA molecules are directed against the same or different RNA targets (such as e.g. mRNAs transcribed from genes of interest).
- RNA targets such as e.g. mRNAs transcribed from genes of interest.
- between 2 and 10, 2 and 8, 2 and 6, 2 and 5, 3 and 5, 3 and 6, or 4 inhibitory RNA molecules are included in the first nucleic acid sequence.
- four inhibitory RNA molecules are included in the first nucleic acid sequence.
- the one or more inhibitor RNA molecules are one or more
- the RNA targets are mRNAs transcribed from genes that are expressed by T cells such as but not limited to PD-l (prevent inactivation); CTLA4 (prevent inactivation); TCRa (safety - prevent autoimmunity); TCRb (safety - prevent autoimmunity); CD3Z (safety - prevent autoimmunity); SOCS1 (prevent inactivation); SMAD2 (prevent inactivation); a miR-l55 target (promote activation); IFN gamma (reduce CRS); cCBL (prolong signaling); TRAIL2 (prevent death); PP2A (prolong signaling); ABCG1 (increase cholesterol microdomain content by limiting clearance of cholesterol).
- T cells such as but not limited to PD-l (prevent inactivation); CTLA4 (prevent inactivation); TCRa (safety - prevent autoimmunity); TCRb (safety - prevent autoimmunity); CD3
- the RNA targets include mRNAs that encode components of the T cell receptor (TCR) complex.
- TCR T cell receptor
- Such components can include components for generation and/or formation of a T cell receptor complex and/or components for proper functioning of a T cell receptor complex.
- At least one of the two or more of inhibitory RNA molecules causes a decrease in the formation and/or function of a TCR complex
- one or more endogenous TCR complexes of a T cell in illustrative embodiments one or more endogenous TCR complexes of a T cell.
- the T cell receptor complex includes TCRa, TCRb, CD3d, CD3e, CD3g, and CD3z. It is known that there is a complex interdependency of these components such that a decrease in the expression of any one subunit will result in a decrease in the expression and function of the complex.
- the RNA target is an mRNA expressing one or more of TCRa, TCRb, CD3d, CD3e, CD3g, and CD3z endogenous to a transduced T cell.
- the RNA target is mRNA transcribed from the endogenous TCRa or TCR gene of the T cell whose genome comprises the first nucleic acid sequence encoding the one or more miRNAs.
- the RNA target is mRNA transcribed from the TCRa gene.
- inhibitory RNA molecules directed against mRNAs transcribed from target genes with similar expected utilities can be combined.
- inhibitory RNA molecules directed against target mRNAs transcribed from target genes with complementary utilities can be combined.
- the two or more inhibitory RNA molecules are directed against the mRNAs transcribed from the target genes CD3Z, PD1, SOCS1, and/or IFN gamma.
- the inhibitory RNA targets mRNA encoding Cbl Proto-Oncogene (RNF55) (also known as cCBL and RNF55) (F1GNC: 1541, Entrez Gene: 867, OMIM: 165360), T-Cell Receptor T3 Zeta Chain (CD3z) (HGNC: 1677, Entrez Gene: 919, OMIM: 186780),
- RNF55 Cbl Proto-Oncogene
- CD3z T3 Zeta Chain
- PD1, CTLA4, T Cell Immunoglobulin Mucin 3 (also known as Hepatitis A Virus Cellular Receptor 2) (HGNC: 18437 Entrez Gene: 84868, OMIM: 606652), Lymphocyte Activating 3 (LAG3) (HGNC: 6476, Entrez Gene: 3902, OMIM: 153337), SMAD2, TNF Receptor Superfamily Member lOb (TNFRSF10B) (HGNC: 11905, Entrez Gene: 8795, OMIM: 603612), Protein Phosphatase 2 Catalytic Subunit Alpha (PPP2CA) (HGNC: 9299, Entrez Gene: 5515, OMIM: 176915), Tumor Necrosis Factor Receptor Superfamily Member 6 (TNFRSF6) (also known as Fas Cell Surface Death Receptor (FAS)) (HGNC: 11920, Entrez Gene: 355, OMIM: 134637), B And T Lymphocyte Associated (BTLA) (BTLA
- HGNC: 348, Entrez Gene: 196, OMIM: 600253 Eomesodermin (EOMES)
- EOMES Eomesodermin
- HGNC: 3372, Entrez Gene: 8320, OMIM: 604615 Eomesodermin
- SMAD3 SMAD Family Member 3
- SMAD4 SMAD Family Member 4
- GPC 6770, Entrez Gene: 4089, OMIM: 600993
- TGFBR2R2D Protein Phosphatase 2 Regulatory Subunit B delta
- PPPP2R2D HGNC: 23732, Entrez Gene: 55844, OMIM: 613992
- TNFSF6 Tumor Necrosis Factor Ligand Superfamily Member 6
- TNFSF6 Tumor Necrosis Factor Ligand Superfamily Member 6
- FASL Tumor Necrosis Factor Ligand Superfamily Member 6
- HGNC 11936, Entrez Gene: 356, OMIM: 134638
- the inhibitory RNA targets mRNA encoding FAS, AHR, CD3z, cCBL, Chromobox 1 (Cbx) (HGNC: 1551, Entrez Gene: 10951, OMIM: 604511), HK2, FASL, SMAD4, or EOMES; or in some illustrative embodiments, the inhibitory RNA, for example miRNA, targets mRNA encoding FAS, AHR, Cbx3,
- a vector or genome herein includes 2 or more, 2-10, 2- 8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 of the inhibitory RNA (e.g. miRNA) identified herein, for example in the paragraph immediately above.
- a vector or genome herein includes 2 or more, 2-10, 2-8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 inhibitory RNA (e.g.
- a vector or genome herein includes 2 or more, 2-10, 2-8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 inhibitory RNA (e.g. miRNA) that target mRNA encoding AHR, Cbx3, EOMES, or HK2, or a combination of 1 or more inhibitory RNA that target such mRNA.
- inhibitory RNA e.g. miRNA
- the two or more inhibitory RNA molecules can be delivered in a single intron, such as but not limited to EFl-a intron A.
- Intron sequences that can be used to harbor miRNAs for the present disclosure include any intron that is processed within a T cell. As indicated herein, one advantage of such an arrangement is that this helps to maximize the ability to include miRNA sequences within the size constraints of a retroviral genome used to deliver such sequences to a T cell in methods provided herein.
- an intron of the first nucleic acid sequence includes all or a portion of a promoter sequence used to express that intron, a CAR sequence, and other functional sequences provided herein, such as lymphoproliferative element(s) that are not inhibitory RNA molecules, in a polycistronic manner. Sequence requirements for introns are known in the art.
- such intron processing is operably linked to a riboswitch, such as any riboswitch disclosed herein.
- the riboswitch can provide a regulatory element for control of expression of the one or more miRNA sequences on the first nucleic acid sequence.
- a riboswitch which can be any of the riboswitches discussed herein.
- inhibitory RNA molecules can be provided on multiple nucleic acid sequences that can be included on the same or a different transcriptional unit.
- a first nucleic acid sequence can encode one or more inhibitory RNA molecules and be expressed from a first promoter and a second nucleic acid sequence can encode one or more inhibitory RNA molecules and be expressed from a second promoter.
- two or more inhibitory RNA molecules are located on a first nucleic acid sequence that is expressed from a single promoter.
- the promoter used to express such miRNAs are typically promoters that are inactive in a packaging cell used to express a retroviral particle that will deliver the miRNAs in its genome to a target T cell, but such promoter is active, either constitutively or in an inducible manner, within a T cell.
- the promoter can be a Pol I, Pol II, or Pol III promoter. In some illustrative embodiments, the promoter is a Pol II promoter.
- the present disclosure provides various methods and compositions that can be used as research reagents in scientific experimentation and for commercial production.
- Such scientific experimentation can include methods for characterization of lymphocytes, such as NK cells and in illustrative
- T cells using methods for genetically modifying, for example transducing lymphocytes provided herein.
- methods for genetically modifying for example transducing lymphocytes provided herein.
- Such methods for example can be used to study activation of lymphocytes and the detailed molecular mechanisms by which activation makes such cells transducible.
- provided herein are genetically modified lymphocytes that will have utility for example, as research tools to better understand factors that influence T cell proliferation and survival.
- lymphocytes such as NK cells and in illustrative embodiments T cells
- lymphocytes can furthermore be used for commercial production, for example for the production of certain factors, such as growth factors and immunomodulatory agents, that can be harvested and tested or used in the production of commercial products.
- T cells and/or NK cells can be transduced with the replication incompetent recombinant retroviral particles provided herein that include polynucleotides.
- transduction of the T cells and/or NK cells can include polynucleotides that include polynucleotides encoding polypeptides of the present disclosure, for example, CARs, lymphoproliferative elements, and/or activation elements.
- the polynucleotides can include inhibitory RNA molecules as discussed elsewhere herein.
- the lymphoproliferative elements can be chimeric lymphoproliferative elements.
- any of the methods for genetically modifying and/or transducing lymphocytes e.g. PBMCs, or T cells and/or NK cells
- any of the methods for genetically modifying and/or transducing lymphocytes e.g. PBMCs, or T cells and/or NK cells
- Embodiments section that includes a contacting step of contacting retroviral particles with lymphocytes (e.g. PBMCs, or T cells and/or NK cells), in certain embodiments, the contacting step can be performed (or can occur) for between 30 seconds and 72 hours, for example, between 1 minute and 12 hours, or between 5 minutes and 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, or 15 minutes.
- lymphocytes e.g. PBMCs, or T cells and/or NK cells
- the contacting can be performed for less than 24 hours, for example, less than 12 hours, less than 8 hours, less than 4 hours, and in illustrative embodiments less than 2 hours, less than 1 hour, less than 30 minutes or less than 15 minutes, but in each case there is at least an initial contacting step in which retroviral particles and cells are brought into contact in suspension in a transduction reaction mixture.
- Such suspension can include allowing cells and retroviral particles to settle or causing such settling through application of a force, such as a centrifugal force, to the bottom of a vessel or chamber.
- a force such as a centrifugal force
- the contacting can be performed (or can occur) for between 30 seconds or 1, 2, 5, 10, 15, 30 or 45 minutes, or 1, 2, 3, 4, 5, 6, 7, or 8 hours on the low end of the range, and between 10 minutes, 15 minutes, 30 minutes, or 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72 hours on the high end of the range.
- the contacting step can be performed for between 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, or 30 minutes on the low end of the range and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours on the high end of the range. In some embodiments, the contacting step is performed for between 30 seconds, 1 minute, and 5 minutes on the low end of the range, and 10 minutes, 15 minutes, 30 minutes, 45 minutes, or 60 minutes on the high end of the range.
- the contacting is performed for between an initial contacting step only (without any further incubating in the reaction mixture including the retroviral particles free in suspension and cells in suspension) without any further incubation in the reaction mixture, or a 5 minute or less, 10 minute or less, 15 minute or less, 30 minute or less, or 1 hour or less incubation in the reaction mixture.
- the replication incompetent recombinant retroviral particles can be immediately washed out after adding them to the cell(s) to be genetically modified and/or transduced such that the contacting time is carried out for the length of time it takes to wash out the replication incompetent recombinant retroviral particles.
- the contacting includes at least an in initial contacting step in which a retroviral particle(s) and a cell(s) are brought into contact in suspension in a transduction reaction mixture.
- Such methods can be performed without prior activation.
- nucleic acid sequence encoding an inhibitory RNA molecule including, but not limited to, aspects and embodiments provided in this Exemplary Embodiments section, unless already stated therein, or incompatible therewith, such nucleic acid sequence is included and such inhibitory RNA molecule, in certain embodiments, targets any of the gene (e.g.
- mRNAs encoding targets identified for example in the Inhibitory RNA Molecules section herein; or in certain embodiments targets TCRa, TCRb, SOCS1, mi R 155 target, IFN gamma, cCBL, TRAIL2, PP2A, ABCG1, cCBL, CD3z, CD3z, PD1, CTLA4, TIM3, LAG3, SMAD2, TNFRSF10B, PPP2CA, TNFRSF6 (FAS), BTFA, TIGIT, A2AR, AHR, EOMES, SMAD3, SMAD4, TGFBR2, PPP2R2D, TNFSF6 (FASE), CASP3, SOCS2, TIEG1, JunB, Cbx3, Tet2, HK2, SHP1, or SHP2; or in certain embodiments targets cCBE, CD3z, CD3z, PD1, CTEA4, TIM3, EAG3, SMAD2, TNFRSF10B, PPP2CA, TNFRSF6
- the inhibitory RNA molecule includes at least one of the sequences of SEQ ID NOs:342-449. In some embodiments, the inhibitory RNA molecule includes at least one of the sequences of SEQ ID NOs:394-40l, 406-409, 438-441, or 446-449.
- nucleic acid sequence encoding an inhibitory RNA molecule including, but not limited to, aspects and embodiments provided in this Exemplary Embodiments section, unless already stated therein, or incompatible therewith, such nucleic acid sequence is included and such inhibitory RNA molecule, in certain embodiments, include 2 or more, 2-10, 2-8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 inhibitory RNA, or of the targeted inhibitory RNA (e.g.
- such polynucleotide includes 2 or more, 2-10, 2-8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 inhibitory RNA (e.g. miRNA) that target mRNA encoding FAS, cCBL, AHR, CD3z, Cbx, EOMES, or HK2, or a combination of 1 or more inhibitory RNA that target such mRNA; or in certain further illustrative embodiments, such polynucleotide includes 2 or more, 2-10, 2-8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 inhibitory RNA (e.g.
- inhibitory RNA e.g. miRNA
- aspects and embodiments provided herein that include a nucleic acid that encodes an inhibitory RNA molecule include, but are not limited to, aspects and embodiments provided herein that are directed to polynucleotides or vectors, for example replication incompetent retroviral particles, or aspects comprising a genome, such as isolated cells or replication incompetent retroviral particles.
- lymphocyte e.g. a T cell or an NK cell
- a method for genetically modifying and/or transducing a lymphocyte comprising contacting blood cells comprising the lymphocyte (e.g. the T cell or NK cell) or the population thereof, ex vivo with a replication incompetent recombinant retroviral particle comprising in its genome a polynucleotide comprising one or more nucleic acid sequences operatively linked to a promoter active in lymphocytes (e.g.
- a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain
- CAR chimeric antigen receptor
- ASTR antigen-specific targeting region
- another of the one or more nucleic acid sequences encodes one or more (e.g. two or more) inhibitory RNA molecules directed against one or more RNA targets
- further optionally another of the one or more nucleic acid sequences encodes a polypeptide lymphoproliferative element, wherein said contacting facilitates genetic modification and/or transduction of the lymphocyte (e.g. T cell or NK cell), or at least some of the lymphocytes (e.g.
- the contacting is typically performed in a reaction mixture, sometimes referred to herein as a transduction reaction mixture, comprising a population of lymphocytes (e.g. T cells and/or NK cells) and contacted with a population of replication incompetent recombinant retroviral particles.
- a transduction reaction mixture comprising a population of lymphocytes (e.g. T cells and/or NK cells) and contacted with a population of replication incompetent recombinant retroviral particles.
- Various contacting times are provided herein, including, but not limited to, in this Exemplary Embodiments section, that can be used in this aspect to facilitate membrane association, and eventual membrane fusion of the lymphocytes (e.g. T cells and/or the NK cells) to the replication incompetent recombinant retroviral particles.
- contacting is performed for less than 15 minutes.
- kits for genetically modifying lymphocytes e.g. T cells or NK cells
- the use of the kit comprises: contacting blood cells comprising the lymphocytes (e.g. T cells and/or the NK cells) ex vivo in a reaction mixture, with the replication incompetent recombinant retroviral particles, wherein the replication incompetent recombinant retroviral particles comprise a pseudotyping element on their surface, wherein the replication incompetent recombinant retroviral particles comprise a polynucleotide comprising one or more nucleic acid sequences, typically
- transcriptional units operatively linked to a promoter active in lymphocytes (e.g. T cells and/or NK cells), wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR), a first polypeptide comprising a lymphoproliferative element (LE), or a first polypeptide comprising an LE and a second polypeptide comprising a CAR, thereby producing the genetically modified lymphocytes (e.g. the genetically modified T cells and/or the genetically modified NK cells).
- CAR chimeric antigen receptor
- LE lymphoproliferative element
- a first polypeptide comprising an LE and a second polypeptide comprising a CAR thereby producing the genetically modified lymphocytes (e.g. the genetically modified T cells and/or the genetically modified NK cells).
- Various contacting times are provided herein, including, but not limited to, in this Exemplary
- Embodiments section that can be used in this aspect to facilitate membrane association, and eventual membrane fusion of the lymphocytes (e.g. T cells and/or the NK cells) to the replication incompetent recombinant retroviral particles.
- contacting is performed for less than 15 minutes.
- a genetically modified lymphocyte made by genetically modifying lymphocytes (e.g. T cells and/or NK cells) according to a method comprising contacting blood cells comprising the T cells or NK cells ex vivo in a reaction mixture, with replication incompetent recombinant retroviral particles, wherein the replication incompetent recombinant retroviral particles comprise a pseudotyping element on their surface, wherein the replication incompetent recombinant retroviral particles comprise a polynucleotide comprising one or more nucleic acid sequences, typically transcriptional units operatively linked to a promoter active in lymphocytes (e.g.
- T cells and/or NK cells wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR), a first polypeptide comprising a lymphoproliferative element (LE), or a first polypeptide comprising an LE and a second polypeptide comprising a CAR, thereby producing the genetically modified lymphocytes (e.g. T cells and/or the genetically modified NK cells).
- CAR chimeric antigen receptor
- LE lymphoproliferative element
- a first polypeptide comprising an LE and a second polypeptide comprising a CAR thereby producing the genetically modified lymphocytes (e.g. T cells and/or the genetically modified NK cells).
- Various contacting times are provided herein, including, but not limited to, in this Exemplary Embodiments section, that can be used in this aspect to facilitate membrane association, and eventual membrane fusion of the lymphocytes (e.g. T cells and/or the NK cells) to the replication in
- a replication incompetent recombinant retroviral particle for use in a method for genetically modifying lymphocyte, for example a T cell and/or NK cell, wherein the method comprises contacting blood cells comprising the lymphocyte, for example T cell and/or NK cell, of the subject in a reaction mixture, ex vivo, with a replication incompetent recombinant retroviral particle comprising in its genome a polynucleotide comprising one or more nucleic acid sequences operatively linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain, and optionally another of the one or more nucleic acid sequences encodes one or more (e.g.
- CAR chimeric antigen receptor
- ASTR antigen-specific targeting
- RNA molecules directed against one or more RNA targets and further optionally another of the one or more nucleic acid sequences encodes a polypeptide lymphoproliferative element, wherein said contacting facilitates transduction of at least some of the resting T cells and/or NK cells by the replication incompetent recombinant retroviral particles, thereby producing a genetically modified T cell and/or NK cell.
- Various contacting times are provided herein, including, but not limited to, in this Exemplary Embodiments section, that can be used in this aspect to facilitate membrane association, and eventual membrane fusion of the lymphocytes (e.g. T cells and/or the NK cells) to the replication incompetent recombinant retroviral particles.
- contacting is performed for less than 15 minutes.
- the method can further include introducing the genetically modified T cell and/or NK cell into a subject.
- the blood cells comprising the lymphocyte e.g. the T cell and/or NK cell
- the introducing is a reintroducing.
- a population of lymphocytes e.g. T cells and/or NK cells
- a replication incompetent recombinant retroviral particle in the manufacture of a kit for genetically modifying a lymphocyte, for example a T cell and/or NK cell of a subject, wherein the use of the kit comprises contacting blood cells comprising the lymphocyte, for example the T cell and/or the NK cell of the subject ex vivo in a reaction mixture, with replication incompetent recombinant retroviral particles comprising in their genome a polynucleotide comprising one or more nucleic acid sequences operatively linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a
- CAR chimeric antigen receptor
- ASTR antigen-specific targeting region
- transmembrane domain and an intracellular activating domain
- another of the one or more nucleic acid sequences encodes one or more (e.g. two or more) inhibitory RNA molecules directed against one or more RNA targets
- another of the one or more nucleic acid sequences encodes a polypeptide lymphoproliferative element
- said contacting facilitates genetic modification of at least some of the T cells and/or NK cells by the replication incompetent recombinant retroviral particles, thereby producing a genetically modified T cell and/or NK cell.
- various contacting times are provided herein, that can be used in this aspect to facilitate membrane association, and eventual membrane fusion of the lymphocyte (e.g.
- the T cell and/or the NK cell to the replication incompetent recombinant retroviral particles.
- contacting is performed for less than 15 minutes.
- the blood cells comprising the lymphocyte e.g. the T cell and/or NK cell
- the introducing is a
- a population of T cells and/or NK cells are contacted in the contacting step, genetically modified and/or transduced, and introduced into the subject in the introducing step.
- a medicament for genetically modifying lymphocytes for example T cells and/or NK cells of a subject
- the use of the medicament comprises:
- CAR chimeric antigen receptor
- ASTR antigen-specific targeting region
- RNA molecules directed against one or more RNA targets and further optionally another of the one or more nucleic acid sequences encodes a polypeptide lymphoproliferative element, wherein said contacting facilitates genetic modification of at least some of the lymphocytes (for example, T cells and/or NK cells) by the replication incompetent recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells; and optionally
- T cell and/or NK cell introducing the genetically modified T cell and/or NK cell into the subject, thereby genetically modifying the lymphocytes, for example T cells and/or NK cells of the subject.
- lymphocytes e.g. T cells and/or the NK cells
- contacting is performed for less than 15 minutes.
- the blood cells, lymphocyte(s) e.g. T cell(s) and/or NK cell(s)
- the genetically modified and/or transduced lymphocyte e.g.
- T cell and/or NK cell or population thereof, is introduced or reintroduced into a subject.
- genetically modified lymphocyte(s) e.g. T cell(s) or NK(s) cell
- methods aspects for genetically modifying and/or transducing a lymphocyte(s) e.g.
- the reaction mixture comprises at least 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, or 99% whole blood and optionally an effective amount of an anticoagulant, or the reaction mixture further comprises at least one additional blood or blood preparation component that is not a PBMC, and in further illustrative embodiments such blood or blood preparation component is one or more of the Noteworthy Non-PBMC Blood or Blood Preparation Components provided herein.
- a reaction mixture comprising replication incompetent recombinant retroviral particles, a T cell activation element, and blood cells, wherein the recombinant retroviral particles comprise a pseudotyping element on their surface, wherein the blood cells comprise T cells and/or NK cells, wherein the replication incompetent recombinant retroviral particles comprise a polynucleotide comprising one or more nucleic acid sequences, typically transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR), a first polypeptide comprising a lymphoproliferative element (LE), and/or one or more inhibitory RNA molecules, and wherein the reaction mixture comprises at least 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, or 99% whole blood.
- the one or more inhibitory RNA molecule(s) comprising replication incompetent re
- a reaction mixture comprising replication incompetent recombinant retroviral particles, and blood cells, wherein the recombinant retroviral particles comprise a pseudotyping element on their surface, wherein the blood cells comprise T cells and/or NK cells, and wherein the reaction mixture comprises at least 10%, 20%, 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% whole blood and optionally an effective amount of an anticoagulant, or wherein the reaction mixture further comprises at least one additional blood or blood preparation component that is not a PBMC, and in illustrative embodiments such blood or blood preparation component is one or more of the Noteworthy Non-PBMC Blood or Blood Preparation Components provided herein.
- a reaction mixture comprising replication incompetent recombinant retroviral particles, a T cell activation element, and blood cells, wherein the recombinant retroviral particles comprise a pseudotyping element on their surface, wherein the blood cells comprise T cells and/or NK cells, wherein the replication incompetent recombinant retroviral particles comprise a polynucleotide comprising one or more nucleic acid sequences, typically transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR), a first polypeptide comprising a lymphoproliferative element (LE), and/or one or more inhibitory RNA molecules, and wherein the reaction mixture comprises at least 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, or 99% whole blood and optionally an effective amount of an anticoagulant, or
- a method for genetically modifying T cells and/or NK cells in blood or a component thereof comprising contacting blood cells comprising the T cells and/or NK cells ex vivo, with replication incompetent recombinant retroviral particles in a reaction mixture, wherein the replication incompetent recombinant retroviral particles comprise a pseudotyping element on their surface, wherein said contacting facilitates association of the T cells and/or NK cells with the replication incompetent recombinant retroviral particles, wherein the recombinant retroviral particles genetically modify and/or transduce the T cells and/or NK cells, and wherein the reaction mixture comprises at least 10% 10%, 20%, 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% whole blood and optionally an effective amount of an anticoagulant, or wherein the reaction mixture further comprises at least one additional blood or blood preparation component that is not a PBMC, and in illustrative
- kits for genetically modifying T cells and/or NK cells of a subject comprising: contacting blood cells comprising the T cells and/or NKs cell ex vivo in a reaction mixture, with the replication incompetent recombinant retroviral particles, wherein the replication incompetent recombinant retroviral particles comprise a pseudotyping element on their surface, wherein said contacting facilitates association of the T cells or NK cells with the replication incompetent recombinant retroviral particles, wherein the recombinant retroviral particles genetically modify and/or transduce the T cells and/or NK cells, and wherein the blood cells comprise T cells, NK cells, and wherein the reaction mixture comprises at least 10%, 20%, 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% whole blood and optionally an effective amount of an
- a genetically modified T cell or NK cell made by genetically modifying T cells and/or NK cells according to a method comprising , contacting blood cells comprising the T cells and/or NK cells ex vivo, with replication incompetent recombinant retroviral particles in a reaction mixture, wherein the replication incompetent recombinant retroviral particles comprise a pseudotyping element on their surface, wherein said contacting facilitates association of the T cells and/or NK cells with the replication incompetent recombinant retroviral particles, wherein the recombinant retroviral particles genetically modify and/or transduce the T cells and/or NK cells, and wherein the reaction mixture comprises at least 10%, 20%, 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% whole blood and optionally an effective amount of an anticoagulant, or wherein the reaction mixture further comprises at least one additional blood or blood preparation component that is not a PBMC,
- the one or more Noteworthy Non-PBMC Blood or Blood Preparation Components are present in certain illustrative embodiments of any of the reaction mixture, use, genetically modified T cell or NK cell, or method for genetically modifying T cells and/or NK cells provided herein, including but not limited to those provided in this Exemplary Embodiments section, because in these certain illustrative embodiments, the reaction mixture comprises at least 10% whole blood.
- the reaction mixture comprises between 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, and 75% on the low end of the range, and 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% on the high end of the range of whole blood, or at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% whole blood.
- the blood cells in the reaction mixture comprise at least 10% neutrophils and at least 0.5% eosinophils, as a percent of the white blood cells in the reaction mixture.
- the reaction mixture comprises at least 20%, 25%, 30%, or 40% neutrophils as a percent of white blood cells in the reaction mixture, or between 20% and 80%, 25% and 75%, or 40% and 60% neutrophils as a percent of white blood cells in the reaction mixture.
- the reaction mixture comprises at least 0.1% eosinophils, or between 0.25% and 8% eosinophils, or between 0.5% and 4% as a percent of white blood cells in the reaction mixture.
- any of the reaction mixture use, genetically modified T cell or NK cell, or method for genetically modifying T cells and/or NK cells provided herein, included but not limited to those provided in this Exemplary Embodiments section, unless incompatible with, or already stated in an aspect or embodiments, the blood cells in the reaction mixture are not subjected to a PBMC enrichment procedure before the contacting.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (20)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA3111084A CA3111084A1 (en) | 2018-09-02 | 2019-09-02 | Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs |
AU2019333324A AU2019333324A1 (en) | 2018-09-02 | 2019-09-02 | Methods and compositions for genetically modifying lymphocytes in blood or in enriched PBMCs |
CN201980067120.4A CN113840911A (en) | 2018-09-02 | 2019-09-02 | Methods and compositions for genetically modifying lymphocytes in blood or in enriched PBMCs |
US17/272,631 US20210317408A1 (en) | 2018-09-02 | 2019-09-02 | Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs |
EP19782802.3A EP3841200A2 (en) | 2018-09-02 | 2019-09-02 | Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs |
JP2021511656A JP2021536245A (en) | 2018-09-02 | 2019-09-02 | Methods and Compositions for Gene Modification of Lymphocytes in Blood or Concentrated PBMCs |
US16/823,319 US20200255864A1 (en) | 2016-03-19 | 2020-03-18 | Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof |
EP20857656.1A EP4022035A4 (en) | 2019-09-01 | 2020-08-31 | Methods and compositions for the modification and delivery of lymphocytes |
CN202080076199.XA CN116057181A (en) | 2019-09-01 | 2020-08-31 | Methods and compositions for modifying and delivering lymphocytes |
BR112022003617A BR112022003617A2 (en) | 2019-09-01 | 2020-08-31 | METHODS AND COMPOSITIONS FOR THE MODIFICATION AND DISTRIBUTION OF LYMPHOCYTES |
AU2020336230A AU2020336230A1 (en) | 2019-09-01 | 2020-08-31 | Methods and compositions for the modification and delivery of lymphocytes |
CA3152780A CA3152780A1 (en) | 2019-09-01 | 2020-08-31 | Methods and compositions for the modification and delivery of lymphocytes |
KR1020227010243A KR20220070449A (en) | 2019-09-01 | 2020-08-31 | Methods and compositions for transformation and delivery of lymphocytes |
JP2022513645A JP2022546101A (en) | 2019-09-01 | 2020-08-31 | Methods and compositions for modification and delivery of lymphocytes |
PCT/US2020/048843 WO2021042072A1 (en) | 2019-09-01 | 2020-08-31 | Methods and compositions for the modification and delivery of lymphocytes |
MX2022002521A MX2022002521A (en) | 2019-09-01 | 2020-08-31 | Methods and compositions for the modification and delivery of lymphocytes. |
US17/110,028 US11325948B2 (en) | 2016-03-19 | 2020-12-02 | Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of MPL |
US18/043,465 US20230357436A1 (en) | 2016-03-19 | 2021-08-31 | Anti-idiotype compositions and methods of use thereof |
US17/684,405 US20230044451A1 (en) | 2016-03-19 | 2022-03-01 | Methods and compositions for the delivery of modified lymphocytes and/or retroviral particles |
US17/659,007 US20220306698A1 (en) | 2016-03-19 | 2022-04-12 | Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of cd79a and cd79b |
Applications Claiming Priority (14)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201862726294P | 2018-09-02 | 2018-09-02 | |
US201862726293P | 2018-09-02 | 2018-09-02 | |
US62/726,293 | 2018-09-02 | ||
US62/726,294 | 2018-09-02 | ||
US201862728056P | 2018-09-06 | 2018-09-06 | |
US62/728,056 | 2018-09-06 | ||
US201862732528P | 2018-09-17 | 2018-09-17 | |
PCT/US2018/051392 WO2019055946A1 (en) | 2017-09-18 | 2018-09-17 | Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof |
USPCT/US2018/051392 | 2018-09-17 | ||
US62/732,528 | 2018-09-17 | ||
US201962821434P | 2019-03-20 | 2019-03-20 | |
US62/821,434 | 2019-03-20 | ||
US201962894853P | 2019-09-01 | 2019-09-01 | |
US62/894,853 | 2019-09-01 |
Related Parent Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2018/020818 Continuation-In-Part WO2018161064A1 (en) | 2016-03-19 | 2018-03-03 | Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof |
US16/490,201 Continuation-In-Part US20200397821A1 (en) | 2016-03-19 | 2018-03-03 | Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof |
PCT/US2018/051392 Continuation-In-Part WO2019055946A1 (en) | 2016-03-19 | 2018-09-17 | Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof |
Related Child Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/823,319 Continuation-In-Part US20200255864A1 (en) | 2016-03-19 | 2020-03-18 | Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof |
PCT/US2020/048843 Continuation-In-Part WO2021042072A1 (en) | 2016-03-19 | 2020-08-31 | Methods and compositions for the modification and delivery of lymphocytes |
US17/110,028 Continuation-In-Part US11325948B2 (en) | 2016-03-19 | 2020-12-02 | Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of MPL |
US18/043,465 Continuation-In-Part US20230357436A1 (en) | 2016-03-19 | 2021-08-31 | Anti-idiotype compositions and methods of use thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2020047527A2 true WO2020047527A2 (en) | 2020-03-05 |
WO2020047527A3 WO2020047527A3 (en) | 2020-05-22 |
Family
ID=69644665
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2019/049259 WO2020047527A2 (en) | 2016-03-19 | 2019-09-02 | Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2020047527A2 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11111505B2 (en) | 2016-03-19 | 2021-09-07 | Exuma Biotech, Corp. | Methods and compositions for transducing lymphocytes and regulating the activity thereof |
US20210324331A1 (en) * | 2020-04-15 | 2021-10-21 | Amgen Inc. | Process for generating genetically engineered autologous t cells |
US11325948B2 (en) | 2016-03-19 | 2022-05-10 | Exuma Biotech Corp. | Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of MPL |
WO2022187289A1 (en) * | 2021-03-01 | 2022-09-09 | Exuma Biotech Corp. | Methods and compositions for the delivery of retroviral particles |
Citations (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993003769A1 (en) | 1991-08-20 | 1993-03-04 | THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES | Adenovirus mediated transfer of genes to the gastrointestinal tract |
WO1993009239A1 (en) | 1991-11-08 | 1993-05-13 | Research Corporation Technologies, Inc. | Adeno-associated virus-2 basal vectors |
WO1993019191A1 (en) | 1992-03-16 | 1993-09-30 | Centre National De La Recherche Scientifique | Defective recombinant adenoviruses expressing cytokines for use in antitumoral treatment |
US5310903A (en) | 1993-03-05 | 1994-05-10 | Merck & Co., Inc. | Imidazolidyl rapamycin derivatives |
WO1994012649A2 (en) | 1992-12-03 | 1994-06-09 | Genzyme Corporation | Gene therapy for cystic fibrosis |
US5362718A (en) | 1994-04-18 | 1994-11-08 | American Home Products Corporation | Rapamycin hydroxyesters |
WO1994028938A1 (en) | 1993-06-07 | 1994-12-22 | The Regents Of The University Of Michigan | Adenovirus vectors for gene therapy sponsorship |
WO1995000655A1 (en) | 1993-06-24 | 1995-01-05 | Mc Master University | Adenovirus vectors for gene therapy |
WO1995011984A2 (en) | 1993-10-25 | 1995-05-04 | Canji, Inc. | Recombinant adenoviral vector and methods of use |
US5525610A (en) | 1994-03-31 | 1996-06-11 | American Home Products Corporation | 42-Epi-rapamycin and pharmaceutical compositions thereof |
WO1996017951A2 (en) | 1994-12-09 | 1996-06-13 | Rpms Technology Limited | Identification of genes responsible for in vivo survival of microorganisms |
US5527907A (en) | 1993-11-19 | 1996-06-18 | Abbott Laboratories | Macrolide immunomodulators |
WO1996041865A1 (en) | 1995-06-07 | 1996-12-27 | Ariad Gene Therapeutics, Inc. | Rapamcycin-based regulation of biological events |
US5677425A (en) | 1987-09-04 | 1997-10-14 | Celltech Therapeutics Limited | Recombinant antibody |
WO1999036553A2 (en) | 1998-01-15 | 1999-07-22 | Ariad Gene Therapeutics, Inc. | Regulation of biological events using multimeric chimeric proteins |
WO2001014387A1 (en) | 1999-08-24 | 2001-03-01 | Ariad Gene Therapeutics, Inc. | 28-epirapalogs |
US6489458B2 (en) | 1997-03-11 | 2002-12-03 | Regents Of The University Of Minnesota | DNA-based transposon system for the introduction of nucleic acid into DNA of a cell |
US20040131637A1 (en) | 2001-03-09 | 2004-07-08 | Chatfield Steven Neville | Salmonella promoter for heterologous gene expression |
US6916846B2 (en) | 2000-05-12 | 2005-07-12 | Merck & Co. Inc. | Coumermycin analogs as chemical dimerizers of chimeric proteins |
US7387896B2 (en) | 2003-03-26 | 2008-06-17 | The Regents Of The University Of Michigan | MicroRNA vectors |
US20120130076A1 (en) | 1994-08-18 | 2012-05-24 | Ariad Pharmaceuticals, Inc. | Synthetic Multimerizing Agents |
US8236925B1 (en) | 2005-08-26 | 2012-08-07 | University Of Minnesota | Protein nanorings |
EP2602315A1 (en) | 2011-12-05 | 2013-06-12 | Pall Corporation | Leukocyte purification |
US8709755B2 (en) | 2009-03-09 | 2014-04-29 | Bioatla, Llc | Mirac proteins |
US8802374B2 (en) | 2009-11-03 | 2014-08-12 | City Of Hope | Truncated epiderimal growth factor receptor (EGFRt) for transduced T cell selection |
WO2016033331A1 (en) | 2014-08-28 | 2016-03-03 | Bioatla, Llc | Conditionally active chimeric antigen receptors for modified t-cells |
WO2017165245A2 (en) | 2016-03-19 | 2017-09-28 | F1 Oncology, Inc. | Methods and compositions for transducing lymphocytes and regulated expansion thereof |
WO2018009923A1 (en) | 2016-07-08 | 2018-01-11 | F1 Oncology, Inc. | Methods and compositions for transducing lymphocytes and regulating the activity thereof |
WO2018161064A1 (en) | 2017-03-03 | 2018-09-07 | F1 Oncology, Inc. | Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof |
WO2019005546A1 (en) | 2017-06-28 | 2019-01-03 | Corning Research & Development Corporation | High fiber count pre-terminated optical distribution assembly |
WO2019055946A1 (en) | 2017-09-18 | 2019-03-21 | F1 Oncology, Inc. | Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2019144A1 (en) * | 2007-07-23 | 2009-01-28 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Vector particles for targeting CD34+ cells |
EP3571229A1 (en) * | 2017-01-18 | 2019-11-27 | F1 Oncology, Inc. | Chimeric antigen receptors against axl or ror2 and methods of use thereof |
-
2019
- 2019-09-02 WO PCT/US2019/049259 patent/WO2020047527A2/en unknown
Patent Citations (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5677425A (en) | 1987-09-04 | 1997-10-14 | Celltech Therapeutics Limited | Recombinant antibody |
WO1993003769A1 (en) | 1991-08-20 | 1993-03-04 | THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES | Adenovirus mediated transfer of genes to the gastrointestinal tract |
WO1993009239A1 (en) | 1991-11-08 | 1993-05-13 | Research Corporation Technologies, Inc. | Adeno-associated virus-2 basal vectors |
WO1993019191A1 (en) | 1992-03-16 | 1993-09-30 | Centre National De La Recherche Scientifique | Defective recombinant adenoviruses expressing cytokines for use in antitumoral treatment |
WO1994012649A2 (en) | 1992-12-03 | 1994-06-09 | Genzyme Corporation | Gene therapy for cystic fibrosis |
US5310903A (en) | 1993-03-05 | 1994-05-10 | Merck & Co., Inc. | Imidazolidyl rapamycin derivatives |
WO1994028938A1 (en) | 1993-06-07 | 1994-12-22 | The Regents Of The University Of Michigan | Adenovirus vectors for gene therapy sponsorship |
WO1995000655A1 (en) | 1993-06-24 | 1995-01-05 | Mc Master University | Adenovirus vectors for gene therapy |
WO1995011984A2 (en) | 1993-10-25 | 1995-05-04 | Canji, Inc. | Recombinant adenoviral vector and methods of use |
US5527907A (en) | 1993-11-19 | 1996-06-18 | Abbott Laboratories | Macrolide immunomodulators |
US5525610A (en) | 1994-03-31 | 1996-06-11 | American Home Products Corporation | 42-Epi-rapamycin and pharmaceutical compositions thereof |
US5362718A (en) | 1994-04-18 | 1994-11-08 | American Home Products Corporation | Rapamycin hydroxyesters |
US20120130076A1 (en) | 1994-08-18 | 2012-05-24 | Ariad Pharmaceuticals, Inc. | Synthetic Multimerizing Agents |
WO1996017951A2 (en) | 1994-12-09 | 1996-06-13 | Rpms Technology Limited | Identification of genes responsible for in vivo survival of microorganisms |
WO1996041865A1 (en) | 1995-06-07 | 1996-12-27 | Ariad Gene Therapeutics, Inc. | Rapamcycin-based regulation of biological events |
US6489458B2 (en) | 1997-03-11 | 2002-12-03 | Regents Of The University Of Minnesota | DNA-based transposon system for the introduction of nucleic acid into DNA of a cell |
WO1999036553A2 (en) | 1998-01-15 | 1999-07-22 | Ariad Gene Therapeutics, Inc. | Regulation of biological events using multimeric chimeric proteins |
WO2001014387A1 (en) | 1999-08-24 | 2001-03-01 | Ariad Gene Therapeutics, Inc. | 28-epirapalogs |
US6916846B2 (en) | 2000-05-12 | 2005-07-12 | Merck & Co. Inc. | Coumermycin analogs as chemical dimerizers of chimeric proteins |
US20040131637A1 (en) | 2001-03-09 | 2004-07-08 | Chatfield Steven Neville | Salmonella promoter for heterologous gene expression |
US7387896B2 (en) | 2003-03-26 | 2008-06-17 | The Regents Of The University Of Michigan | MicroRNA vectors |
US8236925B1 (en) | 2005-08-26 | 2012-08-07 | University Of Minnesota | Protein nanorings |
US8709755B2 (en) | 2009-03-09 | 2014-04-29 | Bioatla, Llc | Mirac proteins |
US8802374B2 (en) | 2009-11-03 | 2014-08-12 | City Of Hope | Truncated epiderimal growth factor receptor (EGFRt) for transduced T cell selection |
EP2602315A1 (en) | 2011-12-05 | 2013-06-12 | Pall Corporation | Leukocyte purification |
WO2016033331A1 (en) | 2014-08-28 | 2016-03-03 | Bioatla, Llc | Conditionally active chimeric antigen receptors for modified t-cells |
WO2017165245A2 (en) | 2016-03-19 | 2017-09-28 | F1 Oncology, Inc. | Methods and compositions for transducing lymphocytes and regulated expansion thereof |
WO2018009923A1 (en) | 2016-07-08 | 2018-01-11 | F1 Oncology, Inc. | Methods and compositions for transducing lymphocytes and regulating the activity thereof |
WO2018161064A1 (en) | 2017-03-03 | 2018-09-07 | F1 Oncology, Inc. | Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof |
WO2019005546A1 (en) | 2017-06-28 | 2019-01-03 | Corning Research & Development Corporation | High fiber count pre-terminated optical distribution assembly |
WO2019055946A1 (en) | 2017-09-18 | 2019-03-21 | F1 Oncology, Inc. | Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof |
Non-Patent Citations (114)
Title |
---|
"GenBank", Database accession no. AF266288.2 |
"Genbank", Database accession no. AY096003.1 |
"NCBI Genbank", Database accession no. AF033819 |
ALEXANDER ET AL., EMBO J., vol. 15, no. 23, 2 December 1996 (1996-12-02), pages 6531 - 40 |
ALI ET AL., HUM GENE THER, vol. 9, 1998, pages 81 86 |
ALI ET AL., HUM MOL GENET, vol. 5, 1996, pages 591 594 |
ALPUCHE-ARANDA ET AL., PNAS, vol. 89, no. 21, 1992, pages 10079 - 83 |
BENIT ET AL., J VIROL, vol. 68, no. 8, August 1994 (1994-08-01), pages 5270 - 4 |
BENNETT ET AL., INVEST OPTHALMOL VIS SCI, vol. 38, 1997, pages 2857 2863 |
BHASKAR THYAGARAJAN ET AL.: "Site-Specific Genomic Integration in Mammalian Cells Mediated by Phage cpC31 Integrase", MOL CELL BIOL., vol. 21, no. 12, June 2001 (2001-06-01), pages 3926 - 3934 |
BIOL RES., vol. 40, no. 2, 2007, pages 97 - 112 |
BURTON, MOLEC. IMMUNOL., vol. 22, 1985, pages 161 - 206 |
C. FERNANDEZ ET AL., J. INTERNAL. MEDICINE, vol. 284, 2018, pages 377 - 387 |
CAVALIERI ET AL., BLOOD, vol. 101, no. 2, 2003, pages 3416 - 1644 |
CHATFIELD ET AL., BIOTECHNOL, vol. 10, 1992, pages 888 - 892 |
CHUNG, K. ET AL., NUCLEIC ACIDS RESEARCH, vol. 34, no. 7, 2006, pages e53 |
COFFIN ET AL.: "Retroviruses", 1997, COLD SPRING HARBOR LABORATORY PRESS, pages: 758 - 763 |
CONSTANTINESCU ET AL., MOL CELL, vol. 7, no. 2, February 2001 (2001-02-01), pages 377 - 85 |
CONSTANTINESCU ET AL., MOL CELL., vol. 7, no. 2, February 2001 (2001-02-01), pages 377 - 85 |
CONSTANTINESCU SN. TRENDS ENDOCRINOL METAB., vol. 10, no. 1, December 1999 (1999-12-01), pages 18 - 23 |
CORNELISSEN C ET AL., EUR J CELL BIOL., vol. 91, no. 6-7, June 2012 (2012-06-01), pages 552 - 66 |
DAVIES ET AL., J BIOL CHEM., vol. 262, no. 23, 15 August 1987 (1987-08-15), pages 10918 - 21 |
DEBOER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 80, 1983, pages 21 - 25 |
DEGUINEBARTON, F1000PRIME REP., vol. 6, 4 November 2014 (2014-11-04), pages 97 |
DIETRICH J ET AL., EMBO J., vol. 13, no. 9, 1 May 1994 (1994-05-01), pages 2156 - 66 |
DRACHMANKAUSHANSKY, PROC NATL ACAD SCI USA., vol. 94, no. 6, 18 March 1997 (1997-03-18), pages 2350 - 5 |
DUNSTAN ET AL., INFECT. IMMUN., vol. 67, 1999, pages 5133 - 5141 |
ECKELHART ET AL., BLOOD, vol. 117, 2011, pages 1565 |
ELGUETA ET AL., IMMUNOL REV., vol. 229, no. 1, May 2009 (2009-05-01), pages 152 - 72 |
FARRAR ET AL., NATURE, vol. 383, 1996, pages 178 - 181 |
FERGUSON MAJKINOSHITA THART GW ET AL.: "Essentials of Glycobiology", 2009, COLD SPRING HARBOR LABORATORY PRESS, article "Glycosylphosphatidylinositol Anchors" |
FLANNERY ET AL., PNAS, vol. 94, 1997, pages 6916 6921 |
FLOTTE ET AL., PNAS, vol. 90, 1993, pages 10613 - 10617 |
FOWLER, D.K. ET AL., NUCLEIC ACIDS RESEARCH, vol. 44, no. 5, 2015, pages e48 |
FRECHA ET AL., BLOOD, vol. 112, no. 13, 2008, pages 4843 - 4852 |
FREITAS, A.A. ET AL., ANNU REV IMMUNOL, vol. 18, 2000, pages 83 - 111 |
FUHRMANN-BENZAKEIN ET AL., PNAS, vol. 28, 2000, pages e99 |
FUKUNAGA ET AL., EMBO J., vol. 10, no. 10, October 1991 (1991-10-01), pages 2855 - 65 |
FUNKE ET AL., MOLECULAR THERAPY, vol. 16, no. 8, 2008, pages 1427 - 1436 |
GAUZZI MC ET AL., PROC NATL ACAD SCI USA., vol. 94, no. 22, 28 October 1997 (1997-10-28), pages 11839 - 44 |
GRINDLEY ET AL., ANNUAL REVIEW OF BIOCHEMISTRY, 2006, pages 567 - 605 |
HARADA ET AL., J EXP MED., vol. 197, no. 2, 20 January 2003 (2003-01-20), pages 257 - 62 |
HARBORNE ET AL., MAL. MICRO., vol. 6, 1992, pages 2805 - 2813 |
HIGASHIMOTO ET AL., GENE THER., vol. 14, 2007, pages 1298 |
HILLEN,W.WISSMANN,A.: "Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction", vol. 10, 1989, MACMILLAN, pages: 143 - 162 |
HITCHCOCK ET AL., BLOOD, vol. 112, no. 6, 15 September 2008 (2008-09-15), pages 2222 - 31 |
HORI, T., INT J HEMATOL., vol. 83, no. 1, January 2006 (2006-01-01), pages 17 - 22 |
HUANG ET AL., MOL CELL, vol. 8, no. 6, December 2001 (2001-12-01), pages 1327 - 38 |
HUCK ET AL., NUCL. ACIDS RES., vol. 14, 1986, pages 1779 |
HUDECEK ET AL., RECENT RESULTS CANCER RES., vol. 209, 2016, pages 37 - 50 |
JAE SEONG LEE ET AL., SCIENTIFIC REPORTS 5, 2015 |
JIANG, QIONG ET AL., MOL. AND CELL. BIOL., vol. 24, no. 14, 2004, pages 6501 - 13 |
JOMARY ET AL., GENE THER, vol. 4, 1997, pages 683 690 |
KAWAMATA, S ET AL., J BIOL CHEM., vol. 273, no. 10, 6 March 1998 (1998-03-06), pages 5808 - 14 |
KORINZACK, JOURNAL OF VIROLOGY, vol. 72, 1998, pages 3161 - 8 |
KOZAK, NUCLEIC ACIDS RES., vol. 15, no. 20, 26 October 1987 (1987-10-26), pages 8125 - 48 |
LAMINET ET AL., J BIOL CHEM., vol. 271, no. 1, 5 January 1996 (1996-01-05), pages 264 - 9 |
LECONTE ET AL., MOL IMMUNOL., vol. 79, November 2016 (2016-11-01), pages 38 - 46 |
LOCKSLEY RM ET AL., CELL, vol. 104, no. 4, 23 February 2001 (2001-02-23), pages 487 - 501 |
LU ET AL., J BIOL CHEM., vol. 278, no. 46, 14 November 2003 (2003-11-14), pages 45414 - 8 |
MANURI ET AL., HUM GENE THER, vol. 21, no. 4, April 2010 (2010-04-01), pages 427 - 37 |
MARRAK, P. ET AL., NAT IMMUNOL, vol. 1, 2000, pages 107 - 111 |
MATZ ET AL., NATURE BIOTECHNOL., vol. 17, 1999, pages 969 - 973 |
MAURICE ET AL., BLOOD, vol. 99, 2002, pages 2342 - 50 |
MAXSON ET AL., J BIOL CHEM., vol. 289, no. 9, 28 February 2014 (2014-02-28), pages 5820 - 7 |
MAY ET AL., FEBS LETT, vol. 394, no. 2, 30 September 1996 (1996-09-30), pages 221 - 6 |
MCKELVIE ET AL., VACCINE, vol. 22, 2004, pages 3243 - 3255 |
MEADS ET AL., J IMMUNOL., vol. 185, no. 3, 1 August 2010 (2010-08-01), pages 1606 - 15 |
MELTON ET AL., NUCL. ACIDS RES., vol. 12, 1984, pages 7035 |
MENDELSON ET AL., VIROL., vol. 166, 1988, pages 154 - 165 |
MILLER, NATURE, vol. 357, 1992, pages 455 - 460 |
MONJEZI ET AL., LEUKEMIA, vol. 31, no. 1, January 2017 (2017-01-01), pages 186 - 194 |
MORELLO ET AL., BLOOD, vol. 86, no. 8, July 1995 (1995-07-01), pages 557 - 71 |
MORGANBOYERINAS, BIOMEDICINES, vol. 4, no. 2, 20 April 2016 (2016-04-20), pages E9 |
MORRIS ET AL.: "The molecular details of cytokine signaling via the JAK/STAT pathway", PROTEIN SCIENCE, vol. 27, 2018, pages 1984 - 2009 |
MURAKAMI ET AL., PROC NATL ACAD SCI U S A., vol. 88, no. 24, 15 December 1991 (1991-12-15), pages 11349 - 53 |
MURAKAMI ET AL., PROC NATL ACAD SCI USA., vol. 88, no. 24, 15 December 1991 (1991-12-15), pages 11349 - 53 |
NGUYEN-JACKSON HT ET AL.: "G-CSF Receptor Structure, Function, and Intracellular Signal Transduction", TWENTY YEARS OF G-CSF, 2011, pages 83 - 105 |
O'NEALLEE, LYMPHOKINE CYTOKINE RES, vol. 12, no. 5, October 1993 (1993-10-01), pages 309 - 12 |
O'NEALLEE, LYMPHOKINE CYTOKINE RES., vol. 12, no. 5, October 1993 (1993-10-01), pages 309 - 12 |
PANDARAVINDRAN: "Isolation of Human PBMCs", BIOPROTOC., vol. 3, no. 3, 2013 |
PECQUET ET AL., BLOOD, vol. 115, no. 5, 4 February 2010 (2010-02-04), pages 1037 - 48 |
PEDROS ET AL., NAT IMMUNOL., vol. 17, no. 7, July 2016 (2016-07-01), pages 825 - 33 |
PLOS ONE, vol. 6, no. 8, 2011, pages e23396 |
PLUCKTHUN: "The Pharmacology of Monoclonal Antibodies", vol. 113, 1994, SPRINGER-VERLAG, pages: 269 - 315 |
PRESKY DH ET AL., PROC NATL ACAD SCI USA., vol. 93, no. 24, 26 November 1996 (1996-11-26) |
PULKKINENMILLER, J. BACTERIAL., vol. 173, no. 1, 1991, pages 86 - 93 |
ROLLING ET AL., HUM GENE THER, vol. 10, 1999, pages 641 648 |
SALMON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 7739 |
SAMULSKI ET AL., J. VIR., vol. 63, 1989, pages 3822 - 3828 |
SAUR ET AL., BLOOD, vol. 115, no. 6, 11 February 2010 (2010-02-11), pages 1254 - 63 |
SCHERAGA, REV. COMPUTATIONAL CHEM., vol. 11173, 1992, pages 142 |
SCHINDLER ET AL., J BIOL CHEM., vol. 282, no. 28, 13 July 2007 (2007-07-13), pages 20059 - 63 |
SEDGERMCDERMOTT, CYTOKINE GROWTH FACTOR REV., vol. 25, no. 4, August 2014 (2014-08-01), pages 453 - 72 |
SHANER ET AL., NAT. METHODS, vol. 2, 2005, pages 905 - 909 |
SHETRON-RAMA ET AL., INFECT. IMMUN., vol. 70, 2002, pages 1087 - 1096 |
STAHL ET AL., SCIENCE, vol. 267, no. 5202, 3 March 1995 (1995-03-03), pages 1349 - 53 |
STREBEL ET AL., BMC MEDICINE, vol. 7, 2009, pages 48 |
TAKAHASHI ET AL., J VIROL, vol. 73, 1999, pages 7812 7816 |
TAN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 162 |
TROPP: "Molecular Biology", 2012, JONES & BARTLETT PUBLISHERS |
TSUKAHARA ET AL., GENE THER., vol. 22, no. 2, February 2015 (2015-02-01), pages 209 - 215 |
UNITED STATES PHARMACOPEIA, vol. 26, 2002, pages 158 |
USACHEVA A ET AL., J BIOL CHEM., vol. 277, no. 50, 13 December 2002 (2002-12-13), pages 48220 - 6 |
VALDIVIAFALKOW, MAL. MICROBIAL., vol. 22, 1996, pages 367 |
VAN DER GEER ET AL., PROC NATL ACAD SCI USA., vol. 93, no. 3, 6 February 1996 (1996-02-06), pages 963 - 8 |
VARGHESE ET AL., FRONT ENDOCRINOL (LAUSANNE, vol. 8, 31 March 2017 (2017-03-31), pages 59 |
VERHOEYEN ET AL., METHODS MOL BIOL., vol. 506, 2009, pages 97 - 114 |
WANG ET AL., J. IMMUNOTHER., vol. 35, no. 9, 2012, pages 689 - 701 |
YAN ET AL., J. BIOL. CHEM., vol. 287, 2012, pages 5891 |
YAO X, CELL RES., vol. 27, no. 6, June 2017 (2017-06-01), pages 801 - 814 |
YE ET AL., SCIENCE, vol. 283, 1999, pages 88 - 91 |
ZAPATA ET AL., PROTEIN ENG., vol. 8, no. 10, 1995, pages 1057 - 1062 |
ZHAO ET AL., CANCER RES., vol. 15, 2010, pages 9053 |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11111505B2 (en) | 2016-03-19 | 2021-09-07 | Exuma Biotech, Corp. | Methods and compositions for transducing lymphocytes and regulating the activity thereof |
US11325948B2 (en) | 2016-03-19 | 2022-05-10 | Exuma Biotech Corp. | Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of MPL |
US20210324331A1 (en) * | 2020-04-15 | 2021-10-21 | Amgen Inc. | Process for generating genetically engineered autologous t cells |
WO2022187289A1 (en) * | 2021-03-01 | 2022-09-09 | Exuma Biotech Corp. | Methods and compositions for the delivery of retroviral particles |
Also Published As
Publication number | Publication date |
---|---|
WO2020047527A3 (en) | 2020-05-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2017292936C1 (en) | Methods and compositions for transducing lymphocytes and regulating the activity thereof | |
EP3735460A1 (en) | Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof | |
WO2018161064A1 (en) | Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof | |
US11325948B2 (en) | Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of MPL | |
US20220340927A1 (en) | Methods and compositions for the modification and delivery of lymphocytes | |
US20230111159A1 (en) | Methods and compositions for the delivery of modified lymphocyte aggregates | |
US20200255864A1 (en) | Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof | |
US20210317408A1 (en) | Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs | |
WO2020047527A2 (en) | Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs | |
US20200397821A1 (en) | Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof | |
WO2022047417A1 (en) | Anti-idiotype compositions and methods of use thereof | |
US20230357436A1 (en) | Anti-idiotype compositions and methods of use thereof | |
US20230044451A1 (en) | Methods and compositions for the delivery of modified lymphocytes and/or retroviral particles | |
US20230392139A1 (en) | Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof | |
JP2024510933A (en) | Methods and compositions for delivery of retroviral particles | |
WO2022187289A1 (en) | Methods and compositions for the delivery of retroviral particles | |
EP4204004A1 (en) | Anti-idiotype compositions and methods of use thereof | |
CN117043346A (en) | Methods and compositions for delivery of retroviral particles |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 19782802 Country of ref document: EP Kind code of ref document: A2 |
|
ENP | Entry into the national phase |
Ref document number: 3111084 Country of ref document: CA Ref document number: 2021511656 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2019782802 Country of ref document: EP Effective date: 20210326 |
|
ENP | Entry into the national phase |
Ref document number: 2019333324 Country of ref document: AU Date of ref document: 20190902 Kind code of ref document: A |