NZ763754B2 - Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy - Google Patents
Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy Download PDFInfo
- Publication number
- NZ763754B2 NZ763754B2 NZ763754A NZ76375415A NZ763754B2 NZ 763754 B2 NZ763754 B2 NZ 763754B2 NZ 763754 A NZ763754 A NZ 763754A NZ 76375415 A NZ76375415 A NZ 76375415A NZ 763754 B2 NZ763754 B2 NZ 763754B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- cells
- cell
- composition
- selection
- sample
- Prior art date
Links
- 238000002560 therapeutic procedure Methods 0.000 title claims abstract description 20
- 210000002865 immune cell Anatomy 0.000 title claims abstract description 18
- 210000004027 cells Anatomy 0.000 claims abstract description 1686
- 239000000203 mixture Substances 0.000 claims abstract description 341
- 210000001744 T-Lymphocytes Anatomy 0.000 claims abstract description 157
- 238000002659 cell therapy Methods 0.000 claims abstract description 42
- 102100008191 CD8A Human genes 0.000 claims description 416
- 101700054655 CD8A Proteins 0.000 claims description 416
- 101700022938 CD4 Proteins 0.000 claims description 414
- 102100013077 CD4 Human genes 0.000 claims description 413
- 230000027455 binding Effects 0.000 claims description 227
- 239000003153 chemical reaction reagent Substances 0.000 claims description 182
- 108090001123 antibodies Proteins 0.000 claims description 169
- 102000004965 antibodies Human genes 0.000 claims description 169
- 108010090804 Streptavidin Proteins 0.000 claims description 92
- 239000000427 antigen Substances 0.000 claims description 68
- 108091007172 antigens Proteins 0.000 claims description 68
- 102000038129 antigens Human genes 0.000 claims description 68
- 239000011324 bead Substances 0.000 claims description 66
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 57
- 230000035693 Fab Effects 0.000 claims description 56
- 201000010099 disease Diseases 0.000 claims description 51
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims description 50
- 102000006306 Antigen Receptors Human genes 0.000 claims description 45
- 108010083359 Antigen Receptors Proteins 0.000 claims description 45
- 108091008153 T cell receptors Proteins 0.000 claims description 45
- 150000001413 amino acids Chemical class 0.000 claims description 41
- 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 claims description 40
- -1 IL- 7 Proteins 0.000 claims description 38
- 235000020958 biotin Nutrition 0.000 claims description 38
- 239000011616 biotin Substances 0.000 claims description 38
- 229960002685 biotin Drugs 0.000 claims description 38
- 210000001266 CD8-Positive T-Lymphocytes Anatomy 0.000 claims description 35
- 230000004936 stimulating Effects 0.000 claims description 35
- 230000000977 initiatory Effects 0.000 claims description 34
- 239000002458 cell surface marker Substances 0.000 claims description 31
- 239000003795 chemical substances by application Substances 0.000 claims description 31
- 230000005291 magnetic Effects 0.000 claims description 25
- 210000003819 Peripheral blood mononuclear cell Anatomy 0.000 claims description 23
- 230000004068 intracellular signaling Effects 0.000 claims description 22
- 210000004369 Blood Anatomy 0.000 claims description 21
- 239000008280 blood Substances 0.000 claims description 21
- 150000001615 biotins Chemical class 0.000 claims description 19
- 239000007787 solid Substances 0.000 claims description 17
- 238000002617 apheresis Methods 0.000 claims description 15
- 230000000694 effects Effects 0.000 claims description 14
- 210000000265 Leukocytes Anatomy 0.000 claims description 13
- 108010002350 Interleukin-2 Proteins 0.000 claims description 12
- 230000002441 reversible Effects 0.000 claims description 12
- 108090001008 Avidin Proteins 0.000 claims description 10
- 230000000139 costimulatory Effects 0.000 claims description 9
- 230000036947 Dissociation constant Effects 0.000 claims description 8
- 108090000172 Interleukin-15 Proteins 0.000 claims description 8
- 108010074108 interleukin-21 Proteins 0.000 claims description 8
- 230000035687 Dissociation Rate Constant Effects 0.000 claims description 4
- 230000003213 activating Effects 0.000 claims description 4
- 239000000178 monomer Substances 0.000 claims description 2
- 238000002955 isolation Methods 0.000 abstract description 62
- 238000010353 genetic engineering Methods 0.000 abstract description 15
- 238000002360 preparation method Methods 0.000 abstract description 14
- 230000002829 reduced Effects 0.000 abstract description 8
- 239000000523 sample Substances 0.000 description 157
- 239000011159 matrix material Substances 0.000 description 88
- 239000003550 marker Substances 0.000 description 86
- 239000012504 chromatography matrix Substances 0.000 description 85
- 238000000926 separation method Methods 0.000 description 64
- 102100008254 SELL Human genes 0.000 description 62
- 101710038663 SELL Proteins 0.000 description 62
- 238000001042 affinity chromatography Methods 0.000 description 52
- 235000001014 amino acid Nutrition 0.000 description 48
- 102100008151 CCR7 Human genes 0.000 description 44
- 101700036258 MECOM Proteins 0.000 description 44
- 230000014509 gene expression Effects 0.000 description 44
- 238000000034 method Methods 0.000 description 42
- 239000011230 binding agent Substances 0.000 description 40
- 230000035492 administration Effects 0.000 description 36
- 102100019461 CD28 Human genes 0.000 description 34
- 101700033362 CD28 Proteins 0.000 description 34
- 102100019459 CD27 Human genes 0.000 description 33
- 101700056583 CD27 Proteins 0.000 description 33
- 239000003446 ligand Substances 0.000 description 33
- 238000004587 chromatography analysis Methods 0.000 description 27
- 239000011534 wash buffer Substances 0.000 description 27
- 239000002245 particle Substances 0.000 description 26
- 239000002699 waste material Substances 0.000 description 23
- 230000004913 activation Effects 0.000 description 22
- 230000037396 body weight Effects 0.000 description 21
- 210000003071 memory T lymphocyte Anatomy 0.000 description 21
- 201000011510 cancer Diseases 0.000 description 19
- 102000005962 receptors Human genes 0.000 description 19
- 108020003175 receptors Proteins 0.000 description 19
- 238000010828 elution Methods 0.000 description 18
- 230000011664 signaling Effects 0.000 description 18
- 206010028980 Neoplasm Diseases 0.000 description 17
- 239000012149 elution buffer Substances 0.000 description 17
- 238000005406 washing Methods 0.000 description 17
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 16
- 239000012530 fluid Substances 0.000 description 15
- 230000001225 therapeutic Effects 0.000 description 14
- 229920000936 Agarose Polymers 0.000 description 13
- 125000003275 alpha amino acid group Chemical group 0.000 description 13
- 239000008194 pharmaceutical composition Substances 0.000 description 13
- 230000000717 retained Effects 0.000 description 13
- 101700027514 CD14 Proteins 0.000 description 12
- 102100003268 CD14 Human genes 0.000 description 12
- 230000001086 cytosolic Effects 0.000 description 12
- 238000000684 flow cytometry Methods 0.000 description 12
- 108010011170 Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Proteins 0.000 description 11
- 230000003993 interaction Effects 0.000 description 11
- 239000000463 material Substances 0.000 description 11
- 230000035755 proliferation Effects 0.000 description 11
- 239000011347 resin Substances 0.000 description 11
- 229920005989 resin Polymers 0.000 description 11
- 230000004083 survival Effects 0.000 description 11
- 101700078950 CD44 Proteins 0.000 description 10
- 102100003735 CD44 Human genes 0.000 description 10
- 210000004698 Lymphocytes Anatomy 0.000 description 10
- 238000009472 formulation Methods 0.000 description 10
- 230000001965 increased Effects 0.000 description 10
- 239000002609 media Substances 0.000 description 10
- 102000004127 Cytokines Human genes 0.000 description 9
- 108090000695 Cytokines Proteins 0.000 description 9
- 238000007792 addition Methods 0.000 description 9
- 239000006249 magnetic particle Substances 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 230000001177 retroviral Effects 0.000 description 9
- 238000010361 transduction Methods 0.000 description 9
- 230000026683 transduction Effects 0.000 description 9
- JTEDVYBZBROSJT-UHFFFAOYSA-N Indole-3-butyric acid Natural products C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 239000003480 eluent Substances 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 108091006822 Human Serum Albumin Proteins 0.000 description 7
- 102000008100 Human Serum Albumin Human genes 0.000 description 7
- 108010002586 Interleukin-7 Proteins 0.000 description 7
- 230000000779 depleting Effects 0.000 description 7
- 238000001415 gene therapy Methods 0.000 description 7
- 230000003834 intracellular Effects 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 230000000638 stimulation Effects 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 102100005826 CD19 Human genes 0.000 description 6
- 101700087100 CD19 Proteins 0.000 description 6
- 102100011842 CEACAM5 Human genes 0.000 description 6
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 6
- 210000003171 Lymphocytes, Tumor-Infiltrating Anatomy 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 201000009910 diseases by infectious agent Diseases 0.000 description 6
- 238000009169 immunotherapy Methods 0.000 description 6
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 6
- 239000011325 microbead Substances 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 230000002572 peristaltic Effects 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000028327 secretion Effects 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 210000001519 tissues Anatomy 0.000 description 6
- AUTOLBMXDDTRRT-JGVFFNPUSA-N (4R,5S)-dethiobiotin Chemical compound C[C@@H]1NC(=O)N[C@@H]1CCCCCC(O)=O AUTOLBMXDDTRRT-JGVFFNPUSA-N 0.000 description 5
- 210000000822 Killer Cells, Natural Anatomy 0.000 description 5
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 5
- 210000003283 T-Lymphocytes, Helper-Inducer Anatomy 0.000 description 5
- 102100009537 TNFRSF9 Human genes 0.000 description 5
- 101710040535 TNFRSF9 Proteins 0.000 description 5
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 5
- 150000007513 acids Chemical class 0.000 description 5
- 230000000735 allogeneic Effects 0.000 description 5
- 239000006143 cell culture media Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000007885 magnetic separation Methods 0.000 description 5
- 230000000284 resting Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 101710042656 BQ2027_MB1231C Proteins 0.000 description 4
- 102100000189 CD22 Human genes 0.000 description 4
- 101700020617 CD22 Proteins 0.000 description 4
- 206010008958 Chronic lymphocytic leukaemia Diseases 0.000 description 4
- 241000701022 Cytomegalovirus Species 0.000 description 4
- 210000003743 Erythrocytes Anatomy 0.000 description 4
- 102100015541 FCGR3A Human genes 0.000 description 4
- 101710044656 FCGR3A Proteins 0.000 description 4
- 101710044657 FCGR3B Proteins 0.000 description 4
- VHLZDSUANXBJHW-UHFFFAOYSA-N Glutaminyl-Phenylalanine Chemical compound NC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 VHLZDSUANXBJHW-UHFFFAOYSA-N 0.000 description 4
- LNCFUHAPNTYMJB-IUCAKERBSA-N His-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CN=CN1 LNCFUHAPNTYMJB-IUCAKERBSA-N 0.000 description 4
- 206010024324 Leukaemias Diseases 0.000 description 4
- 208000000429 Leukemia, Lymphocytic, Chronic, B-Cell Diseases 0.000 description 4
- 206010025323 Lymphomas Diseases 0.000 description 4
- 102100000165 MS4A1 Human genes 0.000 description 4
- 101710010909 MS4A1 Proteins 0.000 description 4
- 210000001616 Monocytes Anatomy 0.000 description 4
- 206010025310 Other lymphomas Diseases 0.000 description 4
- 210000002381 Plasma Anatomy 0.000 description 4
- 210000002307 Prostate Anatomy 0.000 description 4
- 241000580858 Simian-Human immunodeficiency virus Species 0.000 description 4
- LCPVBXOHXMBLFW-JSGCOSHPSA-N Trp-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)=CNC2=C1 LCPVBXOHXMBLFW-JSGCOSHPSA-N 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 239000007825 activation reagent Substances 0.000 description 4
- 239000006172 buffering agent Substances 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 239000001569 carbon dioxide Substances 0.000 description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 239000002771 cell marker Substances 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 4
- 230000003247 decreasing Effects 0.000 description 4
- 230000001809 detectable Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000001963 growth media Substances 0.000 description 4
- 108010085325 histidylproline Proteins 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 230000001404 mediated Effects 0.000 description 4
- 102000003735 mesothelin Human genes 0.000 description 4
- 108090000015 mesothelin Proteins 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 230000001717 pathogenic Effects 0.000 description 4
- 244000052769 pathogens Species 0.000 description 4
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 4
- 230000002335 preservative Effects 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 230000003612 virological Effects 0.000 description 4
- 210000001772 Blood Platelets Anatomy 0.000 description 3
- 210000000481 Breast Anatomy 0.000 description 3
- 101700017647 CD33 Proteins 0.000 description 3
- 102100016493 CD33 Human genes 0.000 description 3
- 102000019034 Chemokines Human genes 0.000 description 3
- 108010012236 Chemokines Proteins 0.000 description 3
- 210000001072 Colon Anatomy 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- 101700053597 FCER2 Proteins 0.000 description 3
- 102100014608 FCER2 Human genes 0.000 description 3
- 210000003714 Granulocytes Anatomy 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 102100006815 IL2RA Human genes 0.000 description 3
- 101700082799 IL2RA Proteins 0.000 description 3
- 101700015336 ISG20 Proteins 0.000 description 3
- 210000000987 Immune System Anatomy 0.000 description 3
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 3
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-Methionine Natural products CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- 210000004185 Liver Anatomy 0.000 description 3
- 210000004072 Lung Anatomy 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241000288906 Primates Species 0.000 description 3
- 210000003324 RBC Anatomy 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- 210000000662 T-Lymphocyte Subsets Anatomy 0.000 description 3
- 239000002269 analeptic agent Substances 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 230000001413 cellular Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 229940079593 drugs Drugs 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 150000004676 glycans Polymers 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 230000036210 malignancy Effects 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 238000009126 molecular therapy Methods 0.000 description 3
- 239000002102 nanobead Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 210000000056 organs Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 150000004804 polysaccharides Polymers 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000010187 selection method Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 210000004881 tumor cells Anatomy 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- 102100007409 APRT Human genes 0.000 description 2
- 108010024223 Adenine Phosphoribosyltransferase Proteins 0.000 description 2
- 241000432074 Adeno-associated virus Species 0.000 description 2
- 206010052747 Adenocarcinoma pancreas Diseases 0.000 description 2
- 229940064005 Antibiotic throat preparations Drugs 0.000 description 2
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 description 2
- 229940042052 Antibiotics for systemic use Drugs 0.000 description 2
- 229940042786 Antitubercular Antibiotics Drugs 0.000 description 2
- 206010003246 Arthritis Diseases 0.000 description 2
- 206010003816 Autoimmune disease Diseases 0.000 description 2
- 208000003950 B-Cell Lymphoma Diseases 0.000 description 2
- 210000003719 B-Lymphocytes Anatomy 0.000 description 2
- 210000000601 Blood Cells Anatomy 0.000 description 2
- 206010005949 Bone cancer Diseases 0.000 description 2
- 108091003117 Bovine Serum Albumin Proteins 0.000 description 2
- 229940098773 Bovine Serum Albumin Drugs 0.000 description 2
- 108060001249 CD24 Proteins 0.000 description 2
- 102100000197 CD24 Human genes 0.000 description 2
- 102100003279 CD38 Human genes 0.000 description 2
- 101700044948 CD38 Proteins 0.000 description 2
- 102100001891 CTAG1A Human genes 0.000 description 2
- 101710004449 CTAG1A Proteins 0.000 description 2
- JGPOSNWWINVNFV-UHFFFAOYSA-N Carboxyfluorescein diacetate succinimidyl ester Chemical compound C=1C(OC(=O)C)=CC=C2C=1OC1=CC(OC(C)=O)=CC=C1C2(C1=C2)OC(=O)C1=CC=C2C(=O)ON1C(=O)CCC1=O JGPOSNWWINVNFV-UHFFFAOYSA-N 0.000 description 2
- 102100010912 EPCAM Human genes 0.000 description 2
- 108060002563 EPCAM Proteins 0.000 description 2
- 102100016692 ESR1 Human genes 0.000 description 2
- 208000006168 Ewing Sarcoma Diseases 0.000 description 2
- 101700010580 FLO11 Proteins 0.000 description 2
- 102100008453 FOLH1 Human genes 0.000 description 2
- 101700036477 FOLH1 Proteins 0.000 description 2
- 230000036809 Fabs Effects 0.000 description 2
- 108010087819 Fc receptors Proteins 0.000 description 2
- 102000009109 Fc receptors Human genes 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 208000005017 Glioblastoma Diseases 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 229940093922 Gynecological Antibiotics Drugs 0.000 description 2
- 208000002672 Hepatitis B Diseases 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N Hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 102100004549 IL13RA2 Human genes 0.000 description 2
- 101710034342 IL13RA2 Proteins 0.000 description 2
- 102100019441 ITGAM Human genes 0.000 description 2
- 101710006572 ITGAM Proteins 0.000 description 2
- 206010061598 Immunodeficiency Diseases 0.000 description 2
- 101710036390 KLRK1 Proteins 0.000 description 2
- 102100012223 KLRK1 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
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-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
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 102100004400 L1CAM Human genes 0.000 description 2
- 210000003563 Lymphoid Tissue Anatomy 0.000 description 2
- 102100008857 MLANA Human genes 0.000 description 2
- 101710012533 MLANA Proteins 0.000 description 2
- 102100006037 MUC1 Human genes 0.000 description 2
- 101700052761 MUC1 Proteins 0.000 description 2
- 102100006044 MUC16 Human genes 0.000 description 2
- 101700008449 MUC16 Proteins 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 206010025650 Malignant melanoma Diseases 0.000 description 2
- 206010026798 Mantle cell lymphomas Diseases 0.000 description 2
- 208000000172 Medulloblastoma Diseases 0.000 description 2
- 206010027406 Mesothelioma Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- LXCFILQKKLGQFO-UHFFFAOYSA-N Methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 2
- 210000000066 Myeloid Cells Anatomy 0.000 description 2
- 241000713883 Myeloproliferative sarcoma virus Species 0.000 description 2
- 210000004693 NK cell Anatomy 0.000 description 2
- 102100016102 NTRK1 Human genes 0.000 description 2
- 101700043017 NTRK1 Proteins 0.000 description 2
- 108010012255 Neural Cell Adhesion Molecule L1 Proteins 0.000 description 2
- 206010029260 Neuroblastoma Diseases 0.000 description 2
- 206010029592 Non-Hodgkin's lymphomas Diseases 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 102100006820 PMEL Human genes 0.000 description 2
- 101700035121 PMEL Proteins 0.000 description 2
- 102100017963 PSCA Human genes 0.000 description 2
- 101700038464 PSCA Proteins 0.000 description 2
- 101700008337 PSMA Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 101700044827 RNMT Proteins 0.000 description 2
- 102100017729 ROR1 Human genes 0.000 description 2
- 101710036428 ROR1 Proteins 0.000 description 2
- 101700058902 RORA Proteins 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 208000006265 Renal Cell Carcinoma Diseases 0.000 description 2
- 206010039073 Rheumatoid arthritis Diseases 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 210000003491 Skin Anatomy 0.000 description 2
- 241000713880 Spleen focus-forming virus Species 0.000 description 2
- 206010042863 Synovial sarcoma Diseases 0.000 description 2
- 101710040533 TNFRSF8 Proteins 0.000 description 2
- 102100009538 TNFRSF8 Human genes 0.000 description 2
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K Tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 229930003756 Vitamin B7 Natural products 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000003432 anti-folate Effects 0.000 description 2
- 201000009596 autoimmune hypersensitivity disease Diseases 0.000 description 2
- 230000001580 bacterial Effects 0.000 description 2
- 230000001588 bifunctional Effects 0.000 description 2
- 230000003115 biocidal Effects 0.000 description 2
- 201000005216 brain cancer Diseases 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 201000009030 carcinoma Diseases 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 108091006028 chimera Proteins 0.000 description 2
- 201000011231 colorectal cancer Diseases 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 238000009295 crossflow filtration Methods 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 150000002009 diols Chemical class 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 210000003162 effector T lymphocyte Anatomy 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000002708 enhancing Effects 0.000 description 2
- 102000017256 epidermal growth factor-activated receptor activity proteins Human genes 0.000 description 2
- 108040009258 epidermal growth factor-activated receptor activity proteins Proteins 0.000 description 2
- 108010038795 estrogen receptors Proteins 0.000 description 2
- 230000001605 fetal Effects 0.000 description 2
- 239000004052 folic acid antagonist Substances 0.000 description 2
- 201000003444 follicular lymphoma Diseases 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000002068 genetic Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000001976 improved Effects 0.000 description 2
- 200000000018 inflammatory disease Diseases 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 229940079866 intestinal antibiotics Drugs 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000011068 load Methods 0.000 description 2
- 230000002934 lysing Effects 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012092 media component Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 230000003278 mimic Effects 0.000 description 2
- 108010045030 monoclonal antibodies Proteins 0.000 description 2
- 102000005614 monoclonal antibodies Human genes 0.000 description 2
- 210000004877 mucosa Anatomy 0.000 description 2
- 201000009251 multiple myeloma Diseases 0.000 description 2
- 229920005615 natural polymer Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 229940005935 ophthalmologic Antibiotics Drugs 0.000 description 2
- 201000008968 osteosarcoma Diseases 0.000 description 2
- 230000002611 ovarian Effects 0.000 description 2
- 201000002094 pancreatic adenocarcinoma Diseases 0.000 description 2
- 230000005298 paramagnetic Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920000023 polynucleotide Polymers 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 102000003998 progesterone receptors Human genes 0.000 description 2
- 108090000468 progesterone receptors Proteins 0.000 description 2
- 230000001737 promoting Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 101710044770 sll1951 Proteins 0.000 description 2
- 239000000021 stimulant Substances 0.000 description 2
- 108010018381 streptavidin-binding peptide Proteins 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 229920001059 synthetic polymer Polymers 0.000 description 2
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 235000011912 vitamin B7 Nutrition 0.000 description 2
- 239000011735 vitamin B7 Substances 0.000 description 2
- VZQHRKZCAZCACO-PYJNHQTQSA-N (2S)-2-[[(2S)-2-[2-[[(2S)-2-[[(2S)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]propanoyl]amino]prop-2-enoylamino]-3-methylbutanoyl]amino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)C(=C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCNC(N)=N VZQHRKZCAZCACO-PYJNHQTQSA-N 0.000 description 1
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N 1,4-Butanediol, dimethanesulfonate Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- GCMNJUJAKQGROZ-UHFFFAOYSA-N 2-Aminoquinoline Chemical compound C1=CC=CC2=NC(N)=CC=C21 GCMNJUJAKQGROZ-UHFFFAOYSA-N 0.000 description 1
- RCCCMIFBKBHSPQ-UHFFFAOYSA-N 2-[2-(2-oxohydrazinyl)phenyl]benzoic acid Chemical compound ON=NC1=CC=CC=C1C1=CC=CC=C1C(O)=O RCCCMIFBKBHSPQ-UHFFFAOYSA-N 0.000 description 1
- JKNCSZDPWAVQAI-UHFFFAOYSA-N 5-(3-amino-4-azaniumylthiolan-2-yl)pentanoate Chemical compound NC1CSC(CCCCC(O)=O)C1N JKNCSZDPWAVQAI-UHFFFAOYSA-N 0.000 description 1
- DEQPBRIACBATHE-FXQIFTODSA-N 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-2-iminopentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCC(=N)C(=O)O)SC[C@@H]21 DEQPBRIACBATHE-FXQIFTODSA-N 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N 5-flurouricil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- AOJJSUZBOXZQNB-TZSSRYMLSA-N ADRIAMYCIN Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 1
- 229960003272 ASPARAGINASE Drugs 0.000 description 1
- 206010000880 Acute myeloid leukaemia Diseases 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 206010059512 Apoptosis Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 102000015790 Asparaginase Human genes 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 229960001230 Asparagine Drugs 0.000 description 1
- 208000006673 Asthma Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 206010060945 Bacterial infection Diseases 0.000 description 1
- 210000003651 Basophils Anatomy 0.000 description 1
- KUVIULQEHSCUHY-XYWKZLDCSA-N Beclometasone dipropionate Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(Cl)[C@@H]1[C@@H]1C[C@H](C)[C@@](C(=O)COC(=O)CC)(OC(=O)CC)[C@@]1(C)C[C@@H]2O KUVIULQEHSCUHY-XYWKZLDCSA-N 0.000 description 1
- 229960000686 Benzalkonium Chloride Drugs 0.000 description 1
- 210000001124 Body Fluids Anatomy 0.000 description 1
- 210000001185 Bone Marrow Anatomy 0.000 description 1
- 210000000988 Bone and Bones Anatomy 0.000 description 1
- 102100016530 CD37 Human genes 0.000 description 1
- 101700044364 CD37 Proteins 0.000 description 1
- 102100005828 CD5 Human genes 0.000 description 1
- 101700066525 CD5 Proteins 0.000 description 1
- 102100019451 CD80 Human genes 0.000 description 1
- 101700080477 CD80 Proteins 0.000 description 1
- 102100016531 CD9 Human genes 0.000 description 1
- 101700017162 CD9 Proteins 0.000 description 1
- 101710043954 CEACAM3 Proteins 0.000 description 1
- 102100011836 CEACAM3 Human genes 0.000 description 1
- 102100002013 CSPG4 Human genes 0.000 description 1
- 101700026160 CSPG4 Proteins 0.000 description 1
- OLESAACUTLOWQZ-UHFFFAOYSA-L Carboplatin Chemical compound O=C1O[Pt]([N]([H])([H])[H])([N]([H])([H])[H])OC(=O)C11CCC1 OLESAACUTLOWQZ-UHFFFAOYSA-L 0.000 description 1
- 229960004562 Carboplatin Drugs 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 210000001175 Cerebrospinal Fluid Anatomy 0.000 description 1
- 210000003679 Cervix Uteri Anatomy 0.000 description 1
- 241000272194 Ciconiiformes Species 0.000 description 1
- 206010011401 Crohn's disease Diseases 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical compound C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N DAUNOMYCIN Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 229960000975 Daunorubicin Drugs 0.000 description 1
- 210000004443 Dendritic Cells Anatomy 0.000 description 1
- 206010012601 Diabetes mellitus Diseases 0.000 description 1
- 229960004679 Doxorubicin Drugs 0.000 description 1
- 108010091358 EC 2.4.2.8 Proteins 0.000 description 1
- 108030007212 EC 3.4.21.79 Proteins 0.000 description 1
- 102000000311 EC 3.5.4.1 Human genes 0.000 description 1
- 108010080611 EC 3.5.4.1 Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 210000001671 Embryonic Stem Cells Anatomy 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 210000003979 Eosinophils Anatomy 0.000 description 1
- 102100016439 FAS Human genes 0.000 description 1
- 101700079540 FAS Proteins 0.000 description 1
- 102100015540 FCGR1A Human genes 0.000 description 1
- 101710003440 FCGR1A Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 229960002949 Fluorouracil Drugs 0.000 description 1
- 102100004391 GZMB Human genes 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N Ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 229960002963 Ganciclovir Drugs 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N Gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 1
- 206010018651 Graft versus host disease Diseases 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 208000003807 Graves Disease Diseases 0.000 description 1
- 201000004779 Graves' disease Diseases 0.000 description 1
- 210000004837 Gut-associated lymphoid tissue Anatomy 0.000 description 1
- 102100004898 HCST Human genes 0.000 description 1
- 101700079958 HCST Proteins 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 102100016790 HPRT1 Human genes 0.000 description 1
- 230000036499 Half live Effects 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 206010020243 Hodgkin's disease Diseases 0.000 description 1
- 201000006743 Hodgkin's lymphoma Diseases 0.000 description 1
- 241000829111 Human polyomavirus 1 Species 0.000 description 1
- 210000004408 Hybridomas Anatomy 0.000 description 1
- 102100006816 IL2RB Human genes 0.000 description 1
- 101700021467 IL2RB Proteins 0.000 description 1
- 101710003546 INPP5K Proteins 0.000 description 1
- 206010021972 Inflammatory bowel disease Diseases 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N Intaxel Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 210000000936 Intestines Anatomy 0.000 description 1
- 102100011339 KLK3 Human genes 0.000 description 1
- 210000003734 Kidney Anatomy 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-2-aminohexanoic acid zwitterion Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N L-serine Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 208000007046 Leukemia, Myeloid, Acute Diseases 0.000 description 1
- 210000001165 Lymph Nodes Anatomy 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 210000002540 Macrophages Anatomy 0.000 description 1
- 210000000138 Mast Cells Anatomy 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010027476 Metastasis Diseases 0.000 description 1
- 108090000393 Muromonab-CD3 Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000063939 Myelois Species 0.000 description 1
- 102100007544 NCAM1 Human genes 0.000 description 1
- 101700077124 NCAM1 Proteins 0.000 description 1
- 210000000440 Neutrophils Anatomy 0.000 description 1
- 210000001672 Ovary Anatomy 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N P-Toluenesulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 102100005499 PTPRC Human genes 0.000 description 1
- 101700059076 PTPRC Proteins 0.000 description 1
- 229960001592 Paclitaxel Drugs 0.000 description 1
- 210000002741 Palatine Tonsil Anatomy 0.000 description 1
- 210000000496 Pancreas Anatomy 0.000 description 1
- 208000006551 Parasitic Disease Diseases 0.000 description 1
- 229960005190 Phenylalanine Drugs 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M Propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N Propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 1
- 206010037075 Protozoal infection Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 229920000970 Repeated sequence (DNA) Polymers 0.000 description 1
- 206010038997 Retroviral infection Diseases 0.000 description 1
- 210000003705 Ribosomes Anatomy 0.000 description 1
- 108010001645 Rituximab Proteins 0.000 description 1
- 101710029276 SPHKAP Proteins 0.000 description 1
- 101700015701 SPI Proteins 0.000 description 1
- 206010039710 Scleroderma Diseases 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 210000002966 Serum Anatomy 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108010070144 Single-Chain Antibodies Proteins 0.000 description 1
- 102000005632 Single-Chain Antibodies Human genes 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 229960003885 Sodium Benzoate Drugs 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M Sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 210000000952 Spleen Anatomy 0.000 description 1
- 210000002784 Stomach Anatomy 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical class OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 210000004243 Sweat Anatomy 0.000 description 1
- 210000001179 Synovial Fluid Anatomy 0.000 description 1
- 101710040448 TNFRSF4 Proteins 0.000 description 1
- 102100013135 TNFRSF4 Human genes 0.000 description 1
- 210000001550 Testis Anatomy 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine Kinase Proteins 0.000 description 1
- 210000001541 Thymus Gland Anatomy 0.000 description 1
- 206010043709 Thyroid disease Diseases 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H Tricalcium phosphate Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K Trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 210000002700 Urine Anatomy 0.000 description 1
- 229960003048 Vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 229960004528 Vincristine Drugs 0.000 description 1
- 206010047461 Viral infection Diseases 0.000 description 1
- 208000001756 Virus Disease Diseases 0.000 description 1
- 230000002378 acidificating Effects 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 1
- 230000001154 acute Effects 0.000 description 1
- 230000003044 adaptive Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 230000001464 adherent Effects 0.000 description 1
- 230000001058 adult Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000001640 apoptogenic Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 244000052616 bacterial pathogens Species 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid group Chemical group C(C1=CC=CC=C1)(=O)O WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000001268 conjugating Effects 0.000 description 1
- 230000000875 corresponding Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001461 cytolytic Effects 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000003111 delayed Effects 0.000 description 1
- 230000001419 dependent Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 201000008325 diseases of cellular proliferation Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000003260 fluorescence intensity Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 239000012595 freezing medium Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- 230000030414 genetic transfer Effects 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 150000002333 glycines Chemical class 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000003394 haemopoietic Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002443 helper T lymphocyte Anatomy 0.000 description 1
- 230000002209 hydrophobic Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 229960001330 hydroxycarbamide Drugs 0.000 description 1
- VSNHCAURESNICA-UHFFFAOYSA-N hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 description 1
- 229940027318 hydroxyurea Drugs 0.000 description 1
- 210000002861 immature T-cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003308 immunostimulating Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000002458 infectious Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000000111 isothermal titration calorimetry Methods 0.000 description 1
- 230000000366 juvenile Effects 0.000 description 1
- 230000002147 killing Effects 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 108020001756 ligand binding domains Proteins 0.000 description 1
- 230000000670 limiting Effects 0.000 description 1
- AGBQKNBQESQNJD-UHFFFAOYSA-N lipoic acid Chemical compound OC(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-N 0.000 description 1
- 235000019136 lipoic acid Nutrition 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
- 210000004962 mammalian cells Anatomy 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 229910052752 metalloid Inorganic materials 0.000 description 1
- 150000002738 metalloids Chemical class 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 230000000813 microbial Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 230000000051 modifying Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 239000002120 nanofilm Substances 0.000 description 1
- 230000001264 neutralization Effects 0.000 description 1
- 210000004882 non-tumor cells Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000003071 parasitic Effects 0.000 description 1
- 230000036961 partial Effects 0.000 description 1
- 230000000737 periodic Effects 0.000 description 1
- 230000002093 peripheral Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000000546 pharmaceutic aid Substances 0.000 description 1
- 230000003285 pharmacodynamic Effects 0.000 description 1
- 230000000275 pharmacokinetic Effects 0.000 description 1
- 239000002952 polymeric resin Substances 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000000770 pro-inflamatory Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002062 proliferating Effects 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 210000001938 protoplasts Anatomy 0.000 description 1
- 201000004681 psoriasis Diseases 0.000 description 1
- 230000002685 pulmonary Effects 0.000 description 1
- 230000001698 pyrogenic Effects 0.000 description 1
- PMZDQRJGMBOQBF-UHFFFAOYSA-N quinolin-4-ol Chemical compound C1=CC=C2C(O)=CC=NC2=C1 PMZDQRJGMBOQBF-UHFFFAOYSA-N 0.000 description 1
- 230000001105 regulatory Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 230000001235 sensitizing Effects 0.000 description 1
- 235000004400 serine Nutrition 0.000 description 1
- 150000003355 serines Chemical class 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 231100000803 sterility Toxicity 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000002459 sustained Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing Effects 0.000 description 1
- 229930003347 taxol Natural products 0.000 description 1
- 229960002663 thioctic acid Drugs 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000002588 toxic Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 102000035402 transmembrane proteins Human genes 0.000 description 1
- 108091005683 transmembrane proteins Proteins 0.000 description 1
- LXZZYRPGZAFOLE-UHFFFAOYSA-L transplatin Chemical compound [H][N]([H])([H])[Pt](Cl)(Cl)[N]([H])([H])[H] LXZZYRPGZAFOLE-UHFFFAOYSA-L 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 235000019798 tripotassium phosphate Nutrition 0.000 description 1
- 239000011778 trisodium citrate Substances 0.000 description 1
- JOPDZQBPOWAEHC-UHFFFAOYSA-H tristrontium;diphosphate Chemical compound [Sr+2].[Sr+2].[Sr+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O JOPDZQBPOWAEHC-UHFFFAOYSA-H 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 244000052613 viral pathogens Species 0.000 description 1
Abstract
The present disclosure relates in some aspects to methods, cells, and compositions for preparing cells and compositions for genetic engineering and cell therapy. Provided in some embodiments are streamlined cell preparation methods, e.g., for isolation, processing, incubation, and genetic engineering of cells and populations of cells. Also provided are cells and compositions produced by the methods and methods of their use. The cells can include immune cells, such as T cells, and generally include a plurality of isolated T cell populations or types. In some aspects, the methods are capable of preparing of a plurality of different cell populations for adoptive therapy using fewer steps and/or resources and/or reduced handling compared with other methods. g of cells and populations of cells. Also provided are cells and compositions produced by the methods and methods of their use. The cells can include immune cells, such as T cells, and generally include a plurality of isolated T cell populations or types. In some aspects, the methods are capable of preparing of a plurality of different cell populations for adoptive therapy using fewer steps and/or resources and/or reduced handling compared with other methods.
Description
METHODS FOR ISOLATING, CULTURING, AND GENETICALLY ENGINEERING
IMMUNE CELL POPULATIONS FOR ADOPTIVE THERAPY
Cross-Reference to Related Applications
This application claims priority from U.S. provisional application No. 61/983,415,
filed April 23, 2014, the contents of which are incorporated by reference in their entirety.
Incorporation By Reference of Sequence Listing
The present application is being filed along with a Sequence Listing in electronic
format. The Sequence Listing is provided as a file entitled 735042000240seqlist.txt, created April
22, 2015, which is 13 kilobytes in size. The information in the electronic format of the Sequence
Listing is incorporated by reference in its entirety.
Field
The present disclosure relates in some aspects to methods, cells, and compositions
for preparing cells, and compositions for genetic engineering and cell therapy. Provided in some
embodiments are streamlined cell preparation methods, e.g., for isolation, processing, incubation,
and genetic engineering of cells and populations of cells. Also provided are cells and compositions
produced by the methods and methods of their use. The cells can include immune cells, such as
T cells, and generally include a plurality of isolated T cell populations or T cell sub-types. In some
aspects, the methods are capable of preparing of a plurality of different cell populations for adoptive
therapy using fewer steps and/or resources and/or reduced handling compared with other methods.
Background
Various methods are available for preparing cells for therapeutic use. For example,
methods are available for isolating, processing, and engineering cells, including T cells and other
immune cells. Methods are available to isolate such cells and to express genetically engineered
antigen receptors, such as high affinity T cell receptors (TCRs) and chimeric antigen receptors
(CARs). Methods are available to adoptively transfer such cells into subjects. Improved methods
are needed for the preparation (e.g., isolation, processing, culturing, and engineering) of cells for
sd-660418
735042000240
use in cell therapy. In particular, methods are needed for the preparation and engineering of cells,
e.g., a plurality of isolated cell types or sub-types, with improved efficiency, safety, variability, and
conservation of resources. Provided are methods, cells, compositions, kits, and systems that meet
such needs.
Summary
Provided are methods for the preparation and engineering of cells and populations of
cells and cells and compositions produced by the methods. In some embodiments, the cells can be
used for immunotherapy, such as in connection with adoptive immunotherapy methods. In some
aspects, the provided methods include isolation, selection or enrichment of CD4+ and CD8+ cells,
or sub-populations thereof, from the same starting sample, such as a single sample, for example a
single apheresis sample, leukapheresis sample or a sample containing peripheral blood mononuclear
cells (PBMCs). In some embodiments, the methods include selection or enrichment of at least two
populations of cells, such as a CD4+ cell population and a CD8+ population, in a single processing
stream in which no negative fraction sample is discarded from a first selection or enrichment of one
of the CD4+ or CD8+ in the process, prior to performing the second selection for the other of the
CD4+ or CD8+ cells. In some embodiments, the first and second selection can occur
simultaneously or sequentially.
In some aspects, the selection, enrichment and/or isolation of both populations of
cells, such as CD4+ and CD8+ cells, is performed simultaneously, such as in the same vessel or
using the same apparatus, or sequentially as part of a system or apparatus in which vessels, e.g.
columns, chambers, used in performing the first and second selection, are operably connected. In
some embodiments, the simultaneous and/or sequential enrichments or selections can occur as a
single process stream without handling of any of the positive or negative fractions prepared as part
of the first and/or second selection or enrichment. In some aspects, the isolation, culture, and/or
engineering of the different populations is carried out from the same starting composition or
material, such as from the same sample.
In some embodiments, the methods include performing a first selection by enriching
from a sample containing primary human T cells one of CD4+ or CD8+ cells to generate a first
sd-660418
735042000240
selected population and a non-selected population, and from the non-selected population performing
a second selection by enriching for the other of CD4+ cell or CD8+ cell, wherein the method
produces a composition of cells containing cells enriched for CD4+ cells and cells enriched for
CD8+.
In some embodiments, the second selection is carried out by enriching for the other
of the T cell subtypes from the non-selected population generated by the first selection. Thus, in
some embodiments, the provided methods differ from other selection methods in that the negative
fraction from a first selection is not discarded but rather is used as the basis for a further selection to
enrich for another cell type. In general, where the T cell subset enriched for in the first selection is
a CD4+ subset (or where the first selection enriches for CD4+ cells), it will follow that the first
selection is designed such that it does not enrich for cells of the other subtype to be enriched for in
the second selection. For example, in some embodiments, the first selection enriches for CD4+ cells
and does not enrich for CD8+ cells, and the second selection enriches for CD8+ cells from the
negative fraction recovered from the first selection. Likewise, in general, where the T cell subset
enriched for in the first selection is a CD8+ subset (or where the first selection enriches for CD8+
cells), it will follow that the first selection is designed such that it does not enrich for cells of the
other subtype to be enriched for in the second selection. For example, in some embodiments, the
first selection enriches for CD8+ cells and does not enrich for CD4+ cells, and the second selection
enriches for CD4+ cells from the negative fraction recovered from the first selection.
In some embodiments, the methods further involve third, fourth, and so-forth further
selections, which may enrich for cells from the selected and/or the non-selected populations from
any previous selection step. For example, in some embodiments, cells from either the selected
population or the non-selected population from a given step (e.g., the second selection step, are
further enriched. For example, in some embodiments, selected CD8+ cells are further enriched for
a subtype of CD8+ cells, such as resting cells or central memory cells.
In some embodiments, provided is a method for producing genetically engineered T
cells, comprising (a) providing a culture-initiation composition, said composition produced by
performing a first selection in a closed system, said first selection comprising enriching for one of
sd-660418
735042000240
CD4 cells and CD8+ cells from a sample containing primary human T cells, thereby generating a
first selected population and a non-selected population, and performing a second selection in the
closed system, said second selection comprising enriching for the other of CD4+ cells and CD8+
cells from the non-selected population, thereby generating a second selected population; (b)
incubating a culture-initiating composition, which comprises cells of the first selected population
and cells of the second selected population, in a culture vessel under stimulating conditions, thereby
generating stimulated cells; and (d) introducing a genetically engineered antigen receptor into
stimulated cells generated in (b), wherein the method thereby generates an output composition
comprising CD4 T cells and CD8 T cells expressing the genetically engineered antigen receptor.
Also provided, in some embodiments, are methods that include a simultaneous
enrichment or selection of a first and second population of cells, such as a CD4+ and CD8+ cell
population. In some embodiments, the method includes contacting cells of a sample containing
primary human T cells with a first immunoaffinity reagent that specifically binds to CD4 and a
second immunoaffinity reagent that specifically binds to CD8 in an incubation composition, under
conditions whereby the immunoaffinity reagents specifically bind to CD4 and CD8 molecules,
respectively, on the surface of cells in the sample, and recovering cells bound to the first and/or the
second immunoaffinity reagent, thereby generating an enriched composition comprising CD4+ cells
and CD8+ cells. In some embodiment, the methods are performed to include in the incubation
composition a concentration of the first and/or second immunoaffinity reagent that is at a sub-
optimal yield concentration so that the enriched composition contains less than 70%, less than 60%,
less than 50%, less than 40%, less than 30%, less than 20% or less of the total CD4+ cells in the
incubation composition or less than 70% less than 60%, less than 50%, less than 40%, less than
%, less than 20% or less of the CD8+ cells in the incubation composition.
In some embodiments, provided is a method for enriching CD4+ and CD8+ T cells,
comprising providing an enriched composition of CD4+ and CD9+ T cells, said enriched
composition produced by contacting cells of a sample containing primary human T cells with a first
immunoaffinity reagent that specifically binds to CD4 and a second immunoaffinity reagent that
specifically binds to CD8 in an incubation composition, under conditions whereby the
sd-660418
735042000240
immunoaffinity reagents specifically bind to CD4 and CD8 molecules, respectively, on the surface
of cells in the sample; and recovering cells bound to the first and/or the second immunoaffinity
reagent, thereby generating an enriched composition comprising CD4+ cells and CD8+ cells at a
culture-initiating ratio, wherein: the first and/or second immunoaffinity reagent are present in the
incubation composition at a sub-optimal yield concentration, whereby the enriched composition
contains less than 70% of the total CD4+ cells in the incubation composition and/or less than 70%
of the CD8+ cells in the incubation composition, thereby producing a composition enriched for
CD4+ and CD8+ T cells.
In some embodiments of any of such provided embodiments, the methods are
performed by immunoaffinity-based selection, such as by contacting cells with an antibody that
specifically binds a cell surface marker, such as CD4, CD8 or other cell surface marker, such as
expressed on naïve, resting or central memory T cells. In some embodiments, the solid support is a
sphere, such as a bead, such as a microbead or nanobead. In some embodiments, the bead can be a
magnetic bead. In some embodiments, the solid support can be a column or other vessel to effect
column chromatography.
In some embodiments, the antibody contains one or more binding partners capable of
forming a reversible bond with a binding reagent immobilized on the solid surface, such as a sphere
or chromatography matrix, wherein the antibody is reversibly mobilized to the solid surface. In
some embodiments, cells expressing a cell surface marker bound by the antibody on said solid
surface are capable of being recovered from the matrix by disruption of the reversible binding
between the binding reagent and binding partner. In some embodiments, the binding reagent is
streptavidin or is a streptavidin analog or mutant, such as a streptavidin, analog or mutant set forth
in any of SEQ ID NOS:11-16 or a sequence of amino acids that exhibits at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in any
of SEQ ID NOS: 11-16 and retains binding to a binding partner, such as biotin or peptide. In some
embodiments, the binding partner is biotin, a biotin analog or a peptide capable of binding to the
binding reagent. In some embodiments, the binding partner is or contains a peptide capable of
binding the binding reagent, such as a streptavidin binding peptide, such as a peptide containing the
sd-660418
735042000240
sequence set forth in any of SEQ ID NOS: 1-10. In some embodiments, the methods include after
contacting cells in the sample to the solid support containing the antibody immobilized thereon as
part of the first and/or second selection, applying a competition reagent to disrupt the bond between
the binding partner and binding reagent, thereby recovering the selected cells from the solid surface.
In some embodiments, the competition reagent is biotin or biotin analog.
In some embodiments, the methods generate a selected or enriched composition
containing selected or enriched CD4+ cells to CD8+ cells, or sub-populations thereof, present at a
culture-initiating ratio. In some embodiments, the culture-initiating ratio of CD4+ to CD8+ cells is
between at or about 10:1 and at or about 1:10, between at or about 5:1 and at or about 1:5 or is
between at or about 2:1 and at or about 1:2, such as is at or about 1:1. It is within the level of a
skilled artisan to choose or select a sufficient amount or a sufficient relative amount of
immunoaffinity-based reagent, such as antibody-coated beads (e.g. magnetic beads) or an affinity
chromatography matrix or matrices, to achieve or produce a culture-initiating ratio in the generated
composition, such as culture-initiation composition, containing cells enriched or selected for CD4,
CD8 or sub-populations thereof. Exemplary of such methods are described in subsections below.
In some embodiments, the cells include cells of the immune system, such as
lymphocytes, e.g., T cells (e.g., CD4+ and CD8+ T cells and isolated subpopulations thereof), and
NK cells. In some embodiments, the cells are present in combinations of multiple cell populations
or cell types, which in some aspects are included in the compositions at particular ratios or numbers
of the cells or cell types. Also provided are methods for optimizing the methods, such as by
selecting or determining appropriate ratios and numbers, such as culture-initiating ratios and desired
output ratios and doses, of the cell types and population, for use in connection with the methods.
Also provided are cells, cell populations, and compositions thereof, for use in and
produced by the methods. Also provided are systems, devices, apparatuses, reagents, compounds,
and kits for carrying out the methods. Also provided are therapeutic methods and uses for cells and
compositions produced by the methods, such as methods for adoptive cell therapy.
In some embodiments, provided are methods for producing cells for adoptive cell
therapy, methods for producing cells for genetic engineering, and methods for producing genetically
sd-660418
735042000240
engineered cells. In some aspects, the cells are T cells, such as CD4 and CD8 T cells and/or sub-
types thereof. In some aspects, the cell therapy is T cell therapy.
In some embodiments, the methods are carried out by (a) isolating cell populations
from a sample, and (b) incubating a culture-initiating composition in a culture vessel containing
cells of the isolated populations. In some aspects, the methods further include genetically
engineering the incubated cells or cells in the culture vessel, such as by (c) introducing a genetically
engineered antigen receptor into cells in the culture vessel.
In some embodiments, the methods are carried out by (a) incubating a culture-
initiating composition in a culture vessel containing a plurality of cell populations at a particular
culture-initiation ratio or particular number of cells; and (b) genetically engineering the cells, such
as by introducing a genetically engineered antigen receptor into cells in the culture vessel.
In some embodiments, the genetically engineered antigen receptor is introduced into
cells in the culture vessel, such as to different cell types or sub-populations within the culture
vessel, e.g., to CD4 and CD8 cells in the culture vessel.
In some aspects, the methods produce an output composition for genetic engineering
or adoptive cell therapy or comprising cells expressing the genetically engineered antigen receptor.
In some embodiments, the isolation includes or is carried out by isolating a CD4
primary human T cell population and/or a CD8 primary human T cell population from the sample.
In some aspects, it includes depleting or enriching for a sub-population of CD4 cells, and/or
depleting or enriching for a sub-population of CD8 cells. Thus, in some aspects, the culture-
initiating composition includes isolated CD4 and CD8 primary human T cells.
In some aspects, the enriching or depleting is carried out by immunoaffinity-based
selection, such as binding to antibodies or other binding molecules recognizing surface markers on
the cells. In some aspects, the antibody or other molecule is coupled to a magnetically responsive or
magnetic particle, e.g., bead. In some aspects, the selection includes positive and/or negative
selection steps.
In some embodiments, the isolation of the CD8 primary human T cell population
comprises depleting or enriching for a sub-population of CD8 cells. In some embodiments, the
sd-660418
735042000240
isolation of the CD4 primary human T cell population comprises depleting or enriching for a sub-
population of CD4 cells. In some aspects, the isolation of a T cell population comprises enriching
for T cells. In some aspects, the isolation of the CD8 and/or the CD4 primary human T cell
population comprises enriching for central memory T (T ) cells. In some aspects, the enrichment
for central memory T (TCM) cells comprises negative selection for cells expressing a surface marker
present on naïve T cells, such as CD45RA, or positive selection for cells expressing a surface
marker present on central memory T cells and not present on naïve T cells, such as CD45RO;
and/or positive selection for cells expressing surface marker present on central memory T (TCM)
cells and not present on another memory T cell sub-population, such as CD62L, CCR7, CD27,
CD127, and/or CD44.
In some embodiments, the isolation includes (i) subjecting the sample to positive
selection based on surface expression of CD4, resulting in a positive and first negative fraction,
where the positive fraction is the isolated CD4 population; and (ii) subjecting the first negative
fraction to negative selection based on surface expression of a non-T cell marker and a surface
marker present on naïve T cells, thereby generating a second negative fraction; and (iii) subjecting
the second negative fraction to positive selection based on surface expression of a marker present on
the surface of central memory T (T ) cells and not present on the surface of another memory T cell
sub-population.
In some aspects, the marker present on naïve T cells includes CD45RA. In some
aspects, the surface marker present on central memory T (T ) cells and not present on another
memory T cell sub-population includes CD62L, CCR7, CD27, CD127, and/or CD44.
In some aspects, the isolation or selections are carried out in the same separation
vessel. In some aspects, the isolation comprises (i) subjecting the sample to a first selection,
thereby generating one of the CD4 and CD8 primary human T cell populations and a non-selected
sample; and (ii) subjecting the non-selected sample to a second selection, thereby generating the
+ + +
other of the CD4 and CD8 primary human T cell populations. In some aspects, the CD4 primary
human T cell population is generated in the first selection and the CD8 primary human T cell
sd-660418
735042000240
population is generated in the second selection. In some aspects, the first and/or second selection
comprises a plurality of positive or negative selection steps.
In some aspects, the isolation of one or more of the populations, such as the primary
human T cell population, the primary human CD4+ T cell population, or the primary human CD8+
T cell population, includes positive selection based on surface expression CD62L, CCR7, CD44, or
CD27. In some aspects, the isolation of one or more of the populations, such as the primary human
T cell population, the primary human CD4 T cell population, or the primary human CD8 T cell
population, includes negative selection based on surface expression of CD45RA or positive
selection based on surface expression of CD45RO.
In some embodiments, the various isolations, such as isolation of the plurality of cell
populations, e.g., the CD4 and the CD8 populations, are carried out in the same separation vessel.
In some aspects, the separation vessel is or includes a tube, tubing set, chamber, unit, well, culture
vessel, bag, and/or column. In some aspects, the separation vessel maintains the cells in a contained
or sterile environment during the separation.
In some embodiments, the incubating is carried out under stimulating conditions. In
some aspects, the culture-initiating composition comprises the CD4 and CD8 primary human T
cell populations at a culture-initiating ratio. In some aspects, the culture-initiating ratio is designed
to yield a desired output ratio of CD4 to CD8 cells (or desired total number of T cells or
number(s) of the sub-population(s)) following said incubation, or at some later time, such as
following engineering, cryopreservation, or at the time just prior to administration, such as at thaw
at bedside.
In some embodiments, the desired output ratio is between at or about 5:1 and at or
about 1:5 (or greater than about 1:5 and less than about 5:1), such as between at or about 1:3 and at
or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between at or about 2:1 and
at or about 1:5 (or greater than about 1:5 and less than about 2:1), or is the range of between at or
about 2:1 and at or about 1:5. In some aspects, the desired output ratio is at or about 3:1, 2.9:1,
2.8:1, 2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1,
sd-660418
735042000240
1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4,
1:4.5, or 1:5.
In some aspects, the method results in a ratio of two different cell types or
populations, such as a ratio of CD4 to CD8 cells, in the output composition that is between at or
about 5:1 and at or about 1:5 (or greater than about 1:5 and less than about 5:1), such as between at
or about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between
at or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1), or that is at or
about 3:1, 2.9:1, 2.8:1, 2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1,
1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2,
1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.
In some aspects, the desired output ratio is 1:1 or is about 1:1.
In some aspects, the methods result in the desired output ratio or cell number(s) in
the output composition, results in a ratio or number in the output composition that is within a certain
tolerated difference or range of error of such a desired output ratio or number, and/or results in such
a ratio or number a certain percentage of the time that the method is performed, such as at least at or
about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% of the time.
In some aspects, the tolerated difference is within about 1%, about 2%, about 3%,
about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 45%, about 50% of the desired ratio. In some aspects, the output ratio is within 20 % of
the desired ratio and/or is within that ratio at least 80 % of the time the methods are performed.
In some embodiments, the tolerated difference and/or the desired output ratio or
number(s) is or has been determined by administering different cell types, such as administering
CD4 and CD8 cells, to one or more subjects at a plurality of test ratios or numbers, and assessing
one or more parameters. In some aspects, the determination of the desired output ratio or number or
tolerated difference includes assessing one or more outcomes following administration to the
subject. In some aspects, the outcomes include those selected from among amelioration of a disease
symptom and outcomes indicating safety and/or low or absence of toxicity.
sd-660418
735042000240
In some embodiments, the culture-initiation ratio is selected based on a proliferation
rate or survival capacity of various cell types isolated, such as the CD4 and/or CD8 primary
human T cell population. In some embodiments, the culture-initiating ratio is selected based on a
source of the sample, such as the subject from which the sample has been derived. For example, in
some aspects, the sample is derived from a subject and the culture-initiating ratio is selected based
on a disease or condition affecting said subject and/or a treatment the subject is receiving, has
received, or will receive, such as a co-treatment for administration with the adoptive cell therapy.
In some aspects, the culture-initiating ratio is selected based on a phenotype of one or more of the
cells or sub-types of cells being isolated or cultured, such as a phenotype of the CD4+ and/or CD8+
cell populations, such as expression of a cell surface marker or synthesis or secretion of one or more
factors, such as cytokines or chemokines.
In some embodiments, the methods comprise selecting the culture-initiation ratio
prior to the incubation step. In some aspects, the selection is carried out by measuring a
proliferation rate or survival capacity of one or more of the isolated cell populations or populations
of cells being incubated, such as the CD4 primary human T cells and/or the isolated CD8 primary
human T cells. In some aspects, the selection is carried out by assessing a phenotype of the isolated
CD4 primary human T cells and/or the isolated CD8 primary human T cells. In some aspects, the
phenotype selected from among expression of a surface marker and secretion of a cytokine or other
factor. In some aspects, the selecting is carried out by assessing the source of the sample, such as
where the culture-initiation ratio is selected based on a disease or condition affecting a subject from
which the sample is derived.
In some embodiments, the methods further include determining an intermediate ratio
or number of cells, such as an intermediate ratio of CD4 to CD8 cells, present in the culture vessel
at a time point subsequent to the initiation of said incubation. In some aspects, the methods further
include adjusting one or more parameters and/or or increasing or decreasing the time for carrying
out the incubation and/or engineering steps, based on said intermediate ratio. In one aspect, the
adjusting comprises increasing or decreasing the number of or enriching for one or more cell
populations in the culture vessel, such as increasing or enriching for CD4 or CD8 cells in the
sd-660418
735042000240
culture vessel, adjusting temperature, adding a stimulant to the culture vessel, adjusting the
concentration of one or more stimulants in the culture vessel, and/or adding and/or removing a sub-
population of cells to or from the culture vessel. In some aspects, the determining and/or adjusting
are carried out while maintaining the composition being incubated within a sterile or contained
environment. In some aspects, the determination and/or adjusting are carried out in an automated
fashion, such as controlled by a computer attached to a device in which the steps are performed.
In some aspects, the isolating, incubating, and/or engineering steps are carried out in
a sterile or contained environment and/or in an automated fashion, such as controlled by a computer
attached to a device in which the steps are performed.
In some aspects, the CD8 population in the culture-initiating composition comprises
at least 50 % central memory T (T ) cells or comprises less than 20 % naïve T (T ) cells.
CM N
In some embodiments, the sample is obtained from a subject. In some aspects, the
subject is a subject to whom said genetically engineered cells, e.g., T cells, or cells for adoptive cell
therapy will be administered or subject in need of such administration. In other aspects, the subject
is a subject other than a subject to whom said genetically engineered cells, e.g., T cells, or cells for
adoptive therapy will be administered or is a subject not in need of such therapy. Among the
samples are blood and blood-derived samples, such as white blood cell samples, apheresis samples,
leukapheresis samples, peripheral blood mononuclear cell (PBMC) samples, and whole blood.
In some aspects, the stimulating conditions for the incubation or engineering include
conditions whereby T cells of the culture-initiating composition proliferate or expand. For example,
in some aspects, the incubation is carried out in the presence of an agent capable of activating one
or more intracellular signaling domains of one or more components of a TCR complex, such as a
CD3 zeta chain, or capable of activating signaling through such a complex or component. In some
aspects, the incubation is carried out in the presence of an anti-CD3 antibody, and anti-CD28
antibody, anti1BB antibody, for example, such antibodies coupled to or present on the surface of
a solid support, such as a bead, and/or a cytokine, such as IL-2, IL-15, IL-7, and/or IL-21.
In some embodiments, the genetically engineered antigen receptor is or includes a T
cell receptor (TCR), such as a high-affinity TCR, or functional non-TCR antigen receptor, such as a
sd-660418
735042000240
chimeric antigen receptor (CAR). In some aspects, the receptor specifically binds to an antigen
expressed by cells of a disease or condition to be treated. In some aspects, the CAR contains an
extracellular antigen-recognition domain. In some aspects, it further contains an intracellular
signaling domain comprising an ITAM-containing sequence and an intracellular signaling domain
of a T cell costimulatory molecule.
Also provided are cells and compositions, including pharmaceutical compositions,
produced by any of the methods or embodiments, including genetically engineered cells and cells
for adoptive cell therapy. Also provided are methods for administering such cells and compositions
to subjects and uses of the cells and compositions in such methods. For example, provided are
methods of treatment carried out by producing cells according to the cell production methods and
administering cells of the output composition or a composition derived therefrom to a subject.
Provided are methods of treatment including administering the provided cells or compositions to a
subject. In some aspects, the sample from which the cells are isolated is derived from the subject to
which the cells are administered. In some aspects, the sample is from a different subject. Thus, the
methods include autologous and allogeneic methods. In some embodiments, the methods
ameliorate, treat, or prevent one or more symptoms of a disease or condition in the subject. In some
aspects, the disease or condition is a cancer or associated symptom. In some embodiments, the
cancers include leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL, non-
Hodgkins lymphoma, acute myeloi d leukemia, multiple myeloma, refractory follicular lymphoma,
mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung,
liver, breast, prostate, ovarian, skin (including melanoma), bone, and brain cancer, ovarian cancer,
epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical
carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma,
osteosarcoma, synovial sarcoma, and/or mesothelioma.
Brief Description of the Drawings
Figure 1A: provides a schematic representation of an embodiment of a closed
system for use in embodiments of the provided methods. The depicted exemplary system includes a
sd-660418
735042000240
cell sample 5, a washing buffer reservoir 6, an elution buffer reservoir 7, a first Fab reservoir 18, a
second Fab reservoir 19, and a pump 8, connected through a series of tubing and valves 13, to a first
chromatography column 1 containing a first matrix 3 operably linked via a series of tubing lines to a
second chromatography column 2 containing a second matrix 4. The second chromatography
column 2 is operably linked to a removal chamber 9. The removal chamber 9 is operably linked to
a valve 13, which directs cells and fluids to a waste container 10 or a culture vessel 12 via a series
of tubing lines. The system is enclosed in an enclosure 14.
Figure 1B: provides a schematic representation of an embodiment of a closed
system for use in embodiments of the provided methods. The depicted exemplary system includes a
cell sample 5, a washing buffer reservoir 6, an elution buffer reservoir 7, a first Fab reservoir 18, a
second Fab reservoir 19, a third Fab reservoir 20, and a pump 8, connected through a series of
tubing to a first chromatography column 1 containing a first matrix 3. Valves 13 operably linked to
the series of tubing direct fluids through the series of tubing. A valve 13 operably linked to the first
chromatography column 1 directs cells and fluids to a second chromatography column 2 containing
a second matrix 4, which is operably linked to a removal chamber 9. The valve 13 operably linked
to the first chromatography column 1 also directs cells and fluids to a removal chamber 9, which is
operably linked to a third chromatography column 15 containing a third matrix 16. The third
chromatography column 15 is operably linked to a removal chamber 9. The removal chambers 9
are operably linked to a first waste container 10 or a culture vessel 12 via the series of tubing lines
and valves 13. The system is enclosed in an enclosure 14.
Detailed Description
Unless defined otherwise, all terms of art, notations and other technical and scientific
terms or terminology used herein are intended to have the same meaning as is commonly
understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some
cases, terms with commonly understood meanings are defined herein for clarity and/or for ready
reference, and the inclusion of such definitions herein should not necessarily be construed to
represent a substantial difference over what is generally understood in the art.
sd-660418
735042000240
All publications, including patent documents, scientific articles and databases,
referred to in this application are incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication were individually incorporated by reference. If a
definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the
patents, applications, published applications and other publications that are herein incorporated by
reference, the definition set forth herein prevails over the definition that is incorporated herein by
reference.
The section headings used herein are for organizational purposes only and are not to
be construed as limiting the subject matter described.
I. Methods and systems for isolating, culturing, and engineering cells for adoptive
therapy
Provided are methods of preparing cells, e.g., T cells, for use in genetic engineering
and therapeutic methods such as adoptive cell therapy. Specifically, in some embodiments, the
methods use or generate compositions that contain a plurality of different cell populations or types
of cells, such as isolated CD4 and CD8 T cell populations and sub-populations. In some
embodiments, the methods include steps for isolating one or more cell populations, generally a
plurality of cell populations. The cells generally are isolated from a sample derived from a subject.
In some aspects, the methods are performed by employing simultaneous or
sequential selections or enrichments in which a plurality of different cell populations, such as CD4+
or CD8+ cells, from a sample, such as a sample containing primary human T cells, are selected,
enriched and/or isolated. In some embodiments, such methods are performed using a single
processing stream in which a first population of cells, such as CD4+ or CD8+ cells, and a second
population of cells, such as the other of the CD4+ or CD8+ cells, are selected, enriched and/or
isolated without discarded any cells from the first population prior to performing the selection of the
second population of cells. In some aspects, the first and second selections can be performed
simultaneously or sequentially. In some embodiments, the methods of selection are performed as a
single process stream by performing a first selection to enrich for a first population of cells, such as
sd-660418
735042000240
one of CD4+ and CD8+ cells from a sample, such as a sample containing primary human T cells,
and using the non-selected cells from the first selection as the source of cells for a second selection,
such as for the other of the CD4+ or CD8+ cells from the sample.
In some embodiments, a further selection or selections can be performed of the first
or second selected cells. In some embodiments, the further selection enriches for a sub-population
of CD4+ or CD8+ cells expressing a marker on central memory T (T ) cells and/or enriches for a
sub-population of cells expressing CD62L, CD45RA, CD45RO, CCR7, CD27, CD127, or CD44.
In some embodiments, the methods of selection are performed in a closed system or
apparatus. In some embodiments, within the closed system or apparatus, a composition, such as a
culture-initiation composition, is generated containing the enriched or selected population of cells,
such as both the enriched or selected CD4+ and CD8+ populations, in the same composition.
For example, in some aspects, the one or more steps are carried out with the plurality
of cell populations combined in the same composition, or present in the same vessel, e.g., in the
same closed system or apparatus, or in the same vessel, unit, or chamber, such as the same column,
e.g., magnetic separation column, tube, tubing set, culture or cultivation chamber, culture vessel,
processing unit, cell separation vessel, centrifugation chamber, or using the same separation matrix,
media, and/or reagents, such as the same magnetic or magnetically responsive matrix, particle, or
bead, the same solid support, e.g., affinity-labeled solid support, and/or the same antibodies and/or
other binding partners, such as fluorescently-labeled antibodies and binding partners, for the
plurality of cell populations. In some embodiments, the one or more vessels, units or chambers,
such as columns, are operably connected in the same closed system or apparatus, so that the
methods of isolation, selection and/or enriching occurs in a single process stream in which a non-
selected cell population from a first selection can be used as a source of cells for a second selection.
In some embodiments, the isolation of or enrichment for one or more specific
populations or sub-populations of cells, e.g., CD4+ and CD8+ T cells to be cultured or engineered
for adoptive therapy, provides one or more advantages. For example, engineering cells enriched for
a plurality of different cell populations or types of cells, such as isolated CD4 and CD8 T cell
populations and sub-populations, can improve efficacy of or reduce or avoid unwanted effects. In
sd-660418
735042000240
some aspects, the isolation or enrichment increases the ability of cells ultimately administered to a
subject to persist, expand, become activated, and/or engraft in vivo or upon administration to a
subject. In some aspects, it improves or increases one or more effector function or activation
phenotype. For example, in some aspects, enriching a T cell population, such as a CD8+ T cell
population, for central memory (TCM) cells can provide such advantages. In some aspects, one or
more of such advantages are provided by combining two or more isolated populations or sub-types,
such as isolated populations or sub-populations of CD8+ and CD4+ T cells, such as a T -enriched
CD8 population and a CD4 population. For example, such advantages can be achieved in some
aspects by administering a CD4+ and a CD8+ population in comparison with a CD8+ population
alone.
The methods in some embodiments include processing of a generated composition
containing a plurality of the isolated or selected cell populations, such as a population of selected or
enriched CD4+ cells and CD8+ cells. In one embodiment, processing of the generated composition
includes incubating the cells under stimulating conditions, for example in some aspects, to activate
the cells for engineering or transduction or for cell expansion. The methods, in some embodiments,
include steps for engineering a plurality of cell types, such as CD4+ cells and CD8+ cells, such as
those isolated and present in the incubated composition, such as the culture-initiation composition.
In some aspects, the engineering is carried out to introduce a genetically engineered antigen
receptor into the cells, such as a TCR, e.g., a high-affinity TCR, or a functional non-TCR antigen
receptor, such as a chimeric antigen receptor (CAR). In some aspects, the methods include further
processing, such as further incubation, for example at or about 37º C ± 2º C, and/or formulating of
cells and compositions containing the same. In some embodiments, the processing produces a
resulting output composition containing genetically engineered cells, such as genetically engineered
CD4+ cells and CD8+ cells. In some embodiments, the resulting processed output composition can
be used in methods for administering cells and compositions prepared by the methods to a patient,
for example, in connection with adoptive cell therapy.
In certain embodiments, the isolation or separation is carried out using a system,
device, or apparatus that carries out one or more of the isolation, cell preparation, separation,
sd-660418
735042000240
processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the
system is used to carry out each of these steps in a closed or sterile environment, for example, to
minimize error, user handling and/or contamination. In one example, the system is a system as
described in International Patent Application, Publication Number WO2009/072003, or US
20110003380 A1. In some embodiments, the system is a closed apparatus or system, such as is
depicted in Figure 1A or Figure 1B. In some embodiments, the system or apparatus is automated
and/or carries out the selection steps to enrich cells according to the methods in an automated
fashion.
In some embodiments, the system or apparatus carries out the isolation, such as
selection or enrichments steps. In some embodiments, a further system or apparatus, such as a
closed system or apparatus, can be used to carry out one or more of the other steps such as cells
preparation, processing, incubation, culture and/or formulation steps of the method. In some
embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing,
engineering, and formulation steps in an integrated or self-contained system, and/or in an automated
or programmable fashion. In some aspects, the system or apparatus includes a computer and/or
computer program in communication with the system or apparatus, which allows a user to program,
control, assess the outcome of, and/or adjust various aspects of the processing, isolation,
engineering, and formulation steps.
In some embodiments, because the enriched composition, such as culture initiation
composition, is generated by the provided methods to contain different cell populations, such as
CD4+ and CD8+, the generated composition can be processed together and simultaneously under
the same conditions, and in some aspects, in a closed system. Thus, in some aspects, the methods
provide a process to produce and process a population of selected, enriched or isolated cells
containing different populations of cells, such as CD4+ and CD8+ cells, in a single process stream.
In some aspects, this differs from prior art methods, including prior art methods that process cells
for engineering for adoptive cell therapy, which typically include a separate process stream for each
different cell population, such as at least two process streams. For example, in some aspects of
existing methods, CD4+ T cells are separately isolated, enriched and/or selected and processed
sd-660418
735042000240
under stimulating conditions for genetic engineering, CD8+ T cells are separately isolated, enriched
and/or selected and processed under stimulating conditions for genetic engineering, and the separate
processed and engineered CD4+ and CD8+ T cells are re-combined prior to administration to a
subject.
In some embodiments, the methods provide one or more advantages compared with
other preparation, isolation, incubation, and engineering methods, such as cost, time, and/or
resource savings. Such advantages can include the ability to isolate, process, e.g., incubate, and/or
engineer the plurality of cell populations, present at or near a desired ratio, with increased efficiency
and/or reduced complexity, time, cost, and/or use of resources, compared with other methods.
In some embodiments, such advantages are achieved by streamlining one or more of
the method steps. For example, in some aspects, isolation, culture, and/or engineering of the
different populations is carried out using the same apparatus or equipment and/or simultaneously.
In some aspects, the isolation, culture, and/or engineering of the different populations is carried out
based on the same starting composition. In some aspects, such features of the methods reduce the
amount of time, method steps, cost, complexity, and/or number of resources, compared to a method
in which the cell populations are isolated, incubated, and/or engineered separately, in separate
vessels, using separate equipment, at separate times, and/or beginning with different starting
compositions.
In some aspects, the methods of isolating, incubating and engineering cells results in
an output composition in which the different cell populations or cell types, such as CD4+ cells and
CD8+ cells, are present at a desired ratio, or within a certain degree of tolerated error of the desired
ratio. Such ratios include those deemed optimal for the therapeutic use, e.g., output ratios deemed
appropriate or optimal for administration to a patient in connection with adoptive cell therapy. Also
provided are methods for administering cells and compositions prepared by the methods to a
patient, for example, in connection with adoptive cell therapy
In some embodiments, the methods of isolation or selection are performed to achieve
selection of cells at a chosen culture-initiation ratio of CD4+ cells to CD8+ cells or a sub-population
thereof. In some aspects, such ratios include ratios deemed optimal starting points for achieving an
sd-660418
735042000240
optimal ratio, such as a desired ratio in the output composition, at the completion of the method or
one or more steps, such as following a culture, incubation, and/or engineering step. In some
embodiments, providing the cells at a culture-initiation ratio, such as a ratio of CD4+ cells to CD8+
cells, accounts for differences in expansion of CD4+ and CD8+ T cells that can occur upon
stimulation or activation with different reagents, in order to achieve a desired ratio in the output
composition.
In some embodiments, the methods generate engineered cells or cells for engineering
at or within a certain percentage of a desired output ratio, or do so at least a certain percentage of
the time. The desired output ratio typically is a ratio that has been determined to be optimal for
administration to a patient via adoptive transfer. In some embodiments, the populations or sub-
types of cells, such as CD8 and CD4 T cells are administered at or within a tolerated difference of
a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a
desired number of cells or a desired number of cells per unit of body weight of the subject to whom
the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a
minimum number of cells or minimum number of cells per unit of body weight. In some aspects,
among the total cells, administered at the desired dose, the individual populations or sub-types are
present at or near a desired output ratio (such as CD4 to CD8 ratio), e.g., within a certain tolerated
difference or error of such a ratio.
In some embodiments, the cells are administered at or within a tolerated difference
of a desired dose of one or more of the individual populations or sub-types of cells, such as a
desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some aspects, the desired dose
is a desired number of cells of the sub-type or population, or a desired number of such cells per unit
of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects,
the desired dose is at or above a minimum number of cells of the population or sub-type, or
minimum number of cells of the population or sub-type per unit of body weight.
Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells
and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual
sub-types or sub-populations. Thus, in some embodiments, the dosage is based on a desired fixed
sd-660418
735042000240
or minimum dose of T cells and a desired ratio of CD4 to CD8 cells, and/or is based on a desired
fixed or minimum dose of CD4 and/or CD8 cells.
In some embodiments, provided are methods for determining such optimal output
ratios and/or desired doses, and/or levels of variance from the desired output ratio or desired dose
that would be tolerated, e.g., a tolerated difference. In some embodiments, in order to achieve the
desired output ratio or dose(s) (or to achieve such a ratio a certain percentage of the time or within a
certain tolerated difference), the cell populations are combined or incubated at ratios (e.g., culture-
initiation ratios) designed to achieve the desired output ratio for adoptive transfer.
Also provided are methods for determining a culture-initiation ratio designed to
achieve the desired output ratio or dose to do so within a certain tolerated difference and/or a certain
percentage of the time. Also provided are methods for assessing interim ratios or numbers of the
cell populations, such as at one or more periods of times over the course of the various method
steps, e.g., during incubation. Also provided are methods for adjusting various conditions, such as
culture conditions, based on such assessments. In some aspects, the adjustments are carried out to
ensure that a particular output ratio or dose is achieved or achieved within a tolerated difference.
In particular embodiments, provided are streamlined methods for preparing a
composition having at or near a desired output ratio of a CD4 T cell population and a CD8 T cell
population (e.g., a CD8 population enriched for a sub-type of T cells such as central memory T
cells), and/or a desired dose (e.g., number or number per unit of body weight) of T cells and/or of
CD4 and CD8 T cells, for introduction of a genetically engineered antigen receptor for use in
adoptive cell therapy, where the cell populations are isolated, incubated, and/or engineered in
combination and the method is associated with increased efficiency and/or reduced complexity,
time, cost, and/or use of resources compared to a method in which the populations are isolated,
incubated, and/or engineered separately.
Also provided are cells and compositions prepared by the methods, including
pharmaceutical compositions and formulations, and kits, systems, and devices for carrying out the
methods. Also provided are methods for use of the cells and compositions prepared by the
sd-660418
735042000240
methods, including therapeutic methods, such as methods for adoptive cell therapy, and
pharmaceutical compositions for administration to subjects.
A. Isolation, isolated cells, and other processing steps
Among the provided embodiments are methods for isolating a plurality of cells and
populations of cells from a sample, as well as isolated, such as enriched, cells produced by such
methods. The isolation can include one or more of various cell preparation and separation steps,
including separation based on one or more properties, such as size, density, sensitivity or resistance
to particular reagents, and/or affinity, e.g., immunoaffinity, to antibodies or other binding partners.
In some aspects, the isolation is carried out using the same apparatus or equipment sequentially in a
single process stream and/or simultaneously. In some aspects, the isolation, culture, and/or
engineering of the different populations is carried out from the same starting composition or
material, such as from the same sample.
In some aspects, the plurality of cell populations are isolated in the same closed
system or apparatus, and/or in the same vessel or set of vessels, e.g., same (or same set of) unit,
chamber, column, e.g., magnetic separation column, tube, tubing set, culture or cultivation chamber,
culture vessel, processing unit, cell separation vessel, centrifugation chamber. For example, in
some cases, the isolation of a plurality of cell populations is carried out a system or apparatus
employing a single or the same isolation or separation vessel or set of vessels, such as a single
column or set of columns, and/or same tube, or tubing set, for example, without requirements to
transfer the cell population, composition, or suspension from one vessel, e.g., tubing set, to another.
In some aspects, such methods are achieved by a single process stream, such as in a
closed system, by employing simultaneous or sequential selections in which a plurality of different
cell populations, such as CD4+ or CD8+ cells, from a sample, such as a sample containing primary
human T cells, are selected, enriched and/or isolated. In one embodiment, a sample containing
cells is subjected to a selection by simultaneous enrichment of both the CD4+ and CD8+
populations. In some aspects, carrying out the separation or isolation in the same vessel or set of
vessels, e.g., tubing set, is achieved by carrying out sequential positive and negative selection steps,
sd-660418
735042000240
the subsequent step subjecting the negative and/or positive fraction from the previous step to further
selection, where the entire process is carried out in the same tube or tubing set. In one embodiment,
a sample containing cells to be selected is subjected to a sequential selection in which a first
selection is effected to enrich for one of the CD4+ or CD8+ populations, and the non-selected cells
from the first selection is used as the source of cells for a second selection to enrich for the other of
the CD4+ or CD8+ populations. In some embodiments, a further selection or selections can be
effected to enrich for sub-populations of one or both of the CD4+ or CD8+ population, for example,
central memory T (TCM) cells.
In a particular aspect, a first selection step is carried out using beads labeled with
CD4-binding molecules, such as antibodies (or secondary reagents that recognize such molecules),
and the positive and negative fractions from the first selection step are retained, followed by further
positive or negative selection of the negative fraction to enrich for CD8+ cells, such as by using
beads labeled with CD8-binding molecules, and optionally selection of a sub-population of cells
from the CD8+ fraction, such as central memory CD8 T cells and/or cells expressing one or more
markers CD62L, CD45RA, CD45RO, CCR7, CD27, CD127, or CD44. In some embodiments, the
order of the selections can be reversed. In some embodiments, a CD4 selection is always performed
first and, from the negative fraction (CD4-), the CD8+ fraction is enriched in a second selection,
e.g., by negative selection for one or more of CD45RA and CD14, and/or positive selection for one
or more of CD62L, CCR7, and/or other markers expressed on central memory cells.
In some embodiments, a plurality of cell populations, e.g., a CD4 T cell population
and a CD8 T cell population, is isolated, for example, to produce a culture-initiating composition
containing the plurality of cell populations. The culture-initiating composition typically contains
the cells at a culture-initiating ratio, which is designed to yield a particular desired output ratio of
two or more cell types, such as a particular CD4 to CD8 ratio, following one or more incubation,
culture, cultivation, and/or engineering steps. The desired output ratio in some embodiments is a
ratio designed to be optimal for administration of the cells to a patient, e.g., in adoptive cell therapy.
In some aspects, isolating the plurality of populations in a single or in the same
isolation or separation vessel or set of vessels, such as a single column or set of columns, and/or
sd-660418
735042000240
same tube, or tubing set or using the same separation matrix or media or reagents, such as the same
magnetic matrix, affinity-labeled solid support, or antibodies or other binding partners, include
features that streamline the isolation, for example, resulting in reduced cost, time, complexity, need
for handling of samples, use of resources, reagents, or equipment. In some aspects, such features
are advantageous in that they minimize cost, efficiency, time, and/or complexity associated with the
methods, and/or avoid potential harm to the cell product, such as harm caused by infection,
contamination, and/or changes in temperature.
In some embodiments the isolated cell populations obtained for use in the methods
herein are sterile. Microbial contamination of cell separation products can in some cases lead to the
infection of the recipient subject, such as an immunocompromised recipient patient unable to fight
the infection. In some embodiments, the cells, cell populations, and compositions are produced
under GMP (good manufacturing practice) conditions. In some embodiments, GMP conditions
comprise stringent batch testing. In certain embodiments, tissue typing is performed prior to
transplantation, e.g., to avoid human leukocyte antigen (HLA) mismatch and prevent problems such
as graft-versus-host disease. In some embodiments, the provided methods reduce handling by
individual users and automate various steps, which in some aspects can increase consistency of the
isolated cell populations and compositions and reduces error, thereby promoting consistency of the
therapy and safety.
1. Cells and populations of cells
In some embodiments, the methods include performing a selection, isolation and/or
enrichment of a cell sample, such as a primary human cell sample. The isolated cell populations
typically include a plurality of cell populations, generally populations of blood or blood-derived
cells, such as hematopoietic cells, leukocytes (white blood cells), peripheral blood mononuclear
cells (PBMCs), and/or cells of the immune system, e.g., cells of the innate or adaptive immunity,
such as myeloid or lymphoid cells, e.g., lymphocytes, typically T cells and/or NK cells. In some
embodiments, the sample is an apheresis or leukapheresis sample. In some embodiments, the
sd-660418
735042000240
selection, isolation and/or enrichment can include positive or negative selection of cells from the
sample.
In some embodiments, the sample is a sample containing primary human T cells,
such as CD4+ and CD8+ T cells. In particular embodiments, the sample is one in which a plurality
of T cell populations is isolated, such as a population of CD4 cells and a population of CD8 T
cells. Thus, in some embodiments, the isolation includes positive selection for cells expressing
CD4 or cells expressing CD8 and/or negative selection for cells expressing non-T cell markers, such
as myeloid or B cell markers, for example, negative selection for cells expressing CD14, CD19,
CD56, CD20, CD11b, and/or CD16.
In some embodiments, the sample is one containing a plurality of populations that
includes a T cell population, such as a whole T cell or CD4+ population, and an NK cell population.
Among the T cell populations that can be enriched, isolated and/or selected are populations of
unfractionated T cells, unfractionated CD4+ cells, unfractionated CD8+ cells, and sub-populations
of CD4+ and/or CD8+ T cells, including subpopulations of T cells generated by enrichment for or
depletion of cells of a particular sub-type or based on a particular surface marker expression profile.
For example, among the sub-types of T cells (e.g., CD4 or CD8 T cells) that can be
enriched, isolated and/or selected are those defined by function, activation state, maturity, potential
for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-
specificity, type of antigen receptor, presence in a particular organ or compartment, marker or
cytokine secretion profile, and/or degree of differentiation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+
T cells that can be enriched, isolated and/or selected are naïve T (TN) cells, effector T cells (TEFF),
memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T
(TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-
infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells,
mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg)
cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,
follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
sd-660418
735042000240
In some embodiments, one or more of the T cell populations enriched, isolated
and/or selected from a sample by the provided methods are cells that are positive for (marker+) or
express high levels (markerhigh) of one or more particular markers, such as surface markers, or that
are negative for (marker-) or express relatively low levels (markerlow) of one or more markers. In
some cases, such markers are those that are absent or expressed at relatively low levels on certain
populations of T cells (such as non-memory cells) but are present or expressed at relatively higher
levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells
(such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected
for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27,
CD44, CD127, and/or CD62L and/or depleted of (e.g., negatively selected for) cells that are
positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched
for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25, CD27,
and/or IL7-R (CD127). In some examples, CD8+ T cells are enriched for cells positive for
CD45RO (or negative for CD45RA) and for CD62L.
In some embodiments, a CD4+ T cell population and a CD8+ T cell sub-population,
e.g., a sub-population enriched for central memory (TCM) cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments,
the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic
cells, mast cells, eosinophils, and/or basophils.
2. Samples
The cells and cell populations typically are isolated from a sample, such as a
biological sample, e.g., one obtained from or derived from a subject, such as one having a particular
disease or condition or in need of a cell therapy or to which cell therapy will be administered. In
some aspects, the subject is a human, such as a subject who is a patient in need of a particular
therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated,
processed, and/or engineered. Accordingly, the cells in some embodiments are primary cells, e.g.,
primary human cells. The samples include tissue, fluid, and other samples taken directly from the
subject, as well as samples resulting from one or more processing steps, such as separation,
sd-660418
735042000240
centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation.
The biological sample can be a sample obtained directly from a biological source or a sample that is
processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma,
serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including
processed samples derived therefrom.
In some aspects, the sample is blood or a blood-derived sample, or is or is derived
from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral
blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor,
leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid
tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas,
breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous
and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The
cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat,
non-human primate, and pig.
3. Cell processing, preparation and non-affinity-based separation
In some embodiments, isolation of the cells or populations includes one or more
preparation and/or non-affinity based cell separation steps. In some examples, cells are washed,
centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove
unwanted components, enrich for desired components, lyse or remove cells sensitive to particular
reagents. In some examples, cells are separated based on one or more property, such as density,
adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by
apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells,
monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets,
and in some aspects contains cells other than red blood cells and platelets.
sd-660418
735042000240
In some embodiments, the blood cells collected from the subject are washed, e.g., to
remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent
processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS).
In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all
divalent cations. In some aspects, a washing step is accomplished a semi-automated flow -
through centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the
manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow
filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are
++ ++
resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca /Mg
free PBS. In certain embodiments, components of a blood cell sample are removed and the cells
directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods,
such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and
centrifugation through a Percoll or Ficoll gradient.
4. Separation based on affinity and/or marker profile
In some embodiments, the isolation methods include the separation of different cell
types based on the expression or presence in the cell of one or more specific molecules, such as
surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments,
any known method for separation based on such markers may be used. In some embodiments, the
separation is affinity- or immunoaffinity-based separation. For example, the isolation in some
aspects includes separation of cells and cell populations based on the cells expression or expression
level of one or more markers, typically cell surface markers, for example, by incubation with an
antibody or binding partner that specifically binds to such markers, followed generally by washing
steps and separation of cells having bound the antibody or binding partner, from those cells having
not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having
bound the reagents are retained for further use, and/or negative selection, in which the cells having
not bound to the antibody or binding partner are retained. In some examples, both fractions are
sd-660418
735042000240
retained for further use. In some aspects, negative selection can be particularly useful where no
antibody is available that specifically identifies a cell type in a heterogeneous population, such that
separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100 % enrichment or removal of a particular cell
population or cells expressing a particular marker. For example, positive selection of or enrichment
for cells of a particular type, such as those expressing a marker, refers to increasing the number or
percentage of such cells, but need not result in a complete absence of cells not expressing the
marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as
those expressing a marker, refers to decreasing the number or percentage of such cells, but need not
result in a complete removal of all such cells. For example, in some aspects, a selection of one of
the CD4+ or CD8+ population enriches for said population, either the CD4+ or CD8+ population,
but also can contain some residual or small percentage of other non-selected cells, which can, in
some cases, include the other of the CD4 or CD8 population still being present in the enriched
population.
In some examples, multiple rounds of separation steps are carried out, where the
positively or negatively selected fraction from one step is subjected to another separation step, such
as a subsequent positive or negative selection. In some examples, a single separation step can
deplete cells expressing multiple markers simultaneously, such as by incubating cells with a
plurality of antibodies or binding partners, each specific for a marker targeted for negative selection.
Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a
plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells
+ + +
positive or expressing high levels of one or more surface markers, e.g., CD28 , CD62L , CCR7 ,
+ + + + + +
CD27 , CD127 , CD4 , CD8 , CD45RA , and/or CD45RO T cells, are isolated by positive or
negative selection techniques.
For example, CD3 , CD28 T cells can be positively selected using CD3/CD28
conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
sd-660418
735042000240
In some embodiments, isolation is carried out by enrichment for a particular cell
population by positive selection, or depletion of a particular cell population, by negative selection.
In some embodiments, positive or negative selection is accomplished by incubating cells with one
or more antibodies or other binding agent that specifically bind to one or more surface markers
+ high
expressed or expressed (marker ) at a relatively higher level (marker ) on the positively or
negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative
selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood
cells, such as CD14. In some embodiments, the methods include isolation, selection and/or
enrichment of CD4+ and CD8+ cells. In one example, to enrich for CD4 cells by negative
selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b,
CD16 and HLA-DR. In one example, to enrich for a CD8 population by negative selection is
carried out by depletion of cells expressing CD14 and/or CD45RA. In some aspects, a CD4 or
CD8 selection step, such as positive selection for CD4 and positive selection for CD8, is used to
separate CD4 helper and CD8 cytotoxic T cells. Such selections in some aspects are carried out
simultaneously and in other aspects are carried out sequentially, in either order.
In some aspects, the methods include a first positive selection for CD4+ cells in
which the non-selected cells (CD4- cells) from the first selection are used as the source of cells for a
second positive selection to enrich for CD8+ cells. In some aspects, the methods include a first
positive selection for CD8+ cells in which the non-selected cells (CD8- cells) from the first
selection are used as the source of cells for a second position selection to enrich for CD4+ cells.
Such CD4 and CD8 populations can be further sorted into sub-populations by positive or negative
selection for markers expressed or expressed to a relatively higher degree on one or more naive,
memory, and/or effector T cell subpopulations.
In some embodiments, CD4+ cells are further enriched for or depleted of naïve,
central memory, effector memory and/or central memory stem cells, such as by positive or negative
selection based on surface antigens associated with the respective population. CD4 T helper cells
are sorted into naïve, central memory, and effector cells by identifying cell populations that have
sd-660418
735042000240
cell surface antigens. CD4 lymphocytes can be obtained by standard methods. In some
+ - + + +
embodiments, naive CD4 T lymphocytes are CD45RO , CD45RA , CD62L , CD4 T cells. In
+ + +
some embodiments, central memory CD4 cells are CD62L and CD45RO . In some embodiments,
effector CD4 cells are CD62L and CD45RO
In some embodiments, CD8 cells are further enriched for or depleted of naive,
central memory, effector memory, and/or central memory stem cells, such as by positive or negative
selection based on surface antigens associated with the respective subpopulation. In some
embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such
as to improve long-term survival, expansion, and/or engraftment following administration, which in
some aspects is particularly robust in such sub-populations. See Terakuraet al. (2012) Blood.1:72–
82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining T -
enriched CD8 T cells and CD4 T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L and CD62L- subsets of
CD8 peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8 and/or
CD62L CD8 fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (T ) cells is based on
positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, CD27 and/or CD 127;
in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA
and/or granzyme B.
In some embodiments, the provided methods include isolation, selection and/or
enrichment of CD8+ cells from a sample, such as by positive selection based on surface expression
of CD8. In some embodiments, the methods can further include enriching for central memory T
(T ) cells. In one aspect, the enriched CD8+ cells can be further enriched for central memory T
(TCM) cells by selecting for one or more markers expressed on central memory T (TCM) cells., such
as one or more of CD45RO, CD62L, CCR7, CD28, CD3, CD27 and/or CD 127. The selection can
be performed prior to or subsequent to isolation, selection and/or enrichment of CD4+ cells. Such
selections in some aspects are carried out simultaneously and in other aspects are carried out
sequentially, in either order.
sd-660418
735042000240
In some aspects, the methods include a first positive selection for CD4+ cells in
which the non-selected cells (CD4- cells) from the first selection are used as the source of cells for a
second selection to enrich for CD8+ cells, and the enriched or selected CD8+ cells are used in a
third selection to further enrich for cells expressing one or more markers expressed on central
memory T (TCM) cells., such as by a third selection to enrich for CD45RO+, CD62L+, CCR7+,
CD28+, CD3+, CD27+ and/or CD127+ cells. In some aspects, the methods include a first positive
selection for CD8+ cells in which the non-selected cells (CD8- cells) from the first selection are
used as the source of cells for the second selection to enrich for CD4+ cells, and the enriched or
selected CD8+ cells from the first selection also are used in a third selection to further enrich for
cells expressing one or more markers expressed on central memory T (T ) cells., such as by a third
selection to enrich for CD45RO+, CD62L+, CCR7+, CD28+, CD3+, CD27+ and/or CD127+ cells.
In some aspects, isolation of a CD8 population enriched for T cells is carried out
by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for
cells expressing CD62L. In one aspect, enrichment for central memory T (T ) cells is carried out
starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a
negative selection based on expression of CD14 and CD45RA, and a positive selection based on
CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are
carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection
step used in preparing the CD8 cell population or subpopulation, also is used to generate the CD4
cell population or sub-population, such that both the positive and negative fractions from the CD4-
based separation are retained and used in subsequent steps of the methods, optionally following one
or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is
subjected to selection of CD4 cells, where both the negative and positive fractions are retained.
The negative fraction then is subjected to negative selection based on expression of CD14 and
CD45RA or CD19, and positive selection based on a marker characteristic of central memory T
cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either
order.
sd-660418
735042000240
In some embodiments, the methods of isolating, selecting and/or enriching for cells,
such as by positive or negative selection based on the expression of a cell surface marker or
markers, for example by any of the methods described above, can include immunoaffinity-based
selections. In some embodiments, the immunoaffinity-based selections include contacting a sample
containing cells, such as primary human T cells containing CD4+ and CD8+ cells, with an antibody
or binding partner that specifically binds to the cell surface marker or markers. In some
embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a sphere
or bead, for example microbeads, nanobeads, including agarose, magnetic bead or paramagnetic
beads, to allow for separation of cells for positive and/or negative selection. In some embodiments,
the spheres or beads can be packed into a column to effect immunoaffinity chromatography, in
which a sample containing cells, such as primary human T cells containing CD4+ and CD8+ cells,
is contacted with the matrix of the column and subsequently eluted or released therefrom.
a. Immunoaffinity Beads
For example, in some embodiments, the cells and cell populations are separated or
isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods
in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and
In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).
In some aspects, the sample or composition of cells to be separated is incubated with
small, magnetizable or magnetically responsive material, such as magnetically responsive particles
or microparticles, such as paramagnetic beads . The magnetically responsive material, e.g.,
particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that
specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of
cells that it is desired to separate, e.g., that it is desired to negatively or positively select. Such
beads are known and are commercially available from a variety of sources including, in some
aspects, Dynabeads® (Life Technologies, Carlsbad, CA), MACS® beads (Miltenyi Biotec, San
Diego, CA) or Streptamer® bead reagents (IBA, Germany).
In some embodiments, the magnetic particle or bead comprises a magnetically
responsive material bound to a specific binding member, such as an antibody or other binding
sd-660418
735042000240
partner. There are many well-known magnetically responsive materials used in magnetic separation
methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773,
and in European Patent Specification EP 452342 B, which are hereby incorporated by reference.
Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et
al., U.S. Pat. No. 5,200,084 are other examples.
The incubation generally is carried out under conditions whereby the antibodies or
binding partners, or molecules, such as secondary antibodies or other reagents, which specifically
bind to such antibodies or binding partners, which are attached to the magnetic particle or bead,
specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having
magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet
and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet
are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some
aspects, a combination of positive and negative selection is performed during the same selection
step, where the positive and negative fractions are retained and further processed or subject to
further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary
antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In
certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies
specific for one or more markers. In certain embodiments, the cells, rather than the beads, are
labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody-
or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain
embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated
primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the
cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the
particles are left attached to the cells for administration to a patient. In some embodiments, the
magnetizable or magnetically responsive particles are removed from the cells. Methods for
sd-660418
735042000240
removing magnetizable particles from cells are known and include, e.g., the use of competing non-
labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In
some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell
sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic Activated Cell Sorting (MACS)
systems are capable of high-purity selection of cells having magnetized particles attached thereto.
In certain embodiments, MACS operates in a mode wherein the non-target and target species are
sequentially eluted after the application of the external magnetic field. That is, the cells attached to
magnetized particles are held in place while the unattached species are eluted. Then, after this first
elution step is completed, the species that were trapped in the magnetic field and were prevented
from being eluted are freed in some manner such that they can be eluted and recovered. In certain
embodiments, the non-target cells are labelled and depleted from the heterogeneous population of
cells.
In some embodiments, the affinity-based selection employs Streptamers®, which are
magnetic beads, such as nanobeads or microbeads, for example 1-2 µM that, in some aspects, are
conjugated to a binding partner immunoaffinity reagent, such as an antibody via a streptavidin
mutant, e.g. Strep-Tactin® or Strep-Tactin XT® (see e.g. U.S. Patent No. 6,103,493, International
Published PCT Appl. Nos. WO/2013011011, ). In some embodiments, the
streptavidin mutant is functionalized, coated and/or immobilized on the bead.
In some embodiments, the streptavidin mutant exhibits a higher binding affinity for a
peptide ligand containing the sequence of amino acids set forth in any of SEQ ID NOS:1-6, such as
for example SEQ ID NO:5 and/or SEQ ID NO:6 (e.g. Strep-tag II®), than an unmodified or wild
type streptavidin, such as an unmodified or wild type streptavidin set forth in SEQ ID NO: 11 or
SEQ ID NO:14. In some embodiments, the streptavidin mutant exhibits a binding affinity as an
affinity constant for such peptides that is greater than the binding affinity of wild type streptavidin
for the same peptide by greater than 5-fold, 10-fold, 50-fold, 100-fold, 200-fold or greater.
The streptavidin mutein contains one or more amino acid differences compared to an
unmodified streptavidin, such as a wild type streptavidin or fragment thereof. The term
sd-660418
735042000240
unmodi fied streptavidin re fers to a starting polypeptide to which one or more modifications are
made. In some embodiments, the starting or unmodified polypeptide may be a wild type
polypeptide set forth in SEQ ID NO:11. In some embodiments, the unmodified streptavidin is a
fragment of wild type streptavidin, which is shortened at the N- and/or C-terminus. Such minimal
streptavidins include any that begin N-terminally in the region of amino acid positions 10 to 16 of
SEQ ID NO:11 and terminate C-terminally in the region of amino acid positions 133 to 142 of SEQ
ID NO:11. In some embodiments, the unmodified streptavidin has the sequence of amino acids set
forth in SEQ ID NO:14. In some embodiments, the unmodified streptavidin, such as set forth in
SEQ ID NO:14, can further contain an N-terminal methionine at a position corresponding to Ala13
with numbering as set forth in SEQ ID NO:11. Reference to number of residues in streptavidin
provided herein is with reference to numbering of residues in SEQ ID NO:11.
The term streptavidin mutein, streptavidin mutant or variations thereof, refers to
a streptavidin protein that contains one or more amino acid differences compared to an unmodified
or wild type streptavidin, such as a streptavidin set forth in SEQ ID NO: 11 or SEQ ID NO:14. The
one or more amino acid differences can be amino acid mutations, such as one or more amino acid
replacements (substitutions), insertions or deletions. In some embodiments, a streptavidin mutein
can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid
differences compared to a wild type or unmodified streptavidin. In some embodiments, the amino
acid replacements (substitutions) are conservative or non-conservative mutations. The streptavidin
mutein containing the one or more amino acid differences exhibits a binding affinity as an affinity
4 -1
constant that is greater than 2.7 x 10 M for the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly;
also called Strep-tag®, set forth in SEQ ID NO:5). In some embodiments, the streptavidin mutant
4 -1
exhibits a binding affinity as an affinity constant that is greater than 1.4 x 10 M for the peptide
ligand (Trp Ser His Pro Gln Phe Glu Lys; also called Strep-tag® II, set forth in SEQ ID NO:6). In
some embodiments, binding affinity can be determined by methods known in the art, such as any
described below.
In some embodiments, the streptavidin mutein contains a mutation at one or more
residues 44, 45, 46, and/or 47. In some embodiments, the streptavidin mutant contains residues
sd-660418
735042000240
Val44-Thr45-Ala46-Arg47, such as set forth in exemplary streptavidin muteins set forth in SEQ ID
NO: 12 or SEQ ID NO:15. In some embodiments, the streptavidin mutein contains residues Ile44-
Gly45-AlaArg47, such as set forth in exemplary streptavidin muteins set forth in SEQ ID NO:
13 or 16. In some embodiments, the streptavidin mutein exhibits the sequence of amino acids set
forth in SEQ ID NO: 12, 13, 15 or 16, or a sequence of amino acids that exhibits at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino
acids set forth in SEQ ID NO:12, 13, 15 or 16, and exhibits a binding affinity that is greater than 2.7
4 -1
x 10 M for the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also called Strep-tag®, set forth
4 -1
in SEQ ID NO:5) and/or greater than 1.4 x 10 M for the peptide ligand (Trp Ser His Pro Gln Phe
Glu Lys; also called Strep-tag® II, set forth in SEQ ID NO:6).
In some embodiment, the streptavidin mutein is a mutant as described in
International Published PCT Appl. Nos. . In some embodiments, the streptavidin
mutein contains at least two cysteine residues in the region of amino acid positions 44 to 53 with
reference to amino acid positions set forth in SEQ ID NO:11. In some embodiments, the cysteine
residues are present at positions 45 and 52 to create a disulfide bridge connecting these amino acids.
In such an embodiment, amino acid 44 is typically glycine or alanine and amino acid 46 is typically
alanine or glycine and amino acid 47 is typically arginine. In some embodiments, the streptavidin
mutein contains at least one mutation or amino acid difference in the region of amino acids residues
115 to 121 with reference to amino acid positions set forth in SEQ ID NO:11. In some
embodiments, the streptavidin mutein contains at least one mutation at amino acid position 117, 120
and 121 and/or a deletion of amino acids 118 and 119 and substitution of at least amino acid
position 121.
In some embodiments, a streptavidin mutein can contain any of the above mutations
in any combination, so long as the resulting streptavidin mutein exhibits a binding affinity that is
4 -1
greater than 2.7 x 10 M for the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also called
4 -1
Strep-tag®, set forth in SEQ ID NO:5) and/or greater than 1.4 x 10 M for the peptide ligand (Trp
Ser His Pro Gln Phe Glu Lys; also called Strep-tag® II, set forth in SEQ ID NO:6).
sd-660418
735042000240
In some embodiments, the binding affinity of a streptavidin mutant for a peptide
4 -1 5 -1 5 -1 6 -1 6 -1
ligand binding reagent is greater than 5 x 10 M , 1 x 10 M , 5x 10 M , 1 x 10 M , 5 x 10 M
7 -1 13 -1 12 -1 11 -1
or 1 x 10 M , but generally is less than 1 x 10 M , 1 x 10 M or 1 x 10 M .
In some embodiments, the streptavidin mutant also exhibits binding to other
streptavidin ligands, such as but not limited to, biotin, iminobiotin, lipoic acid, desthiobiotin,
diaminobiotin, HABA (hydroxyazobenzene-benzoic acid) or/and dimethyl-HABA. In some
embodiments, the streptavidin muteins exhibits a binding affinity for another streptavidin ligand,
such as biotin or desthiobiotin, that is greater than the binding affinity of the streptavidin mutein for
the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also called Strep-tag®, set forth in SEQ ID
NO:5) or the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also called Strep-tag® II, set forth in
SEQ ID NO:6).
In some embodiments, the streptavidin mutein is a multimer. Multimers can be
generated using any methods known in the art, such as any described in published U.S. Patent
Application No. US2004/0082012. In some embodiments, oligomers or polymers of muteins can
be prepared by the introduction of carboxyl residues into a polysaccharide, e.g. dextran. In some
aspects, streptavidin muteins then are coupled via primary amino groups of internal lysine residues
and/or the free N-terminus to the carboxyl groups in the dextran backbone using conventional
carbodiimide chemistry in a second step. In some embodiments, the coupling reaction is performed
at a molar ratio of about 60 moles streptavidin mutant per mole of dextran. In some embodiments,
oligomers or polymers of can also be obtained by crosslinking via bifunctional linkers, such as
glutardialdehyde or by other methods known in the art.
In some aspects,, an immunoaffinity bead, such as a Streptamer® or other
immunoaffinity bead, can contain an antibody produced by or derived from a hybridoma as follows:
OKT3 (C D3) , 13B8.2 (C D4), OKT8 (C D8), FRT5 (C D25), DREG56 (CD62L), MEM56
(CD45RA). In some embodiments, any of the above antibodies can contain one or more mutations
within the framework of heavy and light chain variable regions without targeting the highly variable
CDR regions. Exemplary of such antibodies include, in some aspects, anti-CD4 antibodies as
described in U.S. Patent No. 7,482,000 and Bes et al. (2003) J. Biol. Chem., 278:14265-14273. In
sd-660418
735042000240
some embodiments, an antigen-binding fragment, such as a Fab fragment, can be generated from
such antibodies using methods known in the art, such as, in some aspects, amplification of
hypervariable sequences of heavy and light chains and cloning to allow combination with sequences
coding for an appropriate constant domain. In some embodiments, the constant domain is of human
subclass IgG1/. Such antibodies can be carboxy -terminally fused with a peptide streptavidin
binding molecule, such as set forth in SEQ ID NO:10. Exemplary of such antibodies are described
in Stemberget et al. (2102) PLoS One, 7:35798 and International PCT Application No.
WO2013/011011.
In some embodiments, the antibody specifically binding a cell surface marker
associated with or coated on a bead or other surface is a full-length antibody or is an antigen-
binding fragment thereof, including a (Fab) fragments, F(ab') fragments, Fab' fragments, Fv
fragments, variable heavy chain (V ) regions capable of specifically binding the antigen, single
chain antibody fragments, including single chain variable fragments (scFv), and single domain
antibodies (e.g., sdAb, sdFv, nanobody) fragments. In some embodiments, the antibody is a Fab
fragment. In some embodiments, the antibody can be monovalent, bivalent or multivalent. In
some embodiments, the antibody, such as a Fab, is a multimer. In some embodiments, the antibody,
such as a Fab multimer, forms a multivalent complex with the cell surface marker.
In some embodiments, the antibody, such as a Fab, associated with a Streptamer
exhibits a particular kinetic measure of binding affinity (e.g. dissociation constant, K , association
constant K , off-rate or other kinetic parameter of binding affinity). Such measurements can be
determined using any binding assay known to a skilled artisan. In particular examples, an affinity-
based biosensor technology is utilized as a measure of binding affinity. Exemplary biosensor
technologies include, for example, Biacore technologies, BioRad ProteOn, Reichert, GWC
Technologies, IBIS SPIR Imaging, Nomadics SensiQ, Akubio RAPid, ForteBio Octet, IAsys,
Nanofilm and others (see e.g. Rich et al. (2009) Analytical Biochemistry, 386:194-216). In some
embodiments, binding affinity is determined by fluorescence titration or titration calorimetry.
In some embodiments, the antibody, such as a Fab, exhibits a k rate (also called
dissociation rate constant) for the binding to a cell surface marker on a cell that is greater than
sd-660418
735042000240
-4 -1 -4 -1 -4 -1 -4 -1 -4
about 0.5 x 10 sec , about 1 x 10 sec , about 2 x 10 sec , about 3 x 10 sec , about 4 x 10
-1 -4 -1 -3 -1 -3 -1 -3 -1
sec , about 5 x 10 sec , about 1 x 10 sec , about 1.5 x 10 sec , about 2 x 10 sec , about 3 x
-3 -1 -3 -1 -3 -1 -2 -1 -1 -1
sec , about 4 x 10 sec , about 5 x 10 sec , about 1 x 10 sec , or about 5 x 10 sec or
greater. The particular k rate can determine the rate at which the antibody reagent can dissociate
from the its interaction with a cell via its binding to a cell surface marker (see e.g. International
Published PCT Appl. No. WO/2013011011). For example, in some aspects, the k range can be
chosen within a range, depending, for example, on the particular application or use of a selected or
enriched cell, including factors such as the desire to remove the bound antibody from the cell
surface, the time of the cells in culture or incubation, the sensitivity to the cell and other factors. In
-4 -1
one embodiment, an antibody has a high k rate of, for example, greater than 4.0 x 10 sec , so
that, after the disruption of the multivalent binding complexes, most of the antibody can be removed
within one hour, since, in some aspects taking into account the half-life T1/2 of the complex, within
56 minutes the concentration of the complexes is reduced to 25 % of the original concentration,
assuming that rebinding effects can be neglected due to sufficient dilution). In another embodiment,
-4 -1
an antibody with a lower k0ff rate of, for example, 1.0 x 10 sec , the dissociation may take longer,
for example about or approximately 212 min or about 3 and a half hours to remove 75% of the
antibody from the surface.
In some embodiments, the antibody, such as a Fab, exhibits a dissociation constant
(Ka) for the binding to a cell surface marker on a cell that can be the range of about 10 M to about
-8 -2 -9 -2 -9 -
M, or of about 10 M to about 10 M, or of about 10 M to about 0.8 x 10 M, or of about 10
2 -9 -2 -9 -2
M to about 0.6 x 10 M, or of about 10 M to about 0.4 x 10 M, or of about 10 M to about 0.3 x
-9 -2 -9 -2 -9
M, or of about 10 M to about 0.2 x 10 , or of about 10 M to about 0.15 x 10 M, or of about
-2 -10
to about 10 , In some embodiments, the dissociation constant (Ka) for the binding a cell
-7 -10 -7
surface marker on a cell can be in the range of about 10 M to about 10 M, or of about 10 M to
-9 -7 -9 -7 -9
about 0.8 x 10 M, or of about 10 M to about 0.6 x 10 M, of about 10 M to about 0.3 x 10 M,
-7 -10 -7 -9
of 1.1 x 10 M to about 10 M, or of about 1.1 x 10 M to about 0.15 x 10 M, or of about 1.1 x
-7 -9 -7 -9 -
M to about 0.3 x 10 M, or of about 1.1 x 10 M to about 0.6 x 10 M, or of about 1.1 x 10
7 -9
M to about 0.8 x 10 M.
sd-660418
735042000240
In some embodiments, the immunoaffinity reagent, such as antibody, for example a
Fab, is linked, directly or indirectly, to a peptide ligand, such as a peptide ligand capable of binding
to a streptavidin mutant (see e.g. U.S. Patent No. 5,506,121). In some embodiments, such as
peptide contains the sequence of amino acids set forth in any of SEQ ID NOS:1-6. In some
embodiments, the immunoaffinity reagent, such as antibody, for example a Fab, is linked, directly
or indirectly, to a peptide ligand containing the sequence of amino acids set forth in SEQ ID NO:6.
In some embodiments, the immunoaffinity reagent, such as antibody, for example a
Fab, is fused directly or indirectly with a peptide sequence that contains a sequential arrangement of
at least two streptavidin-binding modules, wherein the distance between the two modules is at least
0 and not greater than 50 amino acids, wherein one binding module has 3 to 8 amino acids and
contains at least the sequence His-Pro-Xaa (SEQ ID NO:1), where Xaa is glutamine, asparagine, or
methionine, and wherein the other binding module has the sequence of the same or different
streptavidin peptide ligand, such as set forth in SEQ ID NO:3 (see e.g. International Published PCT
Appl. No. WO02/077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide ligand
fused, directly or indirectly, to the immunoaffinity reagent, such as antibody, for example Fab,
contains a sequence having the formula set forth in any of SEQ ID NO: 7 or 8. In some
embodiments, the peptide ligand has the sequence of amino acids set forth in any of SEQ ID NOS:
9, 10 or 17-19.
Alternatively, other streptavidin-binding peptides known in the art may be used, e.g.
as described by Wilson et al. (Proc.Natl.Acad.Sci.USA 98 (2001), 3750-3755). In some
embodiments, the peptide is fused to the N- and/or C-terminus of the protein.
In some embodiments, the antibody, such as a Fab, fused to a peptide ligand capable
of binding a streptavidin mutant, is contacted with streptavidin-mutant containing beads to coat the
beads with antibody. In some embodiments, the coated beads can be used in enrichment and
selection methods as described herein by contacting such beads with a sample containing cells to be
enriched or selected.
In some embodiments, the bond between the peptide ligand binding partner and
streptavidin mutein binding reagent is reversible. In some embodiments, the bond between the
sd-660418
735042000240
peptide ligand binding partner and streptavidin mutein binding reagent is high, such as described
above, but is less than the binding affinity of the streptavidin binding reagent for biotin or a biotin
analog. Hence, in some embodiments, biotin (Vitamin H) or a biotin analog can be added to
compete for binding to disrupt the binding interaction between the streptavidin mutein binding
reagent on the bead and the peptide ligand binding partner associated with the antibody specifically
bound to a cell marker on the surface. In some embodiments, the interaction can be reversed in the
presence of low concentrations of biotin or analog, such as in the presence of 0.1 mM to 10 mM ,
0.5 mM to 5 mM or 1 mM to 3 mM, such as generally at least or about at least 1 mM or at least 2
mM, for example at or about 2.5 mM. In some embodiments, incubation in the presence of a
competing agent, such as a biotin or biotin analog, releases the bead from the selected cell.
b. Immunoaffinity Chromatography
In some embodiments, the affinity-based selection employs immunoaffinity
chromatography. Immunoaffinity chromatography methods include, in some aspects, one or more
chromatography matrix as described in U.S. Published Patent Appl. No. US2015/0024411. In
some embodiments, the chromatographic method is a fluid chromatography, typically a liquid
chromatography. In some embodiments, the chromatography can be carried out in a flow through
mode in which a fluid sample containing the cells to be isolated is applied, for example, by gravity
flow or by a pump on one end of a column containing the chromatography matrix and in which the
fluid sample exits the column at the other end of the column. In addition, in some aspects, the
chromatography can be carried out in an up a nd down mode in which a fluid sample containing
the cells to be isolated is applied, for example, by a pipette on one end of a column containing the
chromatography matrix packed within a pipette tip and in which the fluid sample enters and exits
the chromatography matrix/pipette tip at the other end of the column. In some embodiments, the
chromatography can also be carried out in a batch mode in which the chromatography material
(stationary phase) is incubated with the sample that contains the cells, for example, under shaking,
rotating or repeated contacting and removal of the fluid sample, for example, by means of a pipette.
In some embodiments, the chromatography matrix is a stationary phase. In some
embodiments, the chromatography is column chromatography. In some embodiments, any suitable
sd-660418
735042000240
chromatography material can be used. In some embodiments, the chromatography matrix has the
form of a solid or semi-solid phase. In some embodiments, the chromatography matrix can include
a polymeric resin or a metal oxide or a metalloid oxide. In some embodiments, the chromatography
matrix is a non-magnetic material or non-magnetizable material. In some embodiments, the
chromatography matrix is a derivatized silica or a crosslinked gel, such as in the form of a natural
polymer, for example a polysaccharide. In some embodiments, the chromatography matrix is an
agarose gel. Agarose gel for use in a chromatography matrix are known in the art and include, in
some aspects, Superflow agarose or a Sepharose material such as Superflow™ Sepharose®,
which are commercially available in different bead and pore sizes. In some embodiments, the
chromatography matrix is a particular cross-linked agarose matrix to which dextran is covalently
bonded, such as any known in the art, for example in some aspects, Sephadex®, Superdex® or
Sephacryl®, which are available in different bead and pore sizes.
In some embodiments, a chromatography matrix is made of a synthetic polymer,
such as polyacrylamide, a styrene-divinylbenzene gel, a copolymer of an acrylate and a diol or of an
acrylamide and a diol, a co-polymer of a polysacchardie and agarose, e.g. a polyacrylamide/agarose
composite, a polysaccharide and N, N -methylenebiscarylamide, or a derivatized silica coupled to a
synthetic or natural polymer.
In some embodiments, the chromatography matrix, such as agarose beads or other
matrix, has a size of at least or about at least 50 µm, 60 µm, 70 µm, 80 µm, 90 µm, 100 µm, 120 µm
or 150 µm or more. The exclusion limit of the size exclusion chromatography matrix is selected to
be below the maximal width of the target cell in a sample, e.g. T cells. In some embodiments, the
volume of the matrix is at least 0.5 mL, 1 mL, 1.5 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8
mL, 9 mL, 10 mL or more. In some embodiments, the chromatography matrix is packed into a
column.
In some embodiments, the chromatography matrix, which is an immunoaffinity
chromatography matrix, includes an affinity reagent, such as an antibody or antigen-binding
fragment, such as Fab, immobilized thereto. The antibody or antigen-binding fragment, such as a
Fab, can be any as described above, including, in some aspects, known antibodies in the art,
sd-660418
735042000240
antibodies having a particular k rate and/or antibodies having a particular dissociation constant
(Ka).
In some embodiments, the affinity reagent, such as antibody or antigen-binding
fragment, such as a Fab, is immobilized. In some embodiments, the immunoaffinity reagent, such
as an antibody or antigen-binding fragment, such as a Fab, is fused or linked to a binding partner
that interacts with a binding reagent immobilized on the matrix. In some embodiments, the binding
capacity of the chromatography matrix is sufficient to adsorb or is capable of adsorbing at least 1 x
107 cells/mL, 5 x 107 cells/mL, 1 x 108 cells/mL, 5 x 108 cells/mL, 1 x 109 cells/ml or more, in
which said cells are cells expressing a cell surface marker specifically recognized by the affinity
reagent, such as antibody or Fab.
In some embodiments, the interaction between the binding reagent and binding
partner forms a reversible bond, so that binding of the antibody to the matrix is reversible. In some
embodiments, the reversible binding can be mediated by a streptavidin mutant binding partner and a
binding reagent immobilized on the matrix that is streptavidin, a streptavidin analog or mutein,
avidin or an avidin analog or mutein.
In some embodiments, reversible binding of the affinity reagent, such as antibody or
antigen-binding fragment, such as Fab is via a peptide ligand binding reagent and streptavidin
mutein interaction, as described above with respect to immunoaffinity beads. In aspects of the
chromatography matrix, the matrix, such as agarose beads or other matrix, is functionalized or
conjugated with a streptavidin mutein, such as any described above, for example any set forth in
SEQ ID NOS: 12, 13, 15 or 16. In some embodiments, the antibody or antigen-binding fragment,
such as a Fab, is fused or linked, directly or indirectly, to a peptide ligand capable of binding to a
streptavidin mutant, such as any described above. In some embodiments, the peptide ligand is any
as described above, such as a peptide containing the sequence of amino acids set forth in any of
SEQ ID NOS:1-10 or 17-19. In some embodiments, the chromatography matrix column is
contacted with such an affinity reagent, such as an antibody or antigen-binding fragment, such as a
Fab to immobilize or reversibly bind the affinity reagent to the column.
sd-660418
735042000240
In some embodiments, the immunoaffinity chromatography matrix can be used in
enrichment and selection methods as described herein by contacting said matrix with a sample
containing cells to be enriched or selected. In some embodiments, the selected cells are eluted or
released from the matrix by disrupting the interaction of the binding partner/binding reagent. In
some embodiments, binding partner/binding reagents is mediated by a peptide ligand and
streptavidin mutant interaction, and the release or selected cells can be effected due to the presence
of a reversible bond. For example, in some embodiments, the bond between the peptide ligand
binding partner and streptavidin mutein binding reagent is high, such as described above, but is less
than the binding affinity of the streptavidin binding reagent for biotin or a biotin analog. Hence, in
some embodiments, biotin (Vitamin H) or a biotin analog can be added to compete for binding to
disrupt the binding interaction between the streptavidin mutein binding reagent on the matrix and
the peptide ligand binding partner associated with the antibody specifically bound to a cell marker
on the surface. In some embodiments, the interaction can be reversed in the presence of low
concentrations of biotin or analog, such as in the presence of 0.1 mM to 10 mM , 0.5 mM to 5 mM
or 1 mM to 3 mM, such as generally at least or about at least 1 mM or at least 2 mM, for example at
or about 2.5 mM. In some embodiments, elution in the presence of a competing agent, such as a
biotin or biotin analog, releases the selected cell from the matrix.
In some embodiments, immunoaffinity chromatography in the provided methods is
performed using at least two chromatography matrix columns that are operably connected, whereby
an affinity or binding agent to one of CD4 or CD8, such as an antibody, e.g. a Fab, is coupled to a
first chromatography matrix in a first selection column and an affinity or binding agent to the other
of CD4 or CD8, such as an antibody, e.g. a Fab, is coupled to a second chromatography matrix in a
second selection column. In some embodiments, the at least two chromatography matrix columns
are present in a closed system or apparatus, such as a closed system or apparatus that is sterile.
In some embodiments, also provided herein is a closed system or apparatus
containing at least two chromatography matrix columns that are operably connected, whereby an
affinity or binding agent to one of CD4 or CD8, such as an antibody, e.g. a Fab, is coupled to a first
chromatography matrix in a first selection column and an affinity or binding agent to the other of
sd-660418
735042000240
CD4 or CD8, such as an antibody, e.g. a Fab, is coupled to a second chromatography matrix in a
second selection column. Exemplary of such systems and methods are depicted in Figure 1A an
Figure 1B, and in Examples.
In some embodiments, the closed system is automated. In some embodiments,
components associated with the system can include an integrated microcomputer, peristaltic pump,
and various valves, such as pinch valves or stop cocks, to control flow of fluid between the various
parts of the system. The integrated computer in some aspects controls all components of the
instrument and directs the system to perform repeated procedures in a standardized sequence. In
some embodiments, the peristaltic pump controls the flow rate throughout the tubing set and,
together with the pinch valves, ensures the controlled flow of buffer through the system.
With reference to Figure 1A and Figure 1B, in some embodiments, the first affinity
matrix 3 containing a first affinity or binding agent in a first selection column 1 is i) operably
coupled to the second affinity matrix 4 containing a second affinity or binding agent in a second
selection column 2 via tubing and a valve 13 so that cells having passed through the first affinity
chromatography matrix and not being bound to a first affinity or binding agent thereon are capable
of being passed into the second affinity matrix and ii) is operably coupled to an output container,
such as a culture vessel 12, to collect selected cells from the first affinity matrix that bound to the
first affinity or binding agent thereon, such as after elution and release of such cells from the first
affinity matrix. In some embodiments, the second affinity matrix 4 containing a second affinity or
binding agent in a second selection column 2 also is operably coupled to the output container, such
as a culture vessel 12, to collect selected cells from the second affinity matrix that bound to the
second affinity or binding agent thereon, such as after elution and release of such cells from the
second affinity matrix. In some embodiments, the second selection column 2 is operably connected
to the output container, such as culture vessel 12, through a removal chamber 9 that contains a
binding reagent, such as a streptavidin mutant, that is able to bind with high affinity, such as greater
-10 -1
than 10 M , to an elution reagent, such as biotin.
In some embodiments, the size, e.g. length and/or diameter, of the first selection
column 1 and second selection column 2 can be the same or different. In some embodiments, the
sd-660418
735042000240
size, e.g. length and/or diameter, of one of the first column 1 or second column 2 is larger than the
size of the other of the columns by at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 5
times, 7 times, 8 times, 9 times, 10 times or more.
In some embodiments, the first affinity matrix 3 containing a first affinity or binding
agent in a first selection column 1 also is operably connected to a third affinity matrix 17 containing
a third affinity or binding agent in a third selection column 15 via tubing and a valve 13, so that
selected cells from the first affinity matrix that bound to the first affinity or binding agent thereon
are capable of being passed into the third affinity matrix, such as after elution and release of such
cells from the first affinity matrix. In some embodiments, the first selection column 1 is operably
connected to the third selection column 15 through a removal chamber 9 that contains a binding
reagent, such as a streptavidin mutant, that is able to bind with high affinity, such as greater than 10
-1
M , to an elution reagent, such as biotin. In some embodiments, the third affinity matrix 17
containing a third affinity or binding agent in a third selection column 15 also is operably coupled to
the output container, such as a culture vessel 12, to collect selected cells from the third affinity
matrix having bound to the third affinity or binding agent thereon (and previously having bound to
the first affinity or binding agent), such as after elution and release of such cells from the third
affinity matrix (and previously from the first affinity matrix). In some embodiments, the third
selection column 15 is operably connected to the output container, such as culture vessel 12,
through a removal chamber 9 that contains a binding reagent, such as a streptavidin mutant, that is
-10 -1
able to bind with high affinity, such as greater than 10 M , to an elution reagent, such as biotin.
In some embodiments, the size, e.g. length and/or diameter, of the first selection
column 1 and third selection column 15 can be the same or different. In some embodiments, the
size, e.g. length and/or diameter, of one of the first column 1 or third column 15 is larger than the
size of the other of the columns by at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 5
times, 7 times, 8 times, 9 times, 10 times or more.
In some embodiments, the first selection column 1 is operably coupled to a storage
reservoir containing cell sample 5, such as via tubing, valves and a pump 8, in order to provide a
cell sample into the first selection column.
sd-660418
735042000240
In some embodiments, the first selection column 1 also is operably coupled to a
washing reservoir 6 containing wash buffer and/or an elution reservoir 7 containing an eluent, such
as through tubing and valves, in order to permit passage of a washing buffer or an elution buffer,
respectively, into the first chromatography matrix in the first selection column. In some
embodiments, due to the operable connection between the first and second selection column, the
washing buffer and/or elution buffer can operably pass through into the second column. In some
embodiments, due to the operable connection between the first and third selection column, the
washing buffer and/or elution buffer can operably pass through into the third selection column.
In some embodiments, the second selection column 2 also is operably coupled to a
washing reservoir 6 containing wash buffer and/or an elution reservoir 7 containing an eluent, such
as through tubing and valves, in order to permit passage of a washing buffer or an elution buffer,
respectively, into the first chromatography matrix in the first selection column.
The washing buffer can be any physiological buffer that is compatible with cells,
such as phosphate buffered saline. In some embodiments, the washing buffer contains bovine
serum albumin, human serum albumin, or recombinant human serum albumin, such as at a
concentration of 0.1% to 5% or 0.2% to 1%, such as or at about 0.5%. In some embodiments, the
eluent is biotin or a biotin analog, such as desbiotin, for example in an amount that is or is about at
least 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, or 5 mM.
In some embodiments, the first affinity matrix 3 in the first selection column 1 is
operably connected through tubing and valves to a first affinity reagent reservoir (e.g. Fab reservoir)
18 containing the first affinity or binding agent, such as an antibody, e.g. a Fab, such as for
immobilization onto the first affinity matrix. In some embodiments, the second affinity matrix 4 in
the second column 2 is operably connected through tubing and valves to a second affinity or
binding agent reservoir (e.g. Fab reservoir) 19 containing the second affinity or binding agent, such
as an antibody, e.g. a Fab, such as for immobilization onto the second affinity matrix. In some
embodiments, the third affinity matrix 17 in the third selection column 15 is operably connected
through tubing and valves to a third affinity or binding agent reservoir (e.g. Fab reservoir) 20
sd-660418
735042000240
containing the third affinity or binding agent, such as an antibody, e.g. a Fab, such as for
immobilization onto the second affinity matrix.
In some embodiments, the first and/or second affinity reagent specifically binds CD4
or CD8, where the first and second affinity reagent are not the same. In some embodiments, the
third affinity reagent specifically binds a marker on naïve, resting or central memory T cells or
specifically binds a marker that is CD45RO, CD62L, CCR7, CD28, CD3, CD27 and/or CD127.
. Enrichments and Ratios of Generated Compositions
In some embodiments, performing a first and second selection using methods as
described above enriches from a sample a first population of cells expressing a first cell surface
marker and a second population of cells expressing a second cell surface marker, respectively. In
particular examples, the first and/or second population of enriched cells can be a population of cells
enriched for CD4+ cells, and the other of the enriched population of cells, i.e. the other of the first
or second population of cells, can be a population enriched for CD8+. As described above, in some
embodiment, a third, fourth or subsequent selection can be performed to enrich for a further sub-
population of cells from a population of cells previously enriched in the first, second or subsequent
enrichments, such as a sub-population of CD4+ cells and/or a sub-population of CD8+ cells..
In some embodiments, the method produces an enriched composition of cells
containing the first and second population of enriched cells, such as a population of cells enriched
for CD4+ cells and a population of cells enriched for CD8+ cells. In some embodiments, the
enriched composition of cells is designated a culture initiation composition and is used in
subsequent processing steps, such as subsequent processing steps involving incubation, stimulation,
activation, engineering and/or formulation of the enriched cells. In some embodiments, subsequent
to the further processing steps, such as processing steps involving incubation, stimulation,
activation, engineering and/or formulation, and output composition is generated that, in some
aspects, can contain genetically engineered cells containing CD4+ cells and CD8+ cells expressing
a genetically engineered antigen receptor.
sd-660418
735042000240
In some embodiments, the enriched compositions of cells are enriched cells from a
starting sample as describe above, in which the number of cells in the starting sample is at least
greater than the desired number of cells in an enriched composition, such as a culture-initiation
composition. In some embodiments, the number of cells in the starting sample is greater by at least
%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000% or more greater
than the desired number of cells in the enriched composition. In some examples, the desired
number of cells in the enriched population, including enriched CD4+ cells, CD8+ cells or sub-
6 6 6 6 6
populations thereof, is at least 1 x 10 cells, 2 x 10 cells, 4 x 10 cells, , 6 x 10 cells 8 x 10 cells, 1
7 7 7 7 7 8 8 8
x 10 cells, 2 x 10 cells, 4 x 10 cells, 6 x 10 cells, 8 x 10 cell, 1 x 10 cells, 2 x 10 cells, 4 x10
8 8 9
cells, 6 x 10 cells, 8 x 10 cells, 1 x 10 cells or greater. In some embodiments, the number of cells
8 8 9 9 9
in the starting sample, is at least 1 x10 cells, 5 x 10 cells, 1 x 10 cells, 2 x 10 cells, 3 x 10 cells,
9 9 9 9 9 9 10
4 x 10 cells, 5 x 10 cells, 6 x 10 cells, 7 x 10 cells, 8 x 10 cells, 9 x 10 cells, 1 x 10 cells or
more.
In some embodiments, the yield of the first and/or second population or sub-
population thereof, in the enriched composition, i.e. the number of enriched cells in the population
or sub-population compared to the number of the same population or sub-population of cells in the
starting sample, is 10% to 100%, such as 20% to 80%, 20% to 60%, 20% to 40%, 40% to 80%,
40% to 60%, or 60%, to 80%,. In some embodiments, the yield of the first and/or second
population of cells or sub-population thereof is less than 70%, less than 60%, less than 50%, less
than 40%, less than 30% or less than 20%.
In some embodiments, the purity of the first and/or second population of cells or sub-
population of cells thereof in the enriched composition, i.e. the percentage of cells positive for the
selected cell surface marker versus total cells in the population of enriched cells, is at least 90%,
91%, 92%, 93%, 94%, and is generally at least 95%, 96%, 97%, 98%, 99% or greater.
In some embodiments, the enriched composition of cells, such as a culture-initiation
composition, contains a ratio of CD4+ cells to CD8+ cells at a culture-initiation ratio. The culture-
initiation ratio is the ratio or number of cells at which two types of cells or isolated cell populations
are included in a culture-initiating composition, designed to result in the desired output ratio or
sd-660418
735042000240
dose, e.g., ratio or dose for administration to a patient, or within a tolerated error rate or difference
thereof, at the completion of the incubation and/or engineering step or other processing steps and/or
upon thaw and/or just prior to administration to a subject. In embodiments of the methods provided
herein, the first and/or second selections, or selections for sub-populations thereof, can be
performed in a manner to result in a chosen culture-initiation ratio. Exemplary of such methods are
described below and in Examples.
a. Culture-initiation ratios and numbers
In some embodiments, the culture-initiating ratio of CD4 or sub-populations thereof
to CD8 cells or sub-populations thereof is between at or about 10:1 and at or about 1:10, between
at or about 5:1 and at or about 1:5, or between at or about 2:1 and at or about 1:2. In some
embodiments, the culture-initiating ratio of CD4+ cells or sub-populations thereof to CD8+ cells or
sub-populations thereof is at or about 1:1.
In some embodiments, the culture-initiating ratio of CD4+ to CD8+ cells, or sub-
populations thereof, is different than the ratio of CD4+ to CD8+ cells or the sub-populations thereof
in the sample from the subject. In some embodiments, it is reported that the ratio of CD4+ to CD8+
T cells in a sample from subjects, such as a blood sample, is between 1:1 and 14:1 CD4+:CD8+
cells, and generally is between about 1.5:1 and about 2.5:1 CD4+:CD8+ cells. In some
embodiments, the ratio of CD4+ to CD8+ T cells in a sample, such as a blood sample, is about 2:1
CD4+:CD8+ cells In some embodiments, the ratio of CD4+ to CD8+ T cells in a sample, such as a
blood sample, is about 1:1. (See e.g. Amadori, A et al., Nature Med. 1: 1279-1283, 1995;
Chakravarti, A., Nature Med. 1: 1240-1241, 1995; Clementi, M., et al., Hum. Genet. 105: 337-342,
1999.) In some embodiments, a subject ratio is less than 1:1 CD4+:CD8+ cells. (See e.g. Muhonen,
T. J Immunother Emphasis Tumor Immunol. 1994 Jan;15(1):67-73.). In some embodiments, the
culture-initiating ratio of CD4+ to CD8+ cells is at least 10 %, at least 20 %, at least 30 %, at least
40 %, at least 50 %, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least
125%, at least 150, at least 200%, at least 300%, at least 400%, or at least 500% greater or less than
the ratio of CD4+ to CD8+ cells in the sample from the subject.
sd-660418
735042000240
In some embodiments, prior to performing the first and/or second selection, the ratio
of CD4+ to CD8+ T cells in the sample from the subject is determined. Based on the particular
ratio of the CD4+ to CD8+ T cells in the subject, which can vary among subject, the particular
mode of selection can be individualized to the subject, for example by sizing of chromatography
columns or selection of amount or concentration of immunoaffinity reagents, to achieve the desired
or chosen culture-initiating ratio. The relative level or frequency of various cell populations in a
subject can be determined based on assessing surface expression of a marker or markers present on
such populations or sub-populations. A number of well-known methods for assessing expression
level of surface markers or proteins may be used, such as detection by affinity-based methods, e.g.,
immunoaffinity-based methods, e.g., in the context of cell surface proteins, such as by flow
cytometry.
In some contexts, the appropriate culture-initiating ratio for particular cell types can
vary depending on context, e.g., for example, for a particular disease, condition, or prior treatment
of a subject from which cells are derived, and/or a particular antigen-specificity of the cells, relative
representation among cells of a particular type (e.g., CD8 T cells) of various subpopulations, e.g.,
effector versus memory versus naïve cells, and/or one or more conditions under which cells will be
incubated, such as medium, stimulating agents, time of culture, buffers, oxygen content carbon
dioxide content, antigen, cytokine, antibodies, and other components. Thus, it may be that a cell
type which typically or in general is known to proliferate or expand more rapidly than another will
not always have such a property in every context. Thus, in some aspects, the culture-initiation ratio
is determined based on known capacities of cell types in a normal or typical context, coupled with
assessment of phenotypes or states of the cells or subject from which the cells are derived, and/or
empirical evidence.
In some embodiments, the culture-initiation ratios are based on knowledge of one or
more of these features for a particular type of cell known or determined to be in the
concentration. In some embodiments, the ratio of CD4 to CD8 cells in the culture-initiating
composition is greater than or less than 1.5 times, 2, times, 3 times, 4 times, 5 times, 6 times, 7
sd-660418
735042000240
times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80
+ +.
times, 90 times more or less, respectively, than the desired output ratio of CD4 to CD8
In some embodiments, for example, CD4+ cells in some contexts are known to
proliferate or expand to a lesser degree or less rapidly compared with CD8+ cells, when incubated
under certain stimulating conditions. See, e.g., Foulds et al. (2002) J Immunol. 168(4): 1528-1532;
Caggiari et al. (2001) Cytometry. 46(4) 233-237; Hoffman, et al. (2002) Transplantation. 74(6):
836-845; and Rabenstein et al. (2014) J Immunol. Published online before print March 17, 2014,
doi: 10.4049/jimmunol.1302725. Thus, in some examples, the ratio of CD4 to CD8 cells in the
culture-initiating composition is 10 times or 100 times the desired output ratio. For example, if a
1:1 (or 50%/50%) CD4/CD8 output ratio is desired, the CD4 and CD8 populations may in one
example be included in the culture-initiating composition at a ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,
8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1, for example, to account for
differences in expansion rates over a particular period of time. Depending on the differences in
rates or expansion or proliferation of one of the CD4+ or CD8+ cells, or sub-populations thereof,
from the other, a skilled artisan can empirically determine the culture-initiation ratio to achieve a
desired output ratio following under particular conditions of stimulation or activation.
The culture-initiation ratio will not necessarily be identical, or even approximate, the
desired output ratio. For example, if it is desired to administer CD4 and CD8 engineered (e.g.,
CAR-expressing) T cells at or within a certain error of 1:1, the culture-initiation ratio of CD4 and
CD8 cells often is not 1:1. In some embodiments, the culture initiation ratio that results in the
desired output ratio varies depending, e.g., on the source of the cells, the cell types to be cultured,
the patient to whom the cells are to be administered, the subject or subjects from whom the cells
have been isolated or derived, such as what diseases or conditions such a subject has, the disease to
be treated, culture conditions, and other parameters.
In some embodiments, the culture initiation ratio is based on the composition of each
subpopulation of cells. In certain embodiments, the culture initiation ratio is based on the length of
time the populations of cells are culture prior to their being genetically engineered. In some
sd-660418
735042000240
embodiments, the culture initiation ratio is based on the proliferation rate of each population of
cells.
As another aspect, in some embodiments, the methods further include determining a
culture-initiation ratio or number. In some embodiments, the provided methods include methods
and steps for determining appropriate ratios, doses, and numbers of cells, cell types, and populations
of cells. For example, provided are methods for determining ratios of CD4 /CD8 cells,
populations, and/or sub-populations and determining appropriate doses of such cells and sub-types.
In some embodiments, provided are methods for determining appropriate ratios or numbers of cell
types or cell populations to be included in a composition, such as a culture-initiating composition, to
achieve a desired outcome. In some aspects, such ratios or numbers are designed for use in an
incubation or engineering step to achieve a desired output ratio or dose. In some embodiments,
provided are methods for determining desired ratio of the cells, types, or populations, and/or cell
numbers thereof, for administration to a subject or patient.
In some embodiments, the chosen culture initiation ratio is based on the relative
capacity of the different cell types or populations for survival and/or proliferation or expansion rate
of each population of cells or type of cell (such as CD4 versus CD8 cells) in culture when
incubated under such conditions. Thus, in some aspects, proliferation rate, survival, and/or output
ratios are measured or assessed following test incubations, for example, at particular point or points
in time following incubation, and/or following a cryopreservation or freeze step and/or following a
thaw after such procedure, such as just prior to administration, e.g., at the bedside, in order to
determine the optimal ratios for culture initiation. Any of a number of well-known methods for
determination of in vitro or ex vivo cell proliferation rates or survival include flow-cytometry
methods such as labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) or similar
fluorescent dye prior to incubation, followed by assessment of fluorescent intensity by flow
cytometry, and/or assessment of binding of cells to Annexin V or other compound recognizing
markers on or in apoptotic cells and/or uptake of DNA interchelating agents such propidium iodide
or 7AAD, and assessment of uptake and/or cell cycle stages as a measure of proliferation or
apoptosis by flow cytometry.
sd-660418
735042000240
In some embodiments, the culture-initiation ratio is determined by incubating two
isolated populations, e.g., subpopulations, of cells at a range of different ratios in test compositions,
under certain test conditions, and assessing one or more outcomes, such as the output ratio achieved
after a certain period. In some aspects, the conditions are stimulatory conditions, such as those
approximating conditions under which the culture-initiating compositions are to be incubated for
culture and/or engineering steps. For example, the test compositions in some aspects are
administered in the presence of one or more, e.g., each, of the same stimulating agents, media,
buffers, gas content, and/or in the same type of container or vessel, and/or for the same or
approximately the same amount of time as the parameters to be used in the incubation and/or
engineering steps that are to be used in preparing or producing the ultimate composition, such as the
engineered composition for administration.
Exemplary test ratios for the test culture-initiating compositions can include at or
about 90%/10%, 80%/20%, 70%/30%, 60/40%, 50/50%, 40/60%, 30/70%, 20%/80%, and
%/90%, or 0.1:1, 0.5:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:1.1, 1:1.2., 1:1.3, 1:1.4, 1:1.5, or more than at
or about 1:1.5, or at or about 1:0.1, 1:0.4, 1:0.7, 1:0.8, 1:0.8, 1.1:1, 1.2:1, 1.3:1, 1.4:1, or 1.5;1, or
more.
In some contexts, the appropriate culture-initiation ratio of CD4 versus CD8 cells
to achieve a desired CD4:CD8 ratio at the end of production is determined based on the sub-
population of the CD4 and/or CD8 fractions in a particular isolated cell product, such as the
presence and/or percentage of naïve, effector, and various memory compartments are represented in
a particular isolated composition. Such assessment can be by determining the presence or level of
various surface markers on the cells, such as by flow cytometry.
In some embodiments, the culture initiation ratio is based on the phenotype of each
population of cells. In certain embodiments, the culture initiation ratio is based on the culture
conditions (e.g., such as the composition of the media, the presence and/or absence of growth
factors, stimulants, and/or other agents, temperature, aeration conditions, etc.).
In some embodiments of the methods described herein, the culture initiation ratio
produces an output composition that comprises a ratio of CD4 : CD8 cells or a number of such
sd-660418
735042000240
sub-types or total cell number that is within about 10%, about 15%, about 20%, about 25%, about
% about 35%, about 40%, about 45% or about 50% of the desired ratio or dose, including any
range in between these values. In some embodiments of the methods described herein, the culture
initiation ratio produces an output composition having the desired ratio of CD4 :CD8 cells or dose
of such cells at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the time, including any
range between these values. In certain embodiments of the methods, the culture initiation ratio
produces a ratio of CD4 to CD8 cells in the output composition that is within 20 % of the desired
output ratio at least 80 % of the time. In some embodiments of the methods described herein, the
output composition comprises a ratio of CD4 to CD8 cells that is within a tolerated difference of
the desired output ratio, said tolerated difference having been determined as described above, e.g.,
by administering CD4 and CD8 cells to one or more subjects at a plurality of ratios.
b. Output ratios and doses
In some embodiments, the method results an output composition following one or
more processing steps of the culture-initiation composition, such as incubation, stimulation,
activation, engineering and/or formulation of cells. In some embodiments, the method is performed
to achieve or result in a desired ratio of CD4 to CD8 cells, or sub-populations thereof, that is
between at or about 2:1 and at or about 1:5, at or about 2:1 and at or about 1:2, at or about 1.5:1 and
at or about 1:5, at or about 1.5:1 and at or about 1:2, or at or about 1:1 and at or about 1:2. In some
embodiments, the desired output ratio of CD4 to CD8 cells in the output composition is 1:1 or is
about 1:1.
In some embodiments, the method produces or generates an output composition,
such as a composition containing genetically engineered CD4+ T cells and CD8+ T cells or sub-
populations thereof, in which the output ratio of CD4+ to CD8+ cells in the composition, or sub-
populations thereof, is between at or about 2:1 and at or about 1:5, at or about 2:1 and at or about
1:2, at or about 1.5:1 and at or about 1:5, at or about 1.5:1 and at or about 1:2, or at or about 1:1 and
at or about 1:2. In some embodiments, the method produces or generates an output composition
containing an output ratio of CD4 to CD8 cells that is 1:1 or is about 1:1.
sd-660418
735042000240
In some embodiments, the output ratio of CD4+ to CD8+ T cells is a ratio that is
desired as part of a dosage of T cells for immunotherapy, such as in connection with methods of
adoptive immunotherapy.
In some embodiments, desired dosages, such as desired cell numbers and/or desired
output ratios of the cell types or populations (e.g., ratios optimal for therapeutic administration to a
patient), are determined. In some embodiments, the methods include steps for determining tolerated
error or tolerated difference from a desired output or administration ratio or dose, i.e., the margin of
error by which the ratio in a given composition, e.g., engineered composition, can vary from a
desired output ratio, and still achieve a desired outcome, such as an acceptable degree of safety in a
subject or patient, or efficacy in treating a particular disease or condition or other therapeutic effect.
In some embodiments, the desired dose, ratio and/or the tolerated error depends on
the disease or condition to be treated, subject, source of cells, such as whether the cells are from a
subject having a particular condition or disease and whether the cells are for autologous or
allogeneic transplant, e.g., whether they are isolated from a subject who also is to receive the cells
in adoptive cell therapy and/or whether the subject has received or is receiving another treatment
and/or the identity of such treatment. In some embodiments, the ratio, number, and/or tolerated
difference or error depends on one or more other property of the cells, such as proliferation rate,
survival capacity, expression of particular markers or secretion of factors, such as cytokines, or
particular sub-populations isolated prior to incubation and engineering steps. In some examples, the
desired ratio and/or the tolerated error or difference can vary depending on the age, sex, health,
and/or weight of the subject, on biomarkers as an indication of disease trait, on treatments to be co-
administered or having previously been administered to the subject.
In some embodiments, the desired ratio, dose, and/or tolerated error is determined by
administering various test compositions, each containing the cell types or populations of interest at
different ratios or different numbers to a test subject, followed by assessment of one or more
outcome or parameter, such as a parameter indicative of safety, therapeutic efficacy, in vivo
concentration or localization of the cells, and/or other desired outcome.
sd-660418
735042000240
The test subject in some embodiments is a non-human animal, such as a normal
animal or an animal model for disease, such as the disease or condition to be treated by
administration of the cells. In some embodiments, the various test ratios for administration to test
subjects are at or about 90%/10%, 80%/20%, 70%/30%, 60/40%, 50/50%, 40/60%, 30/70%,
%/80%, and 10%/90%, e.g., expressed as percentage of one cell type or sub-type (e.g., CD4 T
cell or sub-type thereof) and another cell type (e.g., CD8 T cell or NK cell or sub-type thereof), and
interim values. The ratios can be expressed as relative percentages or in any other format, such as
ratios of at or about 0.1:1, 0.5:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:1.1, 1:1.2., 1:1.3, 1:1.4, 1:1.5, or more
than at or about 1:1.5, or at or about 1:0.1, 1:0.4, 1:0.7, 1:0.8, 1:0.8, 1.1:1, 1.2:1, 1.3:1, 1.4:1, or
1.5;1, or more, including any range in between these values. In some embodiments, the test subject
is a human. In some embodiments, the desired ratio is the average, mean, or median ratio among
test subjects with a particular optimal effect. In some aspects, the desired ratio is a ratio that
achieved some optimal balance of safety versus efficacy. In some aspects, the desired ratio or dose
is a ratio or dose that achieves the highest efficacy of all test ratios or doses, while still maintaining
a threshold degree of safety. In some aspects, the desired ratio or dose is a ratio or dose that
achieves the highest degree of safety while maintaining a threshold degree of efficacy or within a
range of efficacy. In some cases, the optimal ratio or dose is expressed as a range, such as between
4 9 5 6
1:1 and 1:2 of one cell type to another or between 10 and 10 or between 10 and 10 cells per kg
body weight.
In some embodiments, the tolerated error is determined based on the deviation from
the desired average test subjects are monitored to assess the therapeutic efficacy and/or safety of
each percentage combination. In some embodiments, the tolerated error will be within about 1%,
about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about
%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in
between these ranges.
6. Exemplary Methods of Selecting or Enriching Cells
a. Single Process Stream and/or Simultaneous Selection Using Immunomagnetic
Beads
sd-660418
735042000240
In some embodiments, the separation and/or steps is carried out using
immunomagnetic beads. In some embodiments, a cell sample containing CD4+ and CD8+ cells,
such as a primary human T cell sample, is contacted with magnetic beads containing a first
immunoaffinity reagent that binds to CD4 or CD8 and magnetic beads containing a second
immunoaffinity reagent that binds to the other of the CD4 or CD8. The separation and/or steps can
occur simultaneously and/or sequentially.
In some embodiments, contacting the cells with the magnetic beads is performed
simultaneously, whereby enrichment of cells containing the surface markers CD4 and CD8 also is
performed simultaneously. In some such aspects, the method includes contacting cells of a sample
containing primary human T cells with a first immunoaffinity reagent that specifically binds to CD4
and a second immunoaffinity reagent that specifically binds to CD8 in an incubation composition,
under conditions whereby the immunoaffinity reagents specifically bind to CD4 and CD8
molecules, respectively, on the surface of cells in the sample, and recovering cells bound to the first
and/or the second immunoaffinity reagent, thereby generating an enriched composition including
CD4+ cells and CD8+ cells at a culture-initiating ratio.
In some embodiments, the first and/or second immunoaffinity reagent are present in
the incubation composition at a sub-optimal yield concentration, whereby the enriched composition
contains less than 70% of the total CD4+ cells in the incubation composition and/or less than 70%
of the CD8+ cells in the incubation composition, thereby producing a composition enriched for
CD4+ and CD8+ T cells.
In some embodiments, the suboptimal yield concentration of the affinity reagent is a
concentration below a concentration used or required to achieve an optimal or maximal yield of
bound cells in a given selection or enrichment involving incubating cells with the reagent and
recovering or separating cells having bound to the reagent (yield, for example, being the number
of the cells so-recovered or selected compared to the total number of cells in the incubation that are
targeted by the reagent or to which the reagent is specific or that have a marker for which the
reagent is specific and capable of binding). The suboptimal yield concentration generally is a
concentration or amount of the reagent that in such process or step achieves no more than 70 %
sd-660418
735042000240
yield of bound cells, upon recovery of the cells having bound to the reagent. In some embodiments,
no more than at or about 50 %, 45 %, 40 %, 30 %, or 25 % yield is achieved by the suboptimal
concentration. The concentration may be expressed in terms of number or mass of particles or
surfaces per cell and/or number of mass or molecules of agent (e.g., antibody, such as antibody
fragment) per cell.
For example, in some embodiments, the suboptimal yield concentration is less than
9 9 9
at or about 30 M agent (e.g., antibody) per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells, per 4 x
9 9 9 9 9
cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
incubation composition. In some embodiments, the suboptimal yield concentration is less than at or
9 9 9 9
about 30 M age nt (e.g., antibody) per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells, per 4 x 10 cells,
9 9 9 9
per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the incubation
composition; in some embodiments, the suboptimal yield concentration is less than at or about 20
9 9 9 9 9
M agent (e.g., antibody) per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells, per 4 x 10 cells, per 5 x 10
9 9 9
cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the incubation composition; in
some embodiments, the suboptimal yield concentration is less than at or about 10 M agent (e.g.,
9 9 9 9 9
antibody) per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells, per 4 x 10 cells, per 5 x 10 cells, per 10 x
9 9 9
cells, per 15 x 10 cells, or per 20 x 10 cells in the incubation composition; in some
embodiments, the suboptimal yield concentration is less than at or about 15 M agent (e.g.,
9 9 9 9 9
antibody) per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells, per 4 x 10 cells, per 5 x 10 cells, per 10 x
9 9 9
cells, per 15 x 10 cells, or per 20 x 10 cells in the incubation composition; in some
embodiments, the suboptimal yield concentration is less than at or about 10 M agent (e.g.,
9 9 9 9 9
antibody) per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells, per 4 x 10 cells, per 5 x 10 cells, per 10 x
9 9 9
cells, per 15 x 10 cells, or per 20 x 10 cells in the incubation composition; in some
embodiments, the suboptimal yield concentration is less than at or about 5 M agent (e.g., antibody)
9 9 9 9 9 9
per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells, per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells,
per 15 x 10 cells, or per 20 x 10 cells in the incubation composition; in some embodiments, the
suboptimal yield concentration is less than at or about 1 M agent (e.g., antibody) per 1 x 10 , per 2
9 9 9 9 9 9
x 10 cells, per 3 x 10 cells, per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells,
sd-660418
735042000240
or per 20 x 10 cells in the incubation composition; in some embodiments, the suboptimal yield
concentration is less than at or about 0.5 M agent (e.g., antibody) per 1 x 10 , per 2 x 10 cells, per
9 9 9 9 9
3 x 10 cells, per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x
cells in the incubation composition; in some embodiments, the suboptimal yield concentration is
9 9 9
less than at or about 0.2 M agent (e.g., antibody) per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells, per
9 9 9 9 9
4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
incubation composition.
In some embodiments, the suboptimal yield concentration is less than at or about 15
9 9 9
mg beads, particles, surface, or total regent, per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells, per 4 x
9 9 9 9 9
cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
incubation composition; in some embodiments, the suboptimal yield concentration is less than at or
9 9 9
about 10 mg beads, particles, surface, or total regent, per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells,
9 9 9 9 9
per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
incubation composition; in some embodiments, the suboptimal yield concentration is less than at or
9 9 9
about 5 mg beads, particles, surface, or total regent, per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells,
9 9 9 9 9
per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
incubation composition; in some embodiments, the suboptimal yield concentration is less than at or
9 9 9
about 4 mg beads, particles, surface, or total regent, per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells,
9 9 9 9 9
per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
incubation composition; in some embodiments, the suboptimal yield concentration is less than at or
9 9 9
about 3 mg beads, particles, surface, or total regent, per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells,
9 9 9 9 9
per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
incubation composition; in some embodiments, the suboptimal yield concentration is less than at or
9 9 9
about 2 mg beads, particles, surface, or total regent, per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells,
9 9 9 9 9
per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
incubation composition; in some embodiments, the suboptimal yield concentration is less than at or
9 9 9
about 1 mg beads, particles, surface, or total regent, per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells,
9 9 9 9 9
per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
sd-660418
735042000240
incubation composition; in some embodiments, the suboptimal yield concentration is less than at or
9 9 9
about 0.5 mg beads, particles, surface, or total regent, per 1 x 10 , per 2 x 10 cells, per 3 x 10 cells,
9 9 9 9 9
per 4 x 10 cells, per 5 x 10 cells, per 10 x 10 cells, per 15 x 10 cells, or per 20 x 10 cells in the
incubation composition.
In some embodiments, e.g., when operating in a suboptimal yield concentration for
each or one or more of two or more selection reagents with affinity to two or more markers or cells,
one or more of such reagents is used at a concentration that is higher than one or more of the other
such reagent(s), in order to bias the ratio of the cell type recognized by that reagent as compared to
the cell type(s) recognized by the other(s). For example, the reagent specifically binding to the
marker for which it is desired to bias the ratio may be included at a concentration (e.g., agent or
mass per cells) that is increased by half, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more,
compared to other(s), depending on how much it is desired to increase the ratio.
In some embodiments, employing suboptimal yield concentration to bias one or both
populations of cells can achieve a desired or chosen culture-initiation ratio. In some embodiments,
the selection is performed from a sample, which is a sample containing CD4+ and CD8+ cells,
9 9 9 9
containing high numbers of cells, such as at least 1 x 10 cells, 2 x 10 cells, 3 x 10 cells, 4 x 10
9 9 9 9 10 10 10
cells, 5 x 10 cells, 6 x 10 cells, 7 x 10 cells, 8 x 10 cells, 1 x 10 cells, 2 x 10 cells, 3 x 10
10
cells, 4 x 10 cells, 5 x 10 cells or more. In some embodiments, the high number of cells is
sufficient to ensure saturation of the immunoaffinity reagents in the sample to cells expressing a
marker, such as CD4 or CD8, in which the reagent specifically binds.
In some embodiments, when operating in the suboptimal range and/or with enough
cells to achieve saturation of reagents, the amount of immunoaffinity reagent is proportional to the
approximate yield of enriched cells. In one embodiment, to achieve a culture-initiation ratio of
about or approximately 1:1 of CD4+ cells to CD8+ cells, selection of CD4+ cells and CD8+ cells
can be performed with the same, or about the same, suboptimal yield concentration of
immunoaffinity reagents to CD4 and CD8, respectively. In another exemplary embodiment, to
achieve a culture-initiation ratio of about or approximately 2:1 of CD4+ cells to CD8+ cells,
selection of CD4+ cells can be performed with a suboptimal yield concentration of an
sd-660418
735042000240
immunoaffinity reagent to CD4 that is about or approximately two times greater than the
suboptimal yield concentration of an immunoaffinity reagent to CD8. It is within the level of a
skilled artisan to empirically select or choose an appropriate amount or concentration of
immunoaffinity reagents depending on the desired or chosen culture-initiating ratio of the generated
composition containing enriched or selected cells in view of the above exemplification.
In some embodiments, the separation and/or steps is carried out using magnetic
beads in which immunoaffinity reagents are reversibly bound, such as via a peptide ligand
interaction with a streptavidin mutein as described above. Exemplary of such magnetic beads are
Streptamers®. In some embodiments, the separation and/or steps is carried out using magnetic
beads, such as those commercially available from Miltenyi Biotec.
In some aspects, the separation and/or other steps is carried out for automated
separation of cells on a clinical-scale level in a closed and sterile system. Components can include
an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves.
The integrated computer in some aspects controls all components of the instrument and directs the
system to perform repeated procedures in a standardized sequence. The magnetic separation unit in
some aspects includes a movable permanent magnet and a holder for the selection column. The
peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves,
ensures the controlled flow of buffer through the system and continual suspension of cells. In some
aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotic).
In some embodiments, the automated separation, such as using the CliniMACS
system, in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile,
non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the
cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing
set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set
consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are
for single use only. After initiation of the separation program, the system automatically applies the
cell sample onto the separation column. Labelled cells are retained within the column, while
unlabeled cells are removed by a series of washing steps. In some embodiments, the cell
sd-660418
735042000240
populations for use with the methods described herein are unlabeled and are not retained in the
column. In some embodiments, the cell populations for use with the methods described herein are
labeled and are retained in the column. In some embodiments, the cell populations for use with the
methods described herein are eluted from the column after removal of the magnetic field, and are
collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using a system
equipped with a cell processing unit that permits automated washing and fractionation of cells by
centrifugation. In some aspects, the separation and/or other steps are carried out using the
CliniMACS Prodigy system (Miltenyi Biotec). A system with a cell processing unit can also
include an onboard camera and image recognition software that determines the optimal cell
fractionation endpoint by discerning the macroscopic layers of the source cell product. For
example, peripheral blood is automatically separated into erythrocytes, white blood cells and
plasma layers. A cell processing system, such as the CliniMACS Prodigy system, can also include
an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell
differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for
the sterile removal and replenishment of media and cells can be monitored using an integrated
microscope. See, e.g., Klebanoff et al.(2012) J Immunother. 35(9): 651–660, Terakuraet al. (2012)
Blood.1:72–82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched
(or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried
in a fluidic stream. In some embodiments, a cell population described herein is collected and
enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell
population described herein is collected and enriched (or depleted) by use of
microelectromechanical systems (MEMS) chips in combination with a FACS-based detection
system (see, e.g., , Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al.
(2008) J Biophoton. 1(5):355–376. In both cases, cells can be labeled with multiple markers,
allowing for the isolation of well-defined T cell subsets at high purity.
sd-660418
735042000240
In some embodiments, the antibodies or binding partners are labeled with one or
more detectable marker, to facilitate separation for positive and/or negative selection. For example,
separation may be based on binding to fluorescently labeled antibodies. In some examples,
separation of cells based on binding of antibodies or other binding partners specific for one or more
cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting
(FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS)
chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for
positive and negative selection based on multiple markers simultaneously.
b. Single Process Stream and/or Sequential Selections Using Immunoaffinity
Chromatography
In some embodiments, the first selection or enrichment of a population of cells and
the second selection and/or enrichment of a population of cells are performed using
immunoaffinity-based reagents that include at least a first and second affinity chromatography
matrix, respectively, having immobilized thereon an antibody. In some embodiments, one or both
of the first and/or second selection can employ a plurality of affinity chromatography matrices
and/or antibodies, whereby the plurality of matrices and/or antibodies employed for the same
selection, i.e. the first selection or the second selection, are serially connected. In some
embodiments, the affinity chromatography matrix or matrices employed in a first and/or second
selection adsorbs or is capable of selecting or enriching at least about 50 x 10 cells/mL, 100 x 10
cells/mL, 200 x 10 cells/mL or 400 x 10 cells/mL. In some embodiments, the adsorption capacity
can be modulated based on the diameter and/or length of the column. In some embodiments, the
culture-initiating ratio of the selected or enriched composition is achieved by choosing a sufficient
amount of matrix and/or at a sufficient relative amount to achieve the culture-initiating ratio
assuming based on, for example, the adsorption capacity of the column or columns for selecting
cells.
In one exemplary embodiment, the adsorption capacity of the matrix or matrices is
the same between the first and second selection, e.g. is or is about 1 x 10 cells/mL for both,
whereby enrichment or selection of cells in the first selection and second selection results in a
sd-660418
735042000240
composition containing a CD4+ cells to CD8+ cells at a culture-initiating ratio of or about 1:1. In
another exemplary embodiment, the adsorption capacity of the matrix or matrices used in one of the
first selection or second selection is at least 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold,
7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold or greater than the adsorption capacity of the matrix or
matrices used in the other of the first selection or second selection, thereby resulting in a culture-
initiation ratio in which cells selected with the greater adsorption capacity, e.g. CD4+ cells or CD8+
cells, are present in the culture initiating ratio in an amount that is at least 1.5-fold, 2.0-fold, 3.0-
fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold or greater than the other cell
population. It is within the level of a skilled artisan to select or choose an appropriate volume,
diameter or number of affinity matrix chromatography columns for the first and/or second selection
depending on the desired or chosen culture-initiating ratio of the generated composition containing
enriched or selected cells.
Exemplary processes for carrying out selections by the provided methods are set
forth in Example 2. In some embodiments, such processes achieve a desired culture-initiation ratio
in an enriched or generated composition.
In some embodiments, the first and/or second selection in the provided methods
includes first enriching for one of CD4+ or CD8+ cells, and then enriching for a sub-population of
cells based on, for example, surface expression of a marker expressed on resting, naïve or central
memory T cells, e.g. a marker that is CD28, CD62L, CCR7, CD127 or CD27. In some
embodiments, the first and/or second selection includes enriching for CD8+ cells, said selection
further comprises enriching for central memory T (TCM) cells, where the other of the first and/or
second selection includes enriching for CD4+ cells. In some embodiment, the methods are
performed to enrich or select for CD4+ cells and to enrich or select for a sub-population of cells that
are CD8+/CD28+, CD8+/CD62L+, CD8+/CCR7+, CD8+/CD127+ or CD8+/CD27+. In some
embodiments, the first selection includes enriching for CD8+ cells and the second selection includes
enriching for CD4+ cells, where the first selection, which includes cells enriched for CD8+ cells,
further includes enriching for central memory T (T ) cells or enriching for cells that expresses a
marker that is CD28, CD62L, CCR7, CD127 or CD27, thereby generating a composition, such as a
sd-660418
735042000240
culture-initiation composition, containing CD4+ cells and CD8+ enriched for central memory T
(TCM) cells or a cell expressing a marker that is CD28, CD62L, CCR7, CD127 or CD27. In some
embodiments, the first selection includes enriching for CD4+ cells and the second selection includes
enriching for CD8+ cells, wherein the second selection, which includes cells enriched for CD8+
cells, further includes enriching for central memory T (TCM) cells or enriching for cells that
expresses a marker that is CD28, CD62L, CCR7, CD127 or CD27, thereby generating a
composition, such as a culture-initiation composition, containing CD4+ cells and CD8+ enriched
for central memory T (TCM) cells or a cell expressing a marker that is CD28, CD62L, CCR7, CD127
or CD27.
In some such embodiments involving a further enrichment of a sub-population of
cells, to achieve the culture-initiating ratio of CD4+ cells or a sub-population thereof to CD8+ cells
or a sub-population thereof, the adsorption capacity of a column matrix or matrices is adjusted to
account for differences in the frequency of a sub-population, i.e. CD4+ or CD8+ cells enriched for
resting, naïve, central memory cells or cells expressing a marker that is CD28, CD62L, CCR7,
CD127 or CD27, compared to the frequency of cells of the respective CD4+ or CD8+ parent
population in the starting sample from the subject. The relative level or frequency of various cell
populations in a subject can be determined based on assessing surface expression of a marker or
markers present on such populations or sub-populations. A number of well-known methods for
assessing expression level of surface markers or proteins may be used, such as detection by affinity-
based methods, e.g., immunoaffinity-based methods, e.g., in the context of cell surface proteins,
such as by flow cytometry.
In an exemplary embodiment, a sample is enriched for CD4+ and CD8+/CD62L+
cells to yield a CD4+ to CD8+ ratio of 1:1. In this exemplary embodiment, the first selection can
include enriching for CD8+ cells using a column with an adsorption capacity adjusted for the
relative frequencies of CD4+ cells to CD8+/CD62L+ cells known to be present in the sample, or
using ratios generally estimated to be in such samples. For example, the CD62L+ subpopulation of
CD8+ cells collected from a human subject can sometimes be about 25% of the total CD8+ T cell
fraction. See e.g. Maldonado, Arthritis Res Ther. 2003; 5(2): R91–R96. In such an embodiment,
sd-660418
735042000240
the columns can be arrayed to collect 4-fold more CD8+ cells than CD4+ cells in order to generate a
1:1 culture initiation ratio of the CD4+ cells and the CD8+ sub-population further containing
CD62L+ cells. Thus, assuming a similar adsorption capacity and efficiency for each selection
column, the CD8+ column can be about or approximately 4-fold larger than the CD4 selection
column or the CD62L selection column. The size of the columns can also be adjusted for expected
yield. For example, if each column is only 80% efficient, the size of each column can be adjusted
to account for the efficiency of each subsequent selection.
For example a desired or chosen culture initiation composition can be one that
contains 200 x 10 CD4+ cells and 200 x 10 CD8+/CD62L+ cells. In this example, assuming the
ratios presented above, the sample to be enriched can contain at least or about 200 x 10 CD4+ cells
and at least or about 800 x 10 CD8+ cells, approximately 25% of which (200 x 10 ) may also be
CD62L+. Assuming that each 2 mL of selection matrix can enrich for about or approximately 200
x 10 cells, a CD8 selection column with 8 mL of selection matrix can bind about or approximately
800 x 10 CD8+ cells while the flow-through passes to the CD4 selection column. A CD4 selection
column with 2 mL of selection matrix can bind about or approximately 200 x 10 CD4+ cells. The
CD8+ cells can be further enriched for CD62L by eluting the CD8+ cells into a CD62L selection
column. In this exemplary embodiment, the CD62L selection column can contain 2 mL of selection
matrix, thus enriching for about or approximately 200 x 10 CD8+/CD62L+ cells. The CD4 and
CD8/CD62L columns can be eluted into a culture vessel, yielding the initiation culture composition
or a composition having about or approximately a 1:1 culture-initiation ratio.
In another exemplary embodiment, a sample is enriched for CD4+ and
CD8+/CCR7+ cells to yield a CD4+ to CD8+ ratio of 1:1. In this exemplary embodiment, the first
selection can include enriching for CD8+ cells using a column with an adsorption capacity adjusted
for the relative frequencies of CD4+ cells to CD8+/CCR7+ cells known to be present in the sample,
or using ratios generally estimated to be in such samples. For example, the CCR7+ subpopulation
of CD8+ cells collected from a human subject can sometimes be about 60% of the total CD8+ T cell
fraction. See e.g. Chen, Blood. 2001 Jul 1;98(1):156-64. The columns can be arrayed to collect
3 -fold more CD8+ cells than CD4+ cells in order to generate a 1:1 culture initiation ratio.
sd-660418
735042000240
Assuming a similar adsorption capacity and efficiency for each selection column, the CD8+ column
can be about or approximately 3 -fold larger than the CD4 selection column or the CCR7 selection
column. The size of the columns can also be adjusted for expected yield. For example, if each
column is only 80% efficient, the size of each column can be adjusted to account for the efficiency
of each subsequent selection.
For example, a desired initiation culture can contain 200 x 10 CD4+ cells and 200 x
CD8+/CCR7+ cells. In this example, assuming the ratios presented above, the sample to be
enriched can contain at least 200 x 10 CD4+ cells and at least or about 6.6 x 10 CD8+ cells,
approximately 60% of which (200 x 10 ) may also be CCR7+. Assuming that each 2 mL of
selection matrix can enrich for 200 x 10 cells, a CD8 selection column with 3mL of selection
matrix can bind about or approximately 6.6x10 CD8+ cells while the flow-through passes to the
CD4 selection column. A CD4 selection column with 2 mL of selection matrix can bind about or
approximately 200 x 10 CD4+ cells. The CD8+ cells can be further enriched for CD62L by eluting
the CD8+ cells into a CCR7 selection column. In this exemplary embodiment, the CCR7 selection
column can contain 1mL of selection matrix, thus enriching for about or approximately 200 x 10
CD8+/CCR7+ cells. The CD4 and CCCR7 columns can be eluted into a culture vessel, yielding the
initiation culture containing about or approximately 200 x 10 CD4+ cells and about or
approximately 200 x 10 CD8+/CCR7+ cells, or a 1:1 culture-initiation ratio.
It is within the level of a skilled artisan to empirically select or choose an appropriate
volume, diameter or number of affinity matrix chromatography columns for the first and/or second
and/or third selection depending on the desired or chosen culture-initiating ratio of the generated
composition containing enriched or selected cells, the expected frequency of each sub-population,
the varying efficiencies of each selection column and other factors within the level of the skilled
artisan and in view of the above exemplification.
B. Incubation of isolated cells
In some embodiments, the provided methods include one or more of various steps
for incubating isolated cells and cell populations, such as populations isolated according to the
sd-660418
735042000240
methods herein, such as steps for incubating an isolated CD4+ T cell population, e.g.,
unfractionated CD4+ T cell population or subpopulation(s) thereof, and an isolated CD8+ T cell
population, e.g., isolated unfractionated CD8+ T cell population or subpopulation(s) thereof. In
some embodiments, the cell populations are incubated in a culture-initiating composition.
The plurality of isolated cell populations, e.g., the CD4+ and CD8+ cell populations
(e.g., unfractionated or subpopulations thereof) are generally incubated with the cell populations
combined in a culture-initiating composition in the same culture vessel, such as the same unit,
chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture
or cultivating cells.
In some aspects, the cell populations or cell types are present in the culture-initiating
composition at a culture-initiating ratio, e.g., ratio of CD4 and CD8 cells, designed to achieve a
particular desired output ratio, or a ratio that is within a certain range of tolerated error of such a
desired output ratio, following the incubation and/or engineering steps, or designed to do so a
certain percentage of the time. The output ratio, for example, can be a ratio optimal for achieving
one or more therapeutic effects upon administration to a patient, e.g., via adoptive cell therapy. In
some aspects, the culture-initiating ratio is determined empirically, e.g., using a determination
method as described herein, for example, to determine the optimal culture-initiating ratio for
achieving a desired output ratio in a particular context.
The incubation steps can include culture, cultivation, stimulation, activation,
propagation, including by incubation in the presence of stimulating conditions, for example,
conditions designed to induce proliferation, expansion, activation, and/or survival of cells in the
population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for
the introduction of a genetically engineered antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen
content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or
stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins,
recombinant soluble receptors, and any other agents designed to activate the cells. In one example,
the stimulating conditions include one or more agent, e.g., ligand, which turns on or initiates
sd-660418
735042000240
TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as
those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, anti-
4-1BB, for example, bound to solid support such as a bead, and/or one or more cytokines.
Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti
CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml).
Optionally, the expansion method may further comprise the step of adding IL-2 and/or IL-15 and/or
IL-7 and/or IL-21 to the culture medium (e.g., wherein the concentration of IL-2 is at least about 10
units/m l).
In some aspects, incubation is carried out in accordance with techniques such as
those described in US Patent No. 6,040,1 77 to Riddell et al., Klebanoff et al.(2012) J Immunother.
(9): 651–660, Terakuraet al. (2012) Blood.1:72–82, and/or Wang et al. (2012) J Immunother.
(9):689-701.
In some embodiments, the cell populations, such as CD4 and CD8 populations or
subpopulations, are expanded by adding to the culture-initiating composition feeder cells, such as
non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population
of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in
the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to
expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise
gamma- irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with
gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the
feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the
growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least
about 30 degrees, and generally at or about 37 degrees Celsius. In some embodiments, a
temperature shift is effected during culture, such as from 37 degrees Celsius to 35 degrees Celsius.
Optionally, the incubation may further comprise adding non-dividing EBV-transformed
lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of
sd-660418
735042000240
about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable
amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, populations of CD4 and CD8 that are antigen specific can be
obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example,
antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T
cells from infected subjects and stimulating the cells in vitro with the same antigen. Naive T cells
may also be used.
Interim assessment and adjustment
In some embodiments, the methods include assessment and/or adjustment of the cells
or composition containing the cells, at a time subsequent to the initiation of the incubation or
culture, such as at a time during the incubation. Assessment can include taking one or more
measurements of a composition or vessel containing the cells, such as assessing cells for
proliferation rate, degree of survival, phenotype, e.g., expression of one or more surface or
intracellular markers, such as proteins or polynucleotides, and/or assessing the composition or
vessel for temperature, media component(s), oxygen or carbon dioxide content, and/or presence or
absence or amount or relative amount of one or more factors, agents, components, and/or cell types,
including subtypes. Assessment in some embodiments includes determining an intermediate ratio
of a plurality, e.g., two cell types, such as CD4 and CD8 T cells, in the composition or vessel
being incubated. In some aspects, the assessment is performed in an automated fashion, for
example, using a device as described herein, and/or is set ahead of time to be carried out at certain
time-points during incubation. In some aspects, the outcome of the assessment, such as a
determined interim ratio of two types of cells, indicates that an adjustment should be made, such as
addition or removal of one or more cell types.
Adjustment can include adjusting any cell culture factor or parameter, such as
temperature, length (time) for which incubation or a step thereof will be carried out (duration of
incubation), replenishment, addition and/or removal of one or more components in the composition
being incubated, e.g., media or buffer or components thereof, agents, e.g., nutrients, amino acids,
antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding
sd-660418
735042000240
partners, fusion proteins, recombinant soluble receptors, or cells or cell types or populations of
cells. In some aspects, the removal or addition of various components or other adjustment is carried
out in an automated fashion, for example, using a device or system as described herein. In some
embodiments, the system is programmed such that an adjustment is automatically initiated based on
a certain readout from an interim assessment. For example, in some cases, a system or device is
programmed to carry out one or more assessments at a particular time; the system or device in such
cases can be further programmed such that a particular outcome of such an assessment, such as a
particular ratio of one cell type to another, initiates a particular adjustment, such as addition of one
or more of the cell types.
In some aspects, the adjustment is carried out by addition or removal in a way that
does not disrupt a closed environment containing the cells and compositions, such as by input
and/or removal valves, designed to add or remove components while maintaining sterility, such as
in one or more device or system as described herein.
In a particular embodiment, an interim ratio of CD4 to CD8 T cells is assessed
during the incubation period. In some embodiments, the assessment is carried out after 1, 2, 3, 4, 5,
6, or 7 days, such as between 3 and 5 days, and/or at a time point in which all cells are in or
suspected of being in cell cycle. In some aspects, the interim ratio so-determined indicates that
+ + +
CD4 or CD8 T cells, such as cells of the isolated population of CD4 T cells or isolated
population of CD8 T cells (e.g., sub-population, such as central memory CD8 T cells), should be
added to or enriched in the culture vessel or to the composition being incubated. Thus, in some
aspects, the assessment is followed by such an addition or removal, typically an addition. In some
aspects, multiple assessments and possible adjustments are carried out over the course of
incubation, e.g., in an iterative fashion.
In some embodiments, where cells are engineered, e.g., to introduce a genetically
engineered antigen receptor, the incubation in the presence of one or more stimulating agents
continues during the engineering phase.
In some embodiments, the cells are incubated for at or about 1, 2, 3, 4, 5, 6, 7, 8, 9,
, 11, 12, 13, or 14 days, either in total or prior to engineering.
sd-660418
735042000240
C. Engineering, engineered antigen receptors, and engineered cells
In some embodiments, the methods include genetic engineering of the isolated
and/or incubated cells, such as to introduce into the cells recombinant genes for expression of
molecules, such as receptors, e.g., antigen receptors, useful in the context of adoptive therapy.
Among the genes for introduction are those to improve the efficacy of therapy, such
as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for
selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to
improve safety, for example, by making the cell susceptible to negative selection in vivo as
described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene
Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by
Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a
dominant positive selectable marker with a negative selectable marker. This can be carried out in
accordance with known techniques (see, e.g., Riddell et al., US Patent No. 6,040,177, at columns
14-17) or variations thereof that will be apparent to those skilled in the art based upon the present
disclosure.
The engineering generally includes introduction of gene or genes for expression of a
genetically engineered antigen receptor. Among such antigen receptors are genetically engineered
T cell receptors (TCRs) and components thereof, and functional non-TCR antigen receptors, such as
chimeric antigen receptors (CAR).
The antigen receptor in some embodiments specifically binds to a ligand on a cell or
disease to be targeted, such as a cancer or other disease or condition, including those described
herein for targeting with the provided methods and compositions. Exemplary antigens are orphan
tyrosine kinase receptor RORl, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, and
hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR,
EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-
MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule,
MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100,
oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific
sd-660418
735042000240
antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-
Met, GD-2, and MAGE A3 and/or biotinylated molecules, and/or molecules expressed by HIV,
HCV, HBV or other pathogens.
Antigen Receptors
In one embodiment, the engineered antigen receptors are CARs. The CARs
generally include genetically engineered receptors including an extracellular ligand binding domain
linked to one or more intracellular signaling components. Such molecules typically mimic or
approximate a signal through a natural antigen receptor and/or signal through such a receptor in
combination with a costimulatory receptor.
In some embodiments, CARs are constructed with specificity for a particular marker,
such as a marker expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer
marker. This is achieved in some aspects by inclusion in the extracellular portion of the CAR one
or more antigen binding molecule, such as one or more antigen-binding fragment, domain, or
portion, or one or more antibody variable domains, and/or antibody molecules. In some
embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule,
such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable
light (VL) chains of a monoclonal antibody (mAb).
In some embodiments, the CAR comprises an antibody heavy chain domain that
specifically binds a cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a
cancer cell, such as any of the target antigens described herein or known in the art.
In some embodiments, the tumor antigen or cell surface molecule is a polypeptide.
In some embodiments, the tumor antigen or cell surface molecule is selectively expressed or
overexpressed on tumor cells as compared to non-tumor cells of the same tissue.
In some embodiments, the CAR binds a pathogen-specific antigen. In some
embodiments, the CAR is are specific for viral antigens (such as HIV, HCV, HBV, etc.), bacterial
antigens, and/or parasitic antigens.
In some aspects, the antigen-specific binding, or recognition component is linked to
one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR
sd-660418
735042000240
includes a transmembrane domain fused to the extracellular domain of the CAR. In one
embodiment, the transmembrane domain that naturally is associated with one of the domains in the
CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid
substitution to avoid binding of such domains to the transmembrane domains of the same or
different surface membrane proteins to minimize interactions with other members of the receptor
complex.
The transmembrane domain in some embodiments is derived either from a natural or
from a synthetic source. Where the source is natural, the domain in some aspects is derived from
any membrane-bound or transmembrane protein. Transmembrane regions include those derived
from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-
cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37,
CD64, CD80, CD86, CD 134, CD137, CD 154. Alternatively the transmembrane domain in some
embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises
predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of
phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane
domain.
In some embodiments, a short oligo- or polypeptide linker, for example, a linker of
between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-
serine doublet, is present and forms a linkage between the transmembrane domain and the
cytoplasmic signaling domain of the CAR.
The CAR generally includes intracellular signaling component or components. In
some embodiments, the CAR includes an intracellular component of the TCR complex, such as a
TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in
some aspects, the antigen binding molecule is linked to one or more cell signaling modules. In
some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular
signaling domains, and/or other CD transmembrane domains. In some embodiments, the CAR
further includes a portion of one or more additional molecules such as Fc receptor , CD8, CD4,
sd-660418
735042000240
CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between
CD3-zeta (CD3-) o r Fc receptor and CD8, CD4, CD25 or CD16.
In some embodiments, upon ligation of the CAR, the cytoplasmic domain or
intracellular signaling domain of the CAR activates at least one of the normal effector functions of
the immune cell, e.g., T cell engineered to express the cell. For example, in some contexts, the
CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion
of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling
domain of an antigen receptor component or costimulatory molecule. Such truncated portion in
some aspects is used in place of an intact immunostimulatory chain, for example, if it transduces the
effector function signal. In some embodiments, the intracellular signaling domain or domains
include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of
co-receptors that in the natural context act in concert with such receptor to initiate signal
transduction following antigen receptor engagement, and/or any derivative or variant of such
molecules, and/or any synthetic sequence that has the same functional capability.
In the context of a natural TCR, full activation generally requires not only signaling
through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full
activation, a component for generating secondary or co-stimulatory signal is also included in the
CAR. T cell activation is in some aspects described as being mediated by two classes of
cytoplasmic signaling sequences: those that initiate antigen- dependent primary activation through
the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent
manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling
sequences). In some aspects, the CAR includes one or both of such signaling components.
Primary cytoplasmic signaling sequences can in some aspects regulate primary
activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary
cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs
which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of
ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta,
FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and
sd-660418
735042000240
CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a
cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane
portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS.
In certain embodiments, the intracellular signaling domain comprises a CD28
transmembrane and signaling domain linked to a CD3 intracellular domain. In some embodiments,
the intracellular signaling domain comprises a chimeric CD28 and CD137 co-stimulatory domains,
linked to a CD3 intracellular domain. In some embodiments, a CAR can also include a transduction
marker (e.g., tEGFR). In some embodiments, the intracellular signaling domain of the CD8
cytotoxic T cells is the same as the intracellular signaling domain of the CD4 helper T cells. In
some embodiments, the intracellular signaling domain of the CD8 cytotoxic T cells is different
than the intracellular signaling domain of the CD4 helper T cells.
In some embodiments, the CAR encompasses two or more costimulatory domain
combined with an activation domain, e.g., primary activation domain, in the cytoplasmic portion.
One example is a receptor including intracellular components of CD3-zeta, CD28, and 4-1BB.
CARs and production and introduction thereof can include those described, for
example, by published patent disclosures WO200014257, US6451995, US2002131960,
US7446190, US8252592, EP2537416, US2013287748, and WO2013126726, and/or those
described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388–398; Davila et al. (2013) PLoS
ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al.,
Cancer, 2012 March 18(2): 160-75.
In some embodiments, the T cells are modified with a recombinant T cell receptor.
In some embodiments, the recombinant TCR is specific for an antigen, generally an antigen present
on a target cell, such as a tumor-specific antigen, an antigen expressed on a particular cell type
associated with an autoimmune or inflammatory disease, or an antigen derived from a viral
pathogen or a bacterial pathogen.
In some embodiments, the T cells are engineered to express T-cell receptors (TCRs)
cloned from naturally occurring T cells. In some embodiments, a high-affinity T cell clone for a
sd-660418
735042000240
target antigen (e.g., a cancer antigen) is identified, isolated from a patient, and introduced into the
cells. In some embodiments, the TCR clone for a target antigen has been generated in transgenic
mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or
HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15:169–180 and
Cohen et al. (2005) J Immunol. 175:5799–5808. In some embodiments, phage display is used to
isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390–1395
and Li (2005) Nat Biotechnol. 23:349–354.
In some embodiments, after the T-cell clone is obtained, the TCR alpha and beta
chains are isolated and cloned into a gene expression vector. In some embodiments, the TCR alpha
and beta genes are linked via a picornavirus 2A ribosomal skip peptide so that both chains are
coexpression. In some embodiments, genetic transfer of the TCR is accomplished via retroviral or
lentiviral vectors, or via transposons (see, e.g., Baum et al. (2006) Molecular Therapy: The Journal
of the American Society of Gene Therapy. 13:1050–1063; Frecha et al. (2010) Molecular Therapy:
The Journal of the American Society of Gene Therapy. 18:1748–1757; an Hackett et al. (2010)
Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:674–683.
In some embodiments, gene transfer is accomplished by first stimulating T cell
growth and the activated cells are then transduced and expanded in culture to numbers sufficient for
clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine
or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene
segments that cause the cells to be susceptible to negative selection in vivo, such as upon
administration in adoptive immunotherapy. For example in some aspects, the cells are engineered
so that they can be eliminated as a result of a change in the in vivo condition of the patient to which
they are administered. The negative selectable phenotype may result from the insertion of a gene
that confers sensitivity to an administered agent, for example, a compound. Negative selectable
genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al.,
Cell II :223, I977) which confers ganciclovir sensitivity; the cellular hypoxanthine
sd-660418
735042000240
phosphribosyltransferase (HPRT)gene, the cellular adenine phosphoribosyltransferase (APRT)
gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some aspects, the cells further are engineered to promote expression of cytokines,
such as proinflammatory cytokines, e.g., IL-2, IL-12, IL-7, IL-15, IL-21.
Introduction of the genetically engineered components
Various methods for the introduction of genetically engineered components, e.g.,
antigen receptors, e.g., CARs, are well known and may be used with the provided methods and
compositions. Exemplary methods include those for transfer of nucleic acids encoding the
receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and
electroporation.
In some embodiments, recombinant nucleic acids are transferred into cells using
recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40),
adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are
transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-
retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25;
Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl
Acids 2, e93; Park et al., Trends Biotechnol. 2011 November; 29(11): 550–557.
In some embodiments, the retroviral vector has a long terminal repeat sequence
(LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV),
myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem
cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most
retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses
include those derived from any avian or mammalian cell source. The retroviruses typically are
amphotropic, meaning that they are capable of infecting host cells of several species, including
humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env
sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos.
,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A.
D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al.
sd-660418
735042000240
(1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin.
Genet. Develop. 3:102-109.
Methods of lentiviral transduction are known. Exemplary methods are described in,
e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637–
1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003)
Blood. 102(2): 497-505.
In some embodiments, recombinant nucleic acids are transferred into T cells via
electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al.
(2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are
transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-
437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol
506: 115-126). Other methods of introducing and expressing genetic material in immune cells
include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular
Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated
transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-
777 (1990)); and strontium phosphate DNA co- precipitation (Brash et al., Mol. Cell Biol., 7: 2031-
2034 (1987)).
In some embodiments, the same CAR is introduced into each of CD4 and CD8 T
lymphocytes. In some embodiments, a different CAR is introduced into each of CD4 and CD8 T
lymphocytes. In some embodiments, the CAR in each of these populations has an antigen binding
molecule that specifically binds to the same antigen. In some embodiments, the CAR in each of
these populations binds to a different molecule. In some embodiments, the CAR in each of these
populations has cellular signaling modules that differ. In some embodiments each of the CD4 or
CD8 T lymphocytes have been sorted in to naive, central memory, effector memory or effector
cells prior to transduction.
In other embodiments, the cells, e.g., T cells, are not engineered to express
recombinant receptors, but rather include naturally occurring antigen receptors specific for desired
antigens, such as tumor-infiltrating lymphocytes and/or T cells cultured in vitro or ex vivo, e.g.,
sd-660418
735042000240
during the incubation step(s), to promote expansion of cells having particular antigen specificity.
For example, in some embodiments, the cells are produced for adoptive cell therapy by isolation of
tumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes (TIL). The direct targeting of
human tumors using autologous tumor infiltrating lymphocytes can in some cases mediate tumor
regression (see Rosenberg SA, et al.(1988) N Engl J Med. 319:1676–1680). In some embodiments,
lymphocytes are extracted from resected tumors . In some embodiments, such lymphocytes are
expanded in vitro. In some embodiments, such lymphocytes are cultured with lymphokines (e.g.,
IL-2). In some embodiments, such lymphocytes mediate specific lysis of autologous tumor cells
but not allogeneic tumor or autologous normal cells.
In some aspects, the incubation and/or engineering steps and/or the methods
generally result in a desired output ratio (or a ratio that is within a tolerated error or difference from
such a ratio), or do so within a certain percentage of the time that the methods are performed.
D. Cryopreservation
In some embodiments, the provided methods include steps for freezing, e.g.,
cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some
embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent,
monocytes in the cell population. In some embodiments, the cells are suspended in a freezing
solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known
freezing solutions and parameters in some aspects may be used. One example involves using PBS
containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media.
This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and
4%, respectively. The cells are then frozen to 80° C. at a rate of 1° per minute and stored in the
vapor phase of a liquid nitrogen storage tank.
II. Kits and systems
Also provided are systems, apparatuses, and kits useful in performing the provided
methods. In one example, a single system is provided which carries out one or more of the
isolation, cell preparation, separation, e.g., separation based on density, affinity, sensitivity to one or
sd-660418
735042000240
more components, or other property, washing, processing, incubation, culture, and/or formulation
steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed
or sterile environment, for example, to minimize error, user handling and/or contamination. In one
example, the system is a system as described in International Patent Application, Publication
Number WO2009/072003, or US 20110003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of
the isolation, processing, engineering, and formulation steps in an integrated or self-contained
system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus
includes a computer and/or computer program in communication with the system or apparatus,
which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the
processing, isolation, engineering, and formulation steps.
Also provided are kits for carrying out the provided methods. In some embodiments,
the kits include antibodies or other binding partners, generally coupled to solid supports, for the
isolation, e.g., for immunoaffinity-based separation steps, of the methods.
In some embodiments, the kit comprises antibodies for positive and negative
selection, bound to magnetic beads. In one embodiment, the kit comprises instructions to carry out
selection starting with a sample, such as a PBMC sample, by selecting based on expression of a first
surface marker, recognized by one or more of the antibodies provided with the kit, retaining both
positive and negative fractions. In some aspects, the instructions further include instructions to
carry out one or more additional selection steps, starting with the positive and/or negative fractions
derived therefrom, for example, while maintaining the compositions in a contained environment
and/or in the same separation vessel.
In one embodiment, a kit comprises anti-CD4, anti-CD14, anti-CD45RA, anti-CD14,
and anti-CD62L antibodies, bound to magnetic beads. In one embodiment, the kit comprises
instructions to carry out selection starting with a sample, such as a PBMC sample, by selecting
based on CD4 expression, retaining both positive and negative fractions, and on the negative
fraction, further subjecting the fraction to a negative selection using the anti-CD14, anti-CD45RA
antibodies, and a positive selection using the anti-CD62L antibody, in either order. Alternatively,
sd-660418
735042000240
the components and instructions are adjusted according to any of the separation embodiments
described herein.
In some embodiments, the kit further includes instructions to transfer the cells of the
populations isolated by the selection steps to a culture, cultivation, or processing vessel, while
maintaining the cells in a self-contained system. In some embodiments, the kit includes instructions
to transfer the different isolated cells at a particular ratio.
III. Cells, compositions, and methods of administration
Also provided are cells, cell populations, and compositions (including
pharmaceutical and therapeutic compositions) containing the cells and populations, produced by the
provided methods. Also provided are methods, e.g., therapeutic methods for administrating the
cells and compositions to subjects, e.g., patients.
Provided are methods of administering the cells, populations, and compositions, and
uses of such cells, populations, and compositions to treat or prevent diseases, conditions, and
disorders, including cancers. In some embodiments, the cells, populations, and compositions are
administered to a subject or patient having the particular disease or condition to be treated, e.g., via
adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells and
compositions prepared by the provided methods, such as engineered compositions and end-of-
production compositions following incubation and/or other processing steps, are administered to a
subject, such as a subject having or at risk for the disease or condition. In some aspects, the
methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by
lessening tumor burden in a cancer expressing an antigen recognized by an engineered T cell.
Methods for administration of cells for adoptive cell therapy are known and may be
used in connection with the provided methods and compositions. For example, adoptive T cell
therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to
Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol.
8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al.
(2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
sd-660418
735042000240
In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by
autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who
is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects,
the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following
isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by
allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other
than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In
such embodiments, the cells then are administered to a different subject, e.g., a second subject, of
the same species. In some embodiments, the first and second subjects are genetically identical. In
some embodiments, the first and second subjects are genetically similar. In some embodiments, the
second subject expresses the same HLA class or supertype as the first subject.
In some embodiments, the subject, e.g., patient, to whom the cells, cell populations,
or compositions are administered is a mammal, typically a primate, such as a human. In some
embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any
suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some
embodiments, the subject is a non-primate mammal, such as a rodent.
Also provided are pharmaceutical compositions for use in such methods.
Among the diseases, conditions, and disorders for treatment with the provided
compositions, cells, methods and uses are tumors, including solid tumors, hematologic
malignancies, and melanomas, and infectious diseases, such as infection with a virus or other
pathogen, e.g., HIV, HCV, HBV, CMV, and parasitic disease. In some embodiments, the disease or
condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease. Such diseases
include but are not limited to leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL),
ALL, non-Hodgkins lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular
lymphoma, mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the
colon, lung, liver, breast, prostate, ovarian, skin (including melanoma), bone, and brain cancer,
ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin
sd-660418
735042000240
lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma,
medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma.
In some embodiments, the disease or condition is an infectious disease or condition,
such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency,
Cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In some
embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such
as arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE),
inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves disease,
Crohns disease multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
In some embodiments, the antigen associated with the disease or disorder is selected
from the group consisting of orphan tyrosine kinase receptor RORl, tEGFR, Her2, Ll-CAM, CD19,
CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24,
CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal
acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light
chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA,
NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2,
carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor,
progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3 and/or biotinylated
molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.
In some embodiments, the cells and compositions are administered to a subject in the
form of a pharmaceutical composition, such as a composition comprising the cells or cell
populations and a pharmaceutically acceptable carrier or excipient. The pharmaceutical
compositions in some embodiments additionally comprise other pharmaceutically active agents or
drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin,
daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel,
rituximab, vinblastine, vincristine, etc. In some embodiments, the agents are administered in the
form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid
addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic,
sd-660418
735042000240
phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic,
citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for
example, p-toluenesulphonic acid.
The choice of carrier will in the pharmaceutical composition is determined in part by
the particular engineered CAR or TCR, vector, or cells expressing the CAR or TCR, as well as by
the particular method used to administer the vector or host cells expressing the CAR. Accordingly,
there are a variety of suitable formulations. For example, the pharmaceutical composition can
contain preservatives. Suitable preservatives may include, for example, methylparaben,
propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or
more preservatives is used. The preservative or mixtures thereof are typically present in an amount
of about 0.0001% to about 2% by weight of the total composition.
In addition, buffering agents in some aspects are included in the composition.
Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid,
potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more
buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount
of about 0.001 % to about 4% by weight of the total composition. Methods for preparing
administrable pharmaceutical compositions are known. Exemplary methods are described in more
detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &
Wilkins; 21st ed. (May 1 , 2005).
In certain embodiments, the pharmaceutical composition is formulated as an
inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve
to target the host cells (e.g., T-cells or NK cells) to a particular tissue. Many methods are available
for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys.
Bioeng., 9: 467 (1980), and U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
In some embodiments, the pharmaceutical composition employs time-released,
delayed release, and/or sustained release delivery systems, such that the delivery of the composition
occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types
of release delivery systems are available and known to those of ordinary skill in the art. Such
sd-660418
735042000240
systems in some aspects can avoid repeated administrations of the composition, thereby increasing
convenience to the subject and the physician.
In some embodiments, the he pharmaceutical composition comprises the cells or cell
populations in an amount that is effective to treat or prevent the disease or condition, such as a
therapeutically effective or prophylactically effective amount. Thus, in some embodiments, the
methods of administration include administration of the cells and populations at effective amounts.
Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of
treated subjects. For repeated administrations over several days or longer, depending on the
condition, the treatment is repeated until a desired suppression of disease symptoms occurs.
However, other dosage regimens may be useful and can be determined. The desired dosage can be
delivered by a single bolus administration of the composition, by multiple bolus administrations of
the composition, or by continuous infusion administration of the composition.
In some embodiments, the cells are administered at a desired dosage, which in some
aspects includes a desired dose or number of cells or cell type(s) and/or a desired ratio of cell types.
Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per
kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to
CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or
number per kg of body weight) of cells in the individual populations or of individual cell types. In
some embodiments, the dosage is based on a combination of such features, such as a desired
number of total cells, desired ratio, and desired total number of cells in the individual populations.
In some embodiments, the populations or sub-types of cells, such as CD8 and CD4
T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a
desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired
number of cells per unit of body weight of the subject to whom the cells are administered, e.g.,
cells/kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum
number of cells per unit of body weight. In some aspects, among the total cells, administered at the
desired dose, the individual populations or sub-types are present at or near a desired output ratio
(such as CD4 to CD8 ratio), e.g., within a certain tolerated difference or error of such a ratio.
sd-660418
735042000240
In some embodiments, the cells are administered at or within a tolerated difference
of a desired dose of one or more of the individual populations or sub-types of cells, such as a
desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some aspects, the desired dose
is a desired number of cells of the sub-type or population, or a desired number of such cells per unit
of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects,
the desired dose is at or above a minimum number of cells of the population or sub-type, or
minimum number of cells of the population or sub-type per unit of body weight.
Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells
and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual
sub-types or sub-populations. Thus, in some embodiments, the dosage is based on a desired fixed
or minimum dose of T cells and a desired ratio of CD4 to CD8 cells, and/or is based on a desired
fixed or minimum dose of CD4 and/or CD8 cells.
In certain embodiments, the cells, or individual populations of sub-types of cells, are
administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1
million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million
cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about
40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to
about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells,
about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about
billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion
cells, or a range defined by any two of the foregoing values), and in some cases about 100 million
cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350
million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900
million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in
between these ranges.
In some embodiments, the dose of total cells and/or dose of individual sub-
populations of cells is within a range of between at or about 10 and at or about 10 cells/kilograms
(kg) body weight, such as between 10 and 10 cells / kg body weight, for example, at or about 1 x
sd-660418
735042000240
5 5 6
cells/kg, 1.5 x 10 cells/kg, 2 x 10 cells/kg, or 1 x 10 cells/kg body weight. For example, in
some embodiments, the cells are administered at, or within a certain range of error of, between at or
4 9 5 6
about 10 and at or about 10 T cells/kilograms (kg) body weight, such as between 10 and 10 T
5 5
cells / kg body weight, for example, at or about 1 x 10 T cells/kg, 1.5 x 10 T cells/kg, 2 x 10 T
cells/kg, or 1 x 10 T cells/kg body weight.
In some embodiments, the cells are administered at or within a certain range of error
4 9 + +
of between at or about 10 and at or about 10 CD4 and/or CD8 cells/kilograms (kg) body weight,
6 + +
such as between 10 and 10 CD4 and/or CD8 cells / kg body weight, for example, at or about 1 x
+ + 5 + + 5 + +
CD4 and/or CD8 cells/kg, 1.5 x 10 CD4 and/or CD8 cells/kg, 2 x 10 CD4 and/or CD8
6 + +
cells/kg, or 1 x 10 CD4 and/or CD8 cells/kg body weight.
In some embodiments, the cells are administered at or within a certain range of error
6 6 6 6
of, greater than, and/or at least about 1 x 10 , about 2.5 x 10 , about 5 x 10 , about 7.5 x 10 , or
6 + 6 6 6
about 9 x 10 CD4 cells, and/or at least about 1 x 10 , about 2.5 x 10 , about 5 x 10 , about 7.5 x
6 6 6 6 6
, or about 9 x 10 CD8+ cells, and/or at least about 1 x 10 , about 2.5 x 10 , about 5 x 10 , about
7.5 x 10 , or about 9 x 10 T cells. In some embodiments, the cells are administered at or within a
8 12 10 11
certain range of error of between about 10 and 10 or between about 10 and 10 T cells,
8 12 10 11 + 8
between about 10 and 10 or between about 10 and 10 CD4 cells, and/or between about 10
12 10 11 +
and 10 or between about 10 and 10 CD8 cells.
In some embodiments, the cells are administered at or within a tolerated range of a
desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-
types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios. for
example, in some embodiments, the desired ratio (e.g., ratio of CD4 to CD8 cells) is between at or
about 5:1 and at or about 5:1 (or greater than about 1:5 and less than about 5:1), or between at or
about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between at
or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1, such as at or
about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1,
1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.
In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about
sd-660418
735042000240
%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50% of the desired ratio, including any value in between these ranges.
The cell populations and compositions in some embodiments are administered to a
subject using standard administration techniques, including oral, intravenous, intraperitoneal,
subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository
administration. In some embodiments, the cell populations are administered parenterally. The term
"parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and
intraperitoneal administration. In some embodiments, the cell populations are administered to a
subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous
injection.
The cell populations obtained using the methods described herein in some
embodiments are co-administered with one or more additional therapeutic agents or in connection
with another therapeutic intervention, either simultaneously or sequentially in any order. In some
contexts, the cells are co-administered with another therapy sufficiently close in time such that the
cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In
some embodiments, the cell populations are administered prior to the one or more additional
therapeutic agents. In some embodiments, the cell populations are administered after to the one or
more additional therapeutic agents.
Following administration of the cells, the biological activity of the engineered cell
populations in some embodiments is measured, e.g., by any of a number of known methods.
Parameters to assess include specific binding of an engineered or natural T cell or other immune
cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain
embodiments, the ability of the engineered cells to destroy target cells can be measured using any
suitable method known in the art, such as cytotoxicity assays described in, for example,
Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological
Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is
measured by assaying expression and/or secretion of one or more cytokines, such as CD 107a,
sd-660418
735042000240
IFN, IL -2, and TNF. In some aspects the biological activity is measured by assessing clinical
outcome, such as reduction in tumor burden or load.
In certain embodiments, the engineered cells are further modified in any number of
ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered
CAR or TCR expressed by the population can be conjugated either directly or indirectly through a
linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to
targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1
(1995), and U.S. Patent 5,087,616.
IV. Definitions
As used herein, the singular forms a, an, and the include p lural referents
unless the context clearly dictates otherwise. For example, a or a n means at least one or one
or more.
Throughout this disclosure, various aspects of the claimed subject matter are
presented in a range format. It should be understood that the description in range format is merely
for convenience and brevity and should not be construed as an inflexible limitation on the scope of
the claimed subject matter. Accordingly, the description of a range should be considered to have
specifically disclosed all the possible sub-ranges as well as individual numerical values within that
range. For example, where a range of values is provided, it is understood that each intervening
value, between the upper and lower limit of that range and any other stated or intervening value in
that stated range is encompassed within the claimed subject matter. The upper and lower limits of
these smaller ranges may independently be included in the smaller ranges, and are also
encompassed within the claimed subject matter, subject to any specifically excluded limit in the
stated range. Where the stated range includes one or both of the limits, ranges excluding either or
both of those included limits are also included in the claimed subject matter. This applies regardless
of the breadth of the range.
The term about as used herein refers to the usual error range for the respective
value readily known to the skilled person in this technical field. Reference to about a value or
sd-660418
735042000240
parameter herein includes (and describes) embodiments that are directed to that value or parameter
per se.
As used herein, the singular forms a, an, and the include plural referents
unless the context clearly dictates otherwise. For example, a or a n means at least one or on e
or more.
Throughout this disclosure, various aspects of the claimed subject matter are
presented in a range format. It should be understood that the description in range format is merely
for convenience and brevity and should not be construed as an inflexible limitation on the scope of
the claimed subject matter. Accordingly, the description of a range should be considered to have
specifically disclosed all the possible sub-ranges as well as individual numerical values within that
range. For example, where a range of values is provided, it is understood that each intervening
value, between the upper and lower limit of that range and any other stated or intervening value in
that stated range is encompassed within the claimed subject matter. The upper and lower limits of
these smaller ranges may independently be included in the smaller ranges, and are also
encompassed within the claimed subject matter, subject to any specifically excluded limit in the
stated range. Where the stated range includes one or both of the limits, ranges excluding either or
both of those included limits are also included in the claimed subject matter. This applies regardless
of the breadth of the range.
As used herein, percent (%) amino acid sequence identity a nd pe rcent ide ntity
when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as
the percentage of amino acid residues in a candidate sequence (e.g., a streptavidin mutein) that are
identical with the amino acid residues in the reference polypeptide sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity,
and not considering any conservative substitutions as part of the sequence identity. Alignment for
purposes of determining percent amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly available computer software such as
BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can
sd-660418
735042000240
determine appropriate parameters for aligning sequences, including any algorithms needed to
achieve maximal alignment over the full length of the sequences being compared.
An amino acid substitution may include replacement of one amino acid in a
polypeptide with another amino acid. Amino acids generally can be grouped according to the
following common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
Non-conservative amino acid substitutions will involve exchanging a member of one
of these classes for another class.
As used herein, a subject includes any living organism, such as humans and other
mammals. Mammals include, but are not limited to, humans, and non-human animals, including
farm animals, sport animals, rodents and pets.
As used herein, a composition refers to any mixture of two or more products,
substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a
paste, aqueous, non-aqueous or any combination thereof.
As used herein, depleting when referring to one or more particular cel l type or cell
population, refers to decreasing the number or percentage of the cell type or population, e.g.,
compared to the total number of cells in or volume of the composition, or relative to other cell
types, such as by negative selection based on markers expressed by the population or cell, or by
positive selection based on a marker not present on the cell population or cell to be depleted. The
term does not require complete removal of the cell, cell type, or population from the composition.
As used herein, enriching when referring to one or more particular cell type or cell
population, refers to increasing the number or percentage of the cell type or population, e.g.,
compared to the total number of cells in or volume of the composition, or relative to other cell
sd-660418
735042000240
types, such as by positive selection based on markers expressed by the population or cell, or by
negative selection based on a marker not present on the cell population or cell to be depleted. The
term does not require complete removal of other cells, cell type, or populations from the
composition and does not require that the cells so enriched be present at or even near 100 % in the
enriched composition.
As used herein, the terms treatment, tre at, and treating, refer to complete or
partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect
or outcome, or phenotype associated therewith. In certain embodiments, the effect is therapeutic,
such that it partially or completely cures a disease or condition or adverse symptom attributable
thereto.
As used herein, a therapeutically effective amount of a compound or composition
or combination refers to an amount effective, at dosages and for periods of time necessary, to
achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or
pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount
may vary according to factors such as the disease state, age, sex, and weight of the subject, and the
populations of cells administered.
As used herein, a statement that a cell or population of cells is positive fo r a
particular marker refers to the detectable presence on or in the cell of a particular marker, typically a
surface marker. When referring to a surface marker, the term refers to the presence of surface
expression as detected by flow cytometry, for example, by staining with an antibody that
specifically binds to the marker and detecting said antibody, wherein the staining is detectable by
flow cytometry at a level substantially above the staining detected carrying out the same procedure
with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise
identical conditions and/or at a level substantially similar to that for cell known to be positive for
the marker, and/or at a level substantially higher than that for a cell known to be negative for the
marker.
As used herein, a statement that a cell or population of cells is negative fo r a
particular marker refers to the absence of substantial detectable presence on or in the cell of a
sd-660418
735042000240
particular marker, typically a surface marker. When referring to a surface marker, the term refers to
the absence of surface expression as detected by flow cytometry, for example, by staining with an
antibody that specifically binds to the marker and detecting said antibody, wherein the staining is
not detected by flow cytometry at a level substantially above the staining detected carrying out the
same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control
under otherwise identical conditions, and/or at a level substantially lower than that for cell known to
be positive for the marker, and/or at a level substantially similar as compared to that for a cell
known to be negative for the marker.
In some embodiments, a decrease in expression of one or markers refers to loss of 1
log in the mean fluorescence intensity and/or decrease of percentage of cells that exhibit the
marker of at least about 20% of the cells, 25% of-the cells, 30% of the cells, 35% of the cells, 40%
of the cells, 45% of the cells, 50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells,
70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells, 90% of the cell, 95% of the
cells, and 100% of the cells and any % between 20 and 100% when compared to a reference cell
population. In some embodiments, a cell population positive for one or markers refers to a
percentage of cells that exhibit the marker of at least about 50% of the cells, 55% of the cells, 60%
of the cells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells,
90% of the cell, 95% of the cells, and 100% of the cells and any % between 50 and 100% when
compared to a reference cell population.
V. Exemplary Embodiments
Among the embodiments provided herein are:
1. A method for enriching CD4+ or CD8+ T cells, the method including:
(a) performing a first selection in a closed system, said first selection including enriching
for one of (i) CD4+ cells and (ii) CD8+ cells from a sample containing primary human T cells, the
enrichment thereby generating a first selected population and a non-selected population; and
(b) performing a second selection in the closed system, said second selection including
enriching for the other of (i) CD4+ cells and (ii) CD8+ cells from the non-selected population, the
enrichment thereby generating a second selected population,
sd-660418
735042000240
wherein the method produces an enriched composition, which is enriched for CD4+ cells
and CD8+ cells and includes cells of the first selected population and cells of the second selected
population.
2. The method of embodiment 1, further including (c) combining cells of the first
selected population and cells of the second selected population, thereby producing the enriched
composition and/or wherein the CD4+ and CD8+ cells in the enriched composition are present at a
culture-initiating ratio of CD4+ cells to CD8+ cells.
3. The method of embodiment 2, wherein said combining is performed in the closed
system.
4. A method for producing genetically engineered T cells, the method including:
(a) performing a first selection in a closed system, said first selection including
enriching for one of (i) CD4 cells and (ii) CD8+ cells from a sample containing primary human T
cells, the enrichment thereby generating a first selected population and a non-selected population;
(b) performing a second selection in the closed system, said second selection including
enriching for the other of (i) CD4+ cells and (ii) CD8+ cells from the non-selected population, the
enrichment thereby generating a second selected population;
(c) incubating a culture-initiating composition, which contains cells of the first selected
population and cells of the second selected population, in a culture vessel under stimulating
conditions, thereby generating stimulated cells; and
(d) introducing a genetically engineered antigen receptor into stimulated cells generated in
(c),
wherein the method thereby generates an output composition including CD4 T cells and
CD8 T cells expressing the genetically engineered antigen receptor.
. The method of embodiment 4, further including, prior to step (c), combining cells of
the first and second selected cell populations to produce the culture-initiating composition and/or
wherein the CD4+ and CD8+ cells in the culture-initiating composition are present at a culture-
initiating ratio of CD4+ cells to CD8+ cells.
sd-660418
735042000240
6. The method of embodiment 5, wherein said combining is performed in the closed
system.
7. The method of any of embodiments 1-6, wherein one or more of the steps are carried
out in an automated fashion and/or wherein the closed system is automated.
8. The method of any of embodiments 2-7, wherein the culture-initiating ratio of CD4
to CD8 cells is between at or about 10:1 and at or about 1:10, between at or about 5:1 and at or
about 1:5, or between at or about 2:1 and at or about 1:2.
9. The method of any of embodiments 2-8, wherein the culture-initiating ratio of CD4+
to CD8+ cells is at or about 1:1.
. The method of any of embodiments 2-6, wherein the sample is obtained from a
human subject and:
+ + + +
the culture-initiating ratio of CD4 to CD8 cells is different than the ratio of CD4 to CD8
cells in the sample from the subject; and/or
the culture-initiating ratio of CD4 to CD8 cells is at least 10 %, at least 20 %, at least 30
%, at least 40 %, or at least 50 % greater or less than the ratio of CD4 to CD8 cells in the sample
from the subject.
11. The method of any of embodiments 1-10, wherein enriching cells in the first and/or
second selection includes performing positive selection or negative selection based on expression of
a cell surface marker.
12. The method of embodiment 11, wherein enriching for cells in the first and/or second
selection includes negative selection, which includes depleting cells expressing a non-T cell surface
marker.
13. The method of embodiment 12, wherein the non-T cell marker comprises CD14.
14. The method of any of embodiments 1-13, wherein enriching cells in the first or
second selection includes performing a plurality of positive or negative selection steps based on
expression of a cell surface marker or markers to enrich for CD4+ or CD8+ cells.
. The method of any of embodiments 1-14, wherein the enriching cells in the first
and/or second selection includes immunoaffinity-based selection.
sd-660418
735042000240
16. The method of embodiment 15, wherein the immununoaffinity-based selection is
effected by contacting cells with an antibody capable of specifically binding to a cell surface marker
and recovering cells bound to the antibody, thereby effecting positive selection, or recovering cells
not bound to the antibody, thereby effecting negative selection, wherein the recovered cells are
enriched for the CD4+ cells or the CD8+ cells and antibody is immobilized on a magnetic particle.
17. The method of any of embodiments 1-13, wherein the first selection and second
selection are carried out in separate separation vessels, which are operably connected.
18. The method of embodiment 17, wherein the separation vessels are operably
connected by tubing.
19. The method of any of embodiments 15-18, wherein the immunoaffinity-based
selection is effected by contacting cells with an antibody immobilized on or attached to an affinity
chromatography matrix, said antibody capable of specifically binding to a cell surface marker to
effect positive or negative selection of CD4+ or CD8+ cells.
. The method of embodiment 19, wherein:
the antibody further includes one or more binding partners capable of forming a reversible
bond with a binding reagent immobilized on the matrix, whereby the antibody is reversibly bound
to said matrix during said contacting; and
cells expressing a cell surface marker specifically bound by the antibody on said matrix are
capable of being recovered from the matrix by disruption of the reversible binding between the
binding reagent and binding partner.
21. The method of embodiment 17, wherein:
the binding partner is selected from among biotin, a biotin analog, and a peptide capable of
binding to the binding reagent; and
the binding reagent is selected from among streptavidin, a streptavidin analog or mutein,
avidin and an avidin analog or mutein.
22. The method of embodiment 21, wherein:
the binding partner includes a sequence of amino acids set forth in SEQ ID NO:6; and/or
sd-660418
735042000240
the binding reagent is a streptavidin mutein including the sequence of amino acids set forth
in SEQ ID NO: 12, 13, 15 or 16.
23. The method of any of embodiments 19-21, further including, after contacting cells in
the sample to an affinity chromatography matrix in the first selection and/or second selection,
applying a competition reagent to disrupt the bond between the binding partner and binding reagent,
thereby recovering the selected cells from the matrix.
24. The method of embodiment 23, wherein the competition reagent is biotin or a biotin
analog.
. The method of any of embodiments 20-24, wherein the antibody or antibodies in the
first and/or second selection has a dissociation rate constant (k ) for binding and the cell surface
-5 -1
marker of greater than or greater than about 3 x 10 sec .
26. The method of any of embodiments 20-25, wherein the antibody or antibodies in the
first and/or second selection has an affinity for the cell surface marker of a dissociation constant
-3 -7 -7 -10
(Kd) in the range of about 10 to 10 or in the range of about 10 to about 10 .
27. The method of any of embodiments 20-26, wherein the chromatography matrix of
the first and/or second selection is packed in a separation vessel, which is a column.
28. The method of embodiment 19-27, wherein the affinity chromatography matrix
adsorbs and/or is capable of selecting at least or at least about 50 x 10 cells/mL, 100 x 10
cells/mL, 200 x 10 cells/mL or 400 x 10 cells/mL.
29. The method of any of embodiments 19-28, wherein the first and the second selection
steps comprise the use of the affinity chromatography matrix and the matrix used in the first and
second selection steps are at sufficient relative amounts to achieve the culture-initiation ratio.
. The method of any of embodiments 1-29, wherein the enriching for the CD4+ cells
includes positive selection based on surface expression of CD4.
31. The method of any of embodiments 1-29, wherein the enriching for the CD8+ cells
includes positive selection based on surface expression of CD8.
sd-660418
735042000240
32. The method of any of embodiments 1-29, wherein the one of the first and second
selections that includes enriching for the CD8+ cells further includes enriching for central memory
T (T ) cells; and/or
enriching for cells expressing a marker selected from among CD28, CD62L, CCR7, CD127
and CD27.
33. The method of any of embodiments 1-32, wherein:
the first selection includes enriching for the CD8+ cells and the second selection includes
enriching for the CD4+ cells; and
the first selection further includes enriching for central memory T (T ) cells and/or
enriching for cells expressing a marker selected from among CD28, CD62L, CCR7, CD127 and
CD27.
34. The method of embodiment 32 or embodiment 33 or embodiment 35, wherein
enriching for central memory T (T ) cells and/or enriching for cells expressing the marker
includes:
selecting from the first and/or second selected cell population, which is enriched for CD8
cells, cells expressing CD62L; and/or
selecting, from the first and/or second selected cell population, which is enriched for CD8
cells, cells expressing CD27; and/or
selecting, from the first and/or second selected cell population, which is enriched for CD8
cells, cells expressing CCR7; and/or
selecting, from the first and/or second selected cell population, which is enriched for CD8
cells, cells expressing CD28; and/or
selecting, from the first and/or second selected cell population, which is enriched for CD8
cells, cells expressing CD127.
. The method of any of embodiments 1-32, wherein:
the first selection includes enriching for the CD4+ cells and the second selection includes
enriching for the CD8+ cells; and
the second selection further includes enriching for central memory T (T ) cells.
sd-660418
735042000240
36. The method of embodiment 35, wherein:
(i) the first selection includes enriching CD4+ cells by positive selection based on
surface expression of CD4, thereby generating the first selected population, which is enriched for
CD4+ primary human T cells, and the non-selected sample;
(ii) the second selection includes enriching for the CD8+ cells and further includes
enriching the second selected sample for central memory T (T ) cells, wherein enriching for
central memory T (T ) cells includes:
negative selection to deplete cells expressing a surface marker present on naïve T
cells and positive selection for cells expressing a surface marker present on central memory
T (T ) cells and not present on another memory T cell sub-population; or
positive selection for cells expressing a surface marker present on central memory T
cells and not present on naïve T cells and positive selection for cells expressing a surface
marker present on central memory T (T ) cells and not present on another memory T cell
sub-population;
thereby generating CD8+ primary human T cells enriched for T cells.
37. The method of embodiment 36, wherein:
the marker present on naïve T cells comprises CD45RA; and
enriching for central memory T (T ) cells includes negative selection to deplete cells
expressing CD45RA and positive selection for cells expressing a surface marker present on central
memory T (TCM) cells and not present on another memory T cell sub-population.
38. The method of embodiment 37, wherein:
the surface marker present on central memory T cells and not present on naïve T cells
comprises CD45RO;
the enrichment for central memory T (T ) cells includes positive selection for cells
expressing CD45RO and positive selection for cells expressing surface marker present on central
memory T (TCM) cells and not present on another memory T cell sub-population.
sd-660418
735042000240
39. The method of any of embodiments 36-38, wherein the surface marker present on
central memory T (TCM) cells and not present on another memory T cell sub-population is selected
from the group consisting of CD62L, CCR7, CD27, CD127, and CD44.
40. The method of embodiment 39, wherein the surface marker present on central
memory T (TCM) cells and not present on another memory T cell sub-population is CD62L.
41. The method of any of embodiments 32-40, wherein the CD8 population in the
enriched composition or the culture-initiating composition includes at least 50 % central memory T
(TCM) cells or includes less than 20 % naïve T (TN) cells or includes at least 80 % CD62L+ cells.
42. A method for enriching CD4+ and CD8+ T cells, the method including contacting
cells of a sample containing primary human T cells with a first immunoaffinity reagent that
specifically binds to CD4 and a second immunoaffinity reagent that specifically binds to CD8 in an
incubation composition, under conditions whereby the immunoaffinity reagents specifically bind to
CD4 and CD8 molecules, respectively, on the surface of cells in the sample; and
recovering cells bound to the first and/or the second immunoaffinity reagent, thereby
generating an enriched composition including CD4+ cells and CD8+ cells at a culture-initiating
ratio, wherein:
the first and/or second immunoaffinity reagent are present in the incubation composition at
a sub-optimal yield concentration, whereby the enriched composition contains less than 70% of the
total CD4+ cells in the incubation composition and/or less than 70% of the CD8+ cells in the
incubation composition, thereby producing a composition enriched for CD4+ and CD8+ T cells.
43. A method for producing genetically engineered T cells, the method including:
(a) enriching for primary human T cells from a sample containing primary human T cells
including:
contacting cells of said sample with a first immunoaffinity reagent that specifically
binds to CD4 and a second immunoaffinity reagent that specifically binds to CD8 in an incubation
composition, under conditions whereby the immunoaffinity reagents specifically bind to CD4 and
CD8 molecules, respectively, on the surface of cells in the sample; and
sd-660418
735042000240
recovering cells bound to the first and/or the second immunoaffinity reagent, thereby
generating an enriched composition including CD4+ cells and CD8+ cells at a culture-initiating
ratio, wherein:
the first and/or second immunoaffinity reagent are present in the incubation
composition at a sub-optimal yield concentration, whereby the enriched composition contains less
than 70% of the total CD4+ cells in the incubation composition and/or less than 70% of the CD8+
cells in the incubation composition; and
(b) incubating cells of the enriched composition in a culture-initiation composition in a
culture vessel under stimulating conditions, thereby generating stimulated cells, wherein the cells
are at or substantially at the culture-initiating ratio; and
(c) introducing a genetically engineered antigen receptor into stimulated cells of (b),
thereby generating an output composition including CD4 T cells and CD8 T cells expressing the
genetically engineered antigen receptor.
44. The method of embodiment 42 or embodiment 43, wherein enriching for primary
human T cells is performed in a closed system.
45. The method of any of embodiments 42-44, wherein the first and second
immunoaffinity reagent are present in the incubation composition at a sub-optimal yield
concentration, whereby the enriched composition contains less than 70% of the total CD4+ cells in
the incubation composition and less than 70% of the total CD8+ cells in the incubation composition.
46. The method of any of embodiments 42-45, wherein:
the first immunoaffinity reagent is present in the incubation composition at a sub-optimal
yield concentration, whereby the enriched composition contains less than 60%, less than 50%, less
than 40%, less than 30% or less than 20% of the total CD4+ cells in the incubation composition;
and/or
the second immunoaffinity reagent is present in the incubation composition at a sub-optimal
yield concentration, whereby the enriched composition contains less than 60%, less than 50%, less
than 40%, less than 30% or less than 20% of the total CD8+ cells in the incubation composition.
sd-660418
735042000240
47. The method of embodiment any of embodiments 42-46, wherein the sample contains
at least 1 x 10 CD3+ T cells.
48. The method of any of embodiments 42-47, wherein the concentration of one of the
first and second immunoaffinity reagent in the incubation composition is greater than that of the
other, whereby the greater concentration effects a higher yield in the enriched composition of CD4+
cells or CD8+ cells, respectively, compared to the yield of the other of the CD4+ or CD8+ cells,
thereby producing the culture-initiating ratio in the enriched composition.
49. The method of embodiment 48, wherein:
the concentration of one of the first and second immunoaffinity reagents is greater, as
compared to the concentration of the other of the first and second immunoaffinity reagents, by at
least 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold,
8.0-fold, 9.0-fold or 10-fold; and/or
the higher yield in the enriched concentration is greater by 1.2-fold, 1.4-fold, 1.6-fold, 1.8-
fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold or 10-fold.
50. The method of any of embodiments 42-49, wherein the culture-initiating ratio of
CD4 to CD8 cells is between at or about 10:1 and at or about 1:10, between at or about 5:1 and at
or about 1:5 or is between at or about 2:1 and at or about 1:2.
51. The method of any of embodiments 42-50, wherein the culture-initiating ratio of
CD4+ to CD8+ cells is at or about 1:1.
52. The method of any of embodiments 42-51, wherein:
+ + + +
the culture-initiating ratio of CD4 to CD8 cells is different than the ratio of CD4 to CD8
cells in the sample from the subject; and/or
the culture-initiating ratio of CD4 to CD8 cells is at least 10 %, at least 20 %, at least 30
%, at least 40 %, or at least 50 % greater or less than the ratio of CD4 to CD8 cells in the sample
from the subject.
53. The method of any of embodiments 42-52, wherein greater than 95% or greater than
98% of the cells in the culture-initiation composition are CD4+ cells and CD8+ cells.
sd-660418
735042000240
54. The method of any of embodiments 42-53, wherein each of the immunoaffinity
reagents contains an antibody.
55. The method of embodiment 54, wherein the antibody is immobilized on the outside
surface of a sphere.
56. The method of embodiment 55, wherein the sphere is a magnetic bead.
57. The method of embodiment 55 or embodiment 56, wherein:
the antibody contains one or more binding partners capable of forming a reversible bond
with a binding reagent immobilized on the sphere, whereby the antibody is reversibly immobilized
to said sphere; and
the method further includes after contacting cells in the sample to the first and second
immunoaffinity reagent, applying a competition reagent to disrupt the bond between the binding
partner and binding reagent, thereby recovering the selected cells from the sphere.
58. The method of embodiment 57, wherein:
the binding partner is selected from among biotin, a biotin analog, or a peptide capable of
binding to the binding reagent; and
the binding reagent is selected from among streptavidin, a streptavidin analog or mutein,
avidin, an avidin analog or mutein.
59. The method of embodiment 58, wherein:
the binding partner contains a peptide including the sequence of amino acids set forth in
SEQ ID NO:6 and/or
the binding reagent is streptavidin mutein including the sequence of amino acids set forth in
SEQ ID NO: 12, 13, 15 or 16.
60. The method of embodiment 58 or embodiment 59, wherein:
the binding reagent is a multimer including one or more monomers of streptavidin or a
streptavidin mutein; and
the binding partner is a peptide including a sequential arrangement of at least two modules
each capable of reversibly binding with at least one monomer of the binding reagent.
sd-660418
735042000240
61. The method of embodiment 60, wherein the binding partner contains the sequence of
amino acids set forth in any of SEQ ID NOS: 7-10.
62. The method of any of embodiments 57-61, wherein the competition reagent is biotin
or a biotin analog.
63. The method of any of embodiments 42-63, wherein the antibody or antibodies in the
first and/or second selection has a dissociation rate constant (k ) for binding between the antibody
-5 -1
and cell surface marker of greater than or greater than about 3 x 10 sec .
64. The method of any of embodiments 42-63, wherein the antibody or antibodies in the
first and/or second selection has an affinity with a dissociation constant (K ) in the range of about
-3 -7 -7 -10
to 10 or with a dissociation constant in the range of about 10 to about 10 .
65. The method of any of embodiments 42-63, wherein one or more steps are carried out
in an automated fashion and/or wherein the closed system is automated.
66. The method of any of embodiments 2, 3 and 5-41 , including:
prior to performing the first selection and/or second selection, determining the ratio of
CD4+ to CD8+ T cells in the sample; and
based on the ratio of CD4+ to CD8+ T cells in the sample, adjusting the first and/or second
selection to produce the composition including CD4+ cells and CD8+ cells at the culture-initiating
ratio.
67. The method of embodiment 66, wherein:
the first and/or second selection includes immunoaffinity-based selection including an
affinity chromatography matrix; and
adjusting the first and/or second selection includes choosing the amount of affinity
chromatography matrix in the first and/or second selection sufficient to achieve the culture-
initiation ratio.
68. The method of any of embodiments 42-65, including:
prior to contacting cells of the sample with the first and second immunoaffinity reagent,
determining the ratio of CD4+ to CD8+ T cells in the sample; and
sd-660418
735042000240
based on the ratio of CD4+ to CD8+ T cells in the sample, choosing the concentration of the
first and/or second immunoaffinity reagent to produce the enriched composition including CD4+
cells and CD8+ cells at the culture-initiating ratio.
69. The method of any of embodiments 1-68, wherein the method results an output
composition including a ratio of CD4 to CD8 cells that is between at or about 2:1 and at or about
1:5.
70. The method of embodiment 69, wherein the ratio of CD4 to CD8 cells in the output
composition is 1:1 or is about 1:1.
71. The method of any of embodiments 1-70, wherein the sample is obtained from a
subject.
72. The method of embodiment 71, wherein the subject is a subject to whom said
genetically engineered T cells or cells for adoptive cell therapy will be administered.
73. The method of embodiment 72, wherein the subject is a subject other than a subject
to whom said genetically engineered T cells or cells for adoptive therapy will be administered.
74. The method of any of embodiments 1-73, wherein the sample is blood or a blood-
derived sample.
75. The method of any of embodiments 1-74, wherein the sample is a white blood cell
sample.
76. The method of any of embodiments 1-75, wherein the sample is an apheresis,
peripheral blood mononuclear cell (PBMC), or leukapheresis sample.
77. The method of any of embodiments 4-41 or 43-76, wherein incubating the
composition in a culture vessel under stimulating conditions is performed prior to, during and/or
subsequent to introducing a genetically engineered antigen receptor.
78. The method of any of embodiments 4-41 or 43-76, wherein incubating the
composition in a culture vessel under stimulating conditions is performed prior to, during and
subsequent to introducing a genetically engineered antigen receptor.
79. The method of any of embodiments 4-41 or 43-78, wherein the stimulating
conditions comprise conditions whereby T cells of the composition proliferate.
sd-660418
735042000240
80. The method of any of embodiments 4-41 or 43-79, wherein the stimulating condition
includes an agent capable of activating one or more intracellular signaling domains of one or more
components of a TCR complex.
81. The method of embodiment 80, wherein the one or more components of the TCR
complex contains the CD3 zeta chain.
82. The method of any of embodiments 4-41 or 43-81, wherein the stimulating condition
contains the presence of an anti-CD3 antibody, and anti-CD28 antibody, anti1BB antibody,
and/or a cytokine.
83. The method of embodiment 82, wherein the anti-CD3 antibody and/or the anti-CD28
antibody is present on the surface of a solid support.
84. The method of embodiment 83, wherein the cytokine includes IL-2, IL-15, IL-7,
and/or IL-21.
85. The method of any of embodiments 4-41 or 43-84, wherein the genetically
engineered antigen receptor includes a T cell receptor (TCR) or a functional non-TCR antigen
receptor.
86. The method of embodiment 85, wherein the receptor specifically binds to an antigen
expressed by cells of a disease or condition to be treated.
87. The method of any of embodiment 85 or 86, wherein the antigen receptor is a
chimeric antigen receptor (CAR).
88. The method of embodiment 87, wherein the CAR contains an extracellular antigen-
recognition domain and an intracellular signaling domain including an ITAM-containing sequence
and an intracellular signaling domain of a T cell costimulatory molecule.
89. A method of treatment, said method including:
(a) producing an output composition including CD4 T cells and CD8 T cells according
to any of embodiments 1-88; and
(b) administering cells of the output composition to a subject.
90. The method of embodiment 89, wherein the sample from which the cells are isolated
is derived from the subject to which the cells are administered.
sd-660418
735042000240
91. A composition of cells produced by the method of any of embodiments 1-88.
92. The composition of embodiment 91, including a pharmaceutically acceptable carrier.
93. A method of treatment, said method including administering to a subject a
composition of cells of embodiment 91 or 92.
94. The method of embodiment 93, wherein the genetically engineered antigen receptor
specifically binds to an antigen associated with the disease or condition.
95. The method of treatment of embodiment 94, wherein the disease or condition is a
cancer.
96. The composition of embodiment 91 or embodiment 92 for use in treating a disease or
condition in a subject.
97. Use of a composition of embodiment 91 or embodiment 92 for the manufacture of a
medicament for treating a disease or disorder in a subject.
98. The composition of embodiment 96 or use of embodiment 97, wherein the
genetically engineered antigen receptor specifically binds to an antigen associated with the disease
or condition.
99. The composition or use of any of embodiments 96-98, wherein the disease or
condition is a cancer.
100. A closed apparatus system for purification of target cells, including:
a) a first affinity chromatography matrix including a first binding agent immobilized
thereon, which binding agent specifically binds to a first cell surface marker present on a first cell,
wherein the first affinity chromatography matrix is operably connected to a storage reservoir
including a cell sample via a first operable connection, said first operable connection capable of
permitting passage of cells from the storage reservoir to the first affinity chromatography matrix
and wherein the first affinity chromatography matrix is operably connected to an output vessel via a
second operable connection; and
b) a second affinity chromatography matrix including a second binding agent immobilized
thereon, which second binding agent specifically binds to a second cell surface marker present on a
second cell, wherein the first affinity chromatography matrix is operably connected via a third
sd-660418
735042000240
operable connection to the second affinity chromatography matrix, said third operable connection
capable of permitting passage of cells having passed through the first affinity chromatography
matrix and not bound to the first binding agent to the second affinity chromatography matrix, and
the second affinity chromatography matrix is operably connected via a fourth operable connection
to the output vessel, the fourth operable connection capable of permitting passage of cells having
bound to and been eluted from the first and/or second affinity chromatography matrix, and the
second affinity chromatography matrix is operably linked, via a fifth operable connection, to a
waste vessel, the fifth operable connection capable of permitting passage of cells having passed
through the first affinity chromatography matrix and not bound to the first binding agent and having
passed through the second affinity chromatography matrix and not bound to the second binding
agent;
c) the output vessel; and
d) the waste vessel,
wherein the system is configured to permit, within the closed system, collection of, in the
output vessel in a single composition, (i) cells having bound and been recovered from the first
affinity chromatography matrix and (ii) cells having passed through and not bound to the first
affinity chromatography matrix and having bound and been recovered from the second
chromatography matrix.
101. The closed apparatus of embodiment 100, wherein one or more of said operable
connections contains tubing connecting the storage reservoir, first affinity chromatography matrix,
second affinity chromatography matrix and/or culture vessel.
102. The closed apparatus of embodiment 101, wherein the tubing is connected to a
stopcock, valve or clamp.
103. The closed apparatus system of any of embodiments 100-102, wherein:
(a) the first affinity chromatography matrix is one of a CD4+ affinity chromatography
matrix or a CD8+ affinity chromatography matrix; and
(b) the second affinity chromatography matrix is the other of the CD4+ affinity
chromatography matrix or the CD8+ affinity chromatography matrix.
sd-660418
735042000240
104. The closed apparatus system of any of embodiments 100-103, further including:
d) a third affinity chromatography matrix including a third binding agent immobilized
thereon, which binding agent specifically binds to a third cell surface marker, whereby the third
binding agent is capable of binding cells expressing the third cell surface marker, wherein the third
operable connection further operably connects the third affinity chromatography matrix to the first
matrix and a sixth operable connection operably connects the third affinity chromatography matrix
to the output container, such that the sixth operable connection is capable of permitting passage of
cells having bound to and been recovered from the first matrix and having bound to and been
recovered from the third matrix to the output container, and
the fifth operable connection further operably connects the third affinity chromatography
matrix to the waste container, such that the fifth operable connection is capable of permitting cells
having passed through and not bound to the third column to the waste container.
105. The closed apparatus system of any of embodiments 100-103, further including:
d) a third affinity chromatography matrix including a third binding agent immobilized
thereon, which binding agent specifically binds to a third cell surface marker, whereby the third
affinity chromatography matrix is capable of binding cells expressing the third cell surface marker,
wherein
the second operable connection further operably connects the third affinity chromatography
matrix to the first matrix and to the output container, such that the second operable connection is
capable of permitting cells having bound to and been recovered from the first matrix and having
bound to and been recovered from the third matrix to the output container.
106. The closed apparatus system of embodiment 104 or 105, wherein:
the first affinity chromatography matrix includes a biding agent that specifically binds CD8;
the second affinity chromatography matrix includes a binding agent that specifically binds
CD4; and
the third affinity chromatography matrix includes a binding agent that specifically binds a
marker expressed on central memory T (T ) cells.
sd-660418
735042000240
107. The closed apparatus system of embodiment 106, wherein the binding agent of the
third affinity chromatography matrix selectively binds to a cell surface marker selected from among
CD62L, CD45RA, CD45RO, CCR7, CD27, CD127, and CD44.
108. The closed apparatus system of any of embodiments 100-107, wherein one or more
or all of the affinity chromatography matrices are further operably connected to an elution buffer
reservoir including one or more competition reagents.
109. The closed apparatus system of any of embodiments 100-107, wherein the
competition reagent is one or more of the group consisting of biotin, a biotin analog, and a peptide
capable of binding to the chromatography matrix.
110. The closed apparatus system of any of embodiments 100-107, wherein one or more
or all of the third, fourth, or sixth operable connections further includes a competition agent removal
chamber.
111. The closed apparatus system of embodiment 110, wherein the competition agent
removal chamber further contains a binding reagent.
112. The closed apparatus system of embodiment 111, wherein the binding reagent
includes one or more of the group consisting of streptavidin, a streptavidin analog or mutein, avidin
and an avidin analog or mutein.
113. The closed apparatus system of any of embodiments 100-112, wherein one or more
or all of the binding agents is an antibody.
114. The closed apparatus system of any of embodiments 100-112, wherein the one or
more or all of the binding agents is reversibly bound to the affinity chromatography matrix.
VI. EXAMPLES
The following examples are included for illustrative purposes only and are not
intended to limit the scope of the invention.
Example 1: Generation of a CD4+ and CD8+ T cell Composition Using a Single Process
Stream By Immunomagnetic Separation For Genetic Engineering And Adoptive Cell Therapy
sd-660418
735042000240
In an exemplary method, a CD4 T cell population and a CD8 T cell population,
enriched for central memory T cells, are isolated from an apheresis product sample, and
subsequently incubated and engineered, followed by administration to a subject. The isolation
procedure is carried out using a single process stream by immunomagnetic separation using the
CliniMACS® Prodigy system. The process is streamlined as compared to other methods, in which
CD4 cells are separated from a first apheresis fraction and CD8 cells are separated and further
depleted/enriched, from a second apheresis fraction, using a CliniMACS® Prodigy device and three
separate tubing sets.
The streamlined process is performed using one tubing set for isolation of the CD4
and CD8 cell populations, without transferring the cell populations from one vessel (e.g., tubing
set) to another.
The CD4 cell population is isolated by incubating an apheresis sample with a
CliniMACS® CD4 reagent. The cells are then separated by the CliniMACS® Prodigy device set to
run an enrichment program, and both cell fractions (i.e., the immunomagnetically selected enriched
CD4 cell fraction and the flow-through cell fraction) are retained. The enriched (positive) fraction
is an isolated CD4 T cell population. A population of CD8 T cells is isolated by incubating the
flow-through (negative) fraction with a CliniMACS® CD14 reagent and a CliniMACS® CD45RA
reagent or CD19 reagent. The CliniMACS® Prodigy device is set to run a depletion program. The
cell/reagent mixture then is separated by the CliniMACS® Prodigy device using the same tubing set
used in the first separation step. The flow-through (negative) fraction from which
CD14 /CD45RA or CD14+/CD19+ cells have been depleted is incubated with CliniMACS®
CD62L reagent. The cell/reagent mixture is separated using the CliniMACS® Prodigy device
running an enrichment program using the same tubing set. The positive fraction is an isolated CD8
cell population enriched for central memory cells.
The isolated CD4+ and CD8+ populations are combined in the same culture vessel in
a culture-initiating composition at a culture initiation ratio. The culture-initiation ratio is designed
to achieve a particular desired output ratio, or a ratio that is within a certain range of tolerated error
of such a desired output ratio, following the incubation and/or engineering steps, or designed to do
sd-660418
735042000240
so a certain percentage of the time. The cells are incubated under stimulating conditions, such as
using anti-CD3/anti-CD28 beads in the presence IL-2 (100 IU/mL) for 72 hours at 37 ºC, within the
Prodigy system.
The cell composition is optionally periodically assessed and/or adjusted at one or
more times subsequent to the initiation of the incubation or culture, such as at a time during the
incubation. Assessment includes measuring proliferation rate, measuring degree of survival,
determining phenotype, e.g., expression of one or more surface or intracellular markers, such as
proteins or polynucleotides, and/or adjusting the composition or vessel for temperature, media
component(s), oxygen or carbon dioxide content, and/or presence or absence or amount or relative
amount of one or more factors, agents, components, and/or cell types, including subtypes.
Cells so-incubated then are genetically engineered, by introducing into the cells
recombinant genes for expression of recombinant antigen receptors, such as chimeric antigen
receptors (CARs) or recombinant TCRs. The introduction is performed in the contained
environment within the CliniMACS® Prodigy device, with CD4 and CD8 cells present in the
same composition and vessel. The method results in an output composition with engineered CD4
and CD8 T cells.
Example 2: Generation of a CD4+ and CD8+ T cell Composition by Sequential Purification
in a Closed System
This example demonstrates an exemplary method of enriching or selecting for a
CD4+ T cell population and a CD8+ T cell population from an apheresis product sample obtained
from a subject. The process is performed using a closed system with multiple chromatography
columns for sequential positive selection of the CD4+ and CD8+ T cell populations from the same
starting sample.
A. Enrichment for CD4+ T cell Population and CD8+ T Cell Population
In the exemplary process, depicted in Figure 1A, a series of immunochromatographic
selection columns and removal columns are arranged in a closed apparatus 14 operably connected
sd-660418
735042000240
to each other and a peristaltic pump 8 through various tubing lines and valves 13 to control flow of
liquid phase.
The first selection column 1 contains a chosen volume of an affinity chromatography
matrix 3, such as an agarose resin, such as a resin as described in published U.S. Patent Appl. No.
US2015/0024411 for the isolation of T cells, such as one with an exclusion limit designed to be
greater than the size of a T cell, such as an agarose as obtained from Agarose Beads Technologies,
Madrid, Spain, with a reduced exclusion size compared to Superflow™ Agarose, which has an
exclusion size of 6 x 10 Daltons. The matrix in the first column is bound to multimers of Strep-
Tactin® (e.g. containing a streptavidin mutant set forth in any of SEQ ID NO:12, 13, 15 or 16, IBA
GmbH, Germany or as described in International Published PCT Appl. Nos. ).
The second selection column 2 contains a chosen volume of an affinity chromatography matrix 4,
such as an agarose resin, such as a resin as described above, which is also bound to multimers of
Strep-Tactin®.
A reservoir containing an anti-CD8 Fab fragment 18 is loaded to run through the
pump 8, tubing, and valves 13 such that the anti-CD8 Fab is applied to the first selection column 1,
whereby the anti-CD8 Fab becomes immobilized on the Strep-Tactin® on the affinity matrix 3 with
a Twin Strep-Tag® (e.g. set forth in SEQ ID NO: 10; IBA GmbH) in the Fab Fragment, which is
fused to the carboxy-terminus of its heavy chain (see e.g. published U.S. Patent Appl. No.
US2015/0024411). In some embodiments, a washing buffer from a washing buffer reservoir 6,
such as phosphate buffered saline (PBS) containing 0.5% bovine serum albumin, human serum
albumin, or recombinant human serum albumin, is loaded to run through the first selection column
via operably coupled tubing. The flow-through is directed to the waste container 10 via valves 13
operably connecting the first selection column and waste container. In some embodiments, the
washing step is repeated a plurality of times.
A reservoir containing an anti-CD4 Fab fragment 19 is loaded to run through the
pump 8, tubing, and valves 13 such that the anti-CD4 Fab is applied to the second selection column
2, whereby the anti-CD8 Fab becomes immobilized on the Strep-Tactin® on the affinity matrix 4
with a Twin Strep-Tag®. In some embodiments, a washing buffer from a washing buffer reservoir
sd-660418
735042000240
6 is run through the second selection column via operably coupled tubing. The flow-through is
directed to the waste container 10 via valves 13 operably connecting the second selection column
and waste container. In some embodiments, the washing step is repeated a plurality of times.
The volume of affinity matrix reagent contained in the first selection column and the
second selection column can be the same or different, and can be chosen based on the desired yield
of the selection and/or desired ratio of CD4+ cells to CD8+ cells following selection. The volume
so chosen is based on an assumption of an average yield of 1 x 10 cells being capable of selection
per each 1 mL volume of filled column. In one exemplary process, the column volumes are the
same, for example, using a 2 mL volume for the column for the anti-CD8 Fab fragment and a 2 mL
column for the anti-CD4 Fab fragment. The column length and/or column diameter can be chosen
to fit the desired volume, for example, to achieve a desired culture-initiation ratio of CD4+ cells to
CD8+ cells. In some embodiments, to accommodate the volume of affinity matrix, a plurality of
columns having immobilized thereto the same Fab reagent can be added directly in series and
operably connected to each other via a tubing line.
To achieve selection of cells, an apheresis sample is applied to the first selection
column 1, whereby CD8+ T cells, if present in the sample, remain bound to the resin 3 of the first
column, as unselected cells (containing CD8- cells) pass through the column. The number of cells in
the starting sample used in some examples is chosen to be above the number of cells to be selected
based on the combined volume of the matrices, such as an amount greater than the capacity of the
column for each selection (e.g. an amount having greater than 200 million CD8+ cells and greater
than 200 million CD4+ cells, e.g., greater by at least about 10 % or 20 % or greater). The flow-
through containing unselected cells (negative fraction) then passes from the first column to a second
selection column 2 via operably coupled tubing. CD4+ T cells remain bound to the resin 4 in the
second column. The flow through containing fluid and further unselected cells (negative cells) is
directed to a waste container 10 via valves 13 operably connecting the second selection column and
waste container.
sd-660418
735042000240
In some embodiments, the first selection column can alternatively be an anti-CD4
affinity chromatography matrix and the second selection column can be an anti-CD8 affinity
chromatography matrix.
From a washing buffer reservoir 6, a washing buffer is run through the first column
and the second column via operably coupled tubing. The flow-through containing any cells that are
washed from the first and second columns is directed to the waste container 10 via valves 13
operably connecting the second selection column and waste container. In some embodiments, the
washing step is repeated a plurality of times.
From an elution buffer reservoir 7, a buffer containing an eluent, such as low
concentrations of biotin or an analog thereof, for example 2.5 mM desthiobiotin, is loaded to run
through the first column and the second column via operably coupled tubing. In some
embodiments, the eluent contains cell culture media. The flow-through, containing enriched CD4+
and CD8+ cells and residual biotin or analog, is directed to a removal chamber 9 to remove biotin or
an analog thereof. The removal chamber 9 is a column of Superflow™ Sepharose® beads having
Strep-Tactin® immobilized thereto with a volume sufficient to remove biotin or a biotin analog
from the sample, such as a column having a bed volume of 6 mL with a binding capacity of 300
nanomol biotin/mL. The flow-through containing enriched cells positively selected for CD4+ and
CD8+ is directed to a culture vessel 12, such as a bag, via valves operably connecting the removal
chamber 9 and culture vessel. In some embodiments, the elution step is repeated.
In some embodiments, after performing the elution step, an activation buffer
containing a T-cell activation reagent (e.g. anti-CD3, anti-CD28, IL-2, IL-15, IL-7, and/or IL-21)
replaces the wash buffer and is directed through the removal chamber and flow through directed to
the culture vessel. The positive fraction collected in the culture vessel is an isolated and combined
CD4+ and CD8+ cell population.
B. Enrichment for a CD4+ T cell Population and a CD8+ T Cell Population, Enriched
for Cells Expressing a Marker on Central Memory T (TCM) Cells
In the exemplary process, depicted in Figure 1B, a series of immunochromatographic
selection columns and removal columns are arranged in a closed apparatus 14 operably connected
sd-660418
735042000240
to each other and a peristaltic pump 8 through various tubing lines and valves 13 to control flow of
liquid phase.
The first selection column 1 and second selection column 2 are the same as described
above with respect to Example 2A. In addition, the process further includes a third selection
column 15 that contains a chosen volume of an affinity matrix 17, such as an agarose resin, such as
a resin as described in published U.S. Patent Appl. No. US2015/0024411 for the isolation of T cells,
such as one as described above in Example 2A.
The anti-CD8 Fab and the anti-CD4 Fab are applied to the first column and second
column, respectively, as described in Example 2A. A reservoir containing a further Fab fragment
against a marker expressed on central memory T (T ) cells 20, such as one of CD28, CD62L,
CCR7, CD27 or CD127, is loaded to run through the pump 8, tubing, and valves 13 such that the
further Fab is applied to the third selection column 15, whereby the further Fab becomes conjugated
to the Strep-Tactin® on the affinity matrix 17 with a Twin Strep-Tag® (SEQ ID NO: 10; IBA
GmbH) in the Fab Fragment, which is fused to the carboxy-terminus of its heavy chain, as
described in Example 2A. In some embodiments, a washing buffer, as described in Example 2A, is
loaded to run through the third selection column via operably coupled tubing. The flow-through is
directed to the waste container 10 via valves 13 operably connecting the third selection column and
waste container. In some embodiments, the washing step is repeated a plurality of times.
The volume of affinity matrix reagent contained in the first selection, second and/or
third selection columns can be the same or different, and can be chosen based on the desired yield
of the selection and/or desired ratio of CD4+ cells to CD8+ cells enriched for cells expressing a
marker expressed on central memory T (T ) cells following selection. The volume so-chosen is
based on the assumption of an average yield of 1 x 10 cells being capable of selection per each 1
mL volume of filled column. Also, for a particular chosen culture-initiation ratio, for example, a
culture-initiation ratio of 1:1 of CD4+ cells to a population containing CD8+ cells enriched for a
marker expressed on central memory T (T ) cells (e.g. a population of CD8+ cells enriched based
on further selection for one of CD28, CD62L, CCR7, CD27 or CD127), the volume of the matrix
for selecting the parent population of the enriched population, e.g. the CD8+ cells, is larger in
sd-660418
735042000240
comparison to the matrix used for selecting the other of the CD4+ or CD8+ T cell population, e.g.
the CD4+ population. The amount or extent in which the volume of the matrix used to select for the
parent population of the further enriched population, e.g. the matrix containing anti-CD8 Fab
fragment, is greater than the volume of the other matrix or other matrices is chosen based on the
fraction or percentage of the further enriched population of cells in the sample (e.g. CD8+/CD28+,
CD8+/CD62L+, CD8+/CCR7+, CD8+/CD27+ or CD8+/CD127+) compared to the fraction or
percentage of the parent population, e.g. CD8+ cells, present in the sample. This can be estimated
based on averages among patients or healthy donors, or it can be measured for a given patient on
which selection is being performed prior to determining the size of the columns to use.
In one exemplary process, the column volumes used in the process are, for example,
a 2 mL column with anti-CD4 Fab fragments, a 6 mL column with anti-CD8 Fab fragments, and a 2
mL column with anti-CD62L Fab fragments. The column length and/or column diameter can be
chosen to fit the desired volume, for example, to achieve a desired culture-initiation ratio of CD4+
cells to CD8+ cells enriched for cells expressing a marker expressed on central memory T (T )
cells, e.g.CD8+/CD28+, CD8+/CD62L+, CD8+/CCR7+, CD8+/CD27+ or CD8+/CD127+ cells. In
some embodiments, to accommodate the volume of affinity matrix, a plurality of columns having
immobilized thereto the same Fab reagent can be added directly in series and operably connected to
each other via a tubing line.
To achieve selection of cells, an apheresis sample is applied to the first selection
column 1, whereby CD8+ T cells, if present in the sample, remain bound to the resin of the first
column as unselected cells (containing CD8- cells) pass through the column. The number of cells in
the starting sample used in some examples is chosen to be above the number of cells to be selected
based on the combined volume of the matrices such as an amount greater than the capacity of the
column for each selection (e.g. an amount having greater than 200 million CD8+/CD62L+ cells and
greater than 200 million CD4+ cells, e.g. greater by at least about 10 % or 20 % or greater). The
flow-through containing unselected cells (negative fraction) is directed to pass to the second column
2 via a valve 13 operably connecting the first and second column. CD4+ T cells remain bound to
the resin in the second column. The flow-through containing fluid and further unselected cells
sd-660418
735042000240
(negative cells) is directed to a first waste container 10 via valves 13 operably connecting the
second selection column and negative fraction container.
From a washing buffer reservoir 6, a washing buffer, such as described in Example
2A, is loaded to run through the first column and the second column via a valve and tubes operably
connecting the columns. The flow-through containing any cells that are washed from the first and
second columns is directed to the waste container 10 via valves 13 operably connecting the second
selection column and waste container. In some embodiments, the washing step is repeated a
plurality of times.
From an elution buffer reservoir 7, a buffer containing an eluent, such as biotin or an
analog thereof, for example 2.5 mM desthiobiotin, is loaded to run through the second column 2 via
valves 13 and tubing operably connecting the elution buffer reservoir and second column. In some
embodiments, the elution buffer contains cell culture media. The flow-through, containing enriched
CD4+ cells and residual biotin, is directed to a removal chamber 9 to remove biotin, such as
described in Example 2A, which is operably connected to the second column via a valve 13 and
tubing. The flow-through containing enriched cells positively selected for CD4+ is directed to a
culture vessel 12, such as a bag, via valves 13 operably connecting the removal chamber 9 and
culture vessel. In some embodiments, the elution step is repeated. In some embodiments, after
performing the elution step, an activation buffer containing a T-cell activation reagent (e.g. anti-
CD3, anti-CD28, IL-2, IL-15, IL-7, and/or IL-21) replaces the wash buffer and is directed via
valves and tubing from the washing buffer reservoir to the second column, removal chamber and
into the culture vessel. The positive fraction collected in the culture vessel is an isolated CD4+ cell
population.
From an elution buffer reservoir 7, a buffer containing an eluent, such as biotin, is
loaded to run through the first column 1 via a valve 13 and tubing operably connecting the elution
buffer reservoir and first column. In some embodiments, the elution buffer contains cell culture
media. The flow-through, containing enriched CD8+ cells and residual biotin or an analog thereof,
is directed to a removal chamber 9 to remove biotin or a biotin analog, such as described in
Example 2A, which is operably connected to the first column via a valve 13 and tubing. The flow-
sd-660418
735042000240
through containing enriched cells positively selected for CD8+ is directed to pass to a third column
via a valve 13 operably connecting the removal chamber 9 and third column. A CD62L+ subset
of the CD8+ cells remain bound to the resin in the third column. The flow through containing fluid
and further unselected cells (negative cells) is directed to a second waste container 11 via valves 13
operably connecting the third selection column and second waste container.
From a washing buffer reservoir 6, a washing buffer, such as described in Example
2A, is loaded to run through the third column 15 via valves 13 and tubing operably connecting the
wash buffer reservoir and third column. The flow through containing any cells that are washed
from the third column is directed to the second waste container 11 via valves 13 operably
connecting the third selection column and second waste container. In some embodiments, the
washing step is repeated a plurality of times.
From an elution buffer reservoir 7, a buffer containing an eluent, such as biotin or an
analog thereof, for example 2.5 mM desthiobiotin, is loaded to run through the third column 15 via
a valve 13 and tubing operably connecting the elution buffer reservoir and third column. In some
embodiments, the elution buffer contains cell culture media. The flow-through, containing CD8+
cells enriched for central memory T (T ) cells expressing one of CD28, CD62L, CCR7, CD27 or
CD127,and residual biotin or analog thereof, is directed to a removal chamber 9 to remove biotin or
a biotin analog, such as described in Example 2A, which is operably connected to the third column
via a valve 13 and tubing. The flow-through containing enriched T cell memory cells positively
selected for CD8 and a marker expressed on central memory T (T ) cells, such as one of CD28,
CD62L, CCR7, CD27 or CD127, is directed to the culture vessel 12, such as a bag, via valves 13
operably connecting the removal chamber 9 and culture vessel. In some embodiments, the culture
vessel contains a T-cell activation reagent, a cell culture media, or both. In some embodiments, the
elution step is repeated. In some embodiments, after performing the elution step, an activation
buffer containing a T-cell activation reagent (e.g. anti-CD3, anti-CD28, IL-2, IL-15, IL-7, and/or
IL-21) replaces the wash buffer and is directed via valves and tubing from the washing buffer
reservoir to the first and/or third column, removal chamber and into the culture vessel. The positive
fraction containing CD8+ cells and cells positive for a marker on central memory T (T ) cells,
sd-660418
735042000240
such as one of CD28, CD62L, CCR7, CD27 or CD127, is collected in the culture vessel with the
CD4+ positive fraction previously collected. In some embodiments, the steps of the process can be
performed in a different order, such as by first enriching for cells containing CD8+ cells and cells
positive for a marker on central memory T (T ) cells, such as one of CD28, CD62L, CCR7, CD27
or CD127 before enriching CD4+ cells.
Example 3: Generation of a CD4+ and CD8+ T cell Composition by Sequential Purification
in a Closed System For Use in Genetic Engineering and Adoptive Cell Therapy
This example demonstrates a procedure for selecting and generating a composition
of cells containing CD4+ and CD8+ T cells, such as CD4+ and CD8+ T cells present in a culture-
initiation ratio, for incubation/activation and transduction in methods associated with genetic
engineering of cells for use in connection with adoptive cell therapy.
A composition of cells generated by selection of CD4+ and CD8+ cells, performed
as described in either Example 2A (CD4+ and CD8+) or Example 2B (CD4+ and CD8+ enriched
for CD62L+), is incubated under stimulating conditions, such as using anti-CD3/anti-CD28 in the
presence IL-2 (100 IU/mL), for example, for 72 hours at 37 ºC. Stimulated cells then are
genetically engineered by introducing into the cells recombinant genes for expression of
recombinant antigen receptors, such as chimeric antigen receptors (CARs) or recombinant TCRs,
for example, by viral transduction. In some embodiments, following the introduction, cells are
further incubated, generally at 37 degrees C, for example, to allow for cell expansion.
The method results in an output composition with engineered CD4 and CD8 T
cells. In some embodiments, based on the chosen volume of the selection columns, the ratio of
CD4+ to CD8+ cells in the composition that is incubated under the stimulating conditions prior to
engineering (culture-initiation ratio) results in a particular desired output ratio of CD4+ to CD8+
cells or of engineered CD4+ cells to engineered CD8+ cells, or such a ratio that is within a certain
range of tolerated error of such a desired output ratio, following the incubation, stimulation and/or
engineering steps. In some embodiments, such a desired output ratio or ratio within the range of
tolerated error is achieved a certain tolerated percentage of the time.
sd-660418
735042000240
Example 4: Selection of Cells using Sub-Optimal Yield Concentrations of Fab-Coated
Surfaces
Human apheresis-derived PBPC samples, at a range of different cell numbers, were
incubated in a single composition with magnetic microbeads conjugated to anti-CD4 Fabs and
magnetic microbeads conjugated to anti-CD8 Fabs, with gentle mixing for about 30 minutes. This
incubation was followed by elution of non-selected cells, and recovery using magnetic field carried
out as described above. Incubation using between 0.05 and 1.5 mL of each of microbead reagent
per million cells produced low yields of CD4+ or CD8+ cells, respectively (cells recovered for the
respective selection compared with positive cells in the incubation), which in this study were
generally in the range of 15-70 %). On average, greater yields were observed at higher
concentrations of reagents per cell using such suboptimal yield concentrations.
Accordingly, in one example, a selection is carried out, in which PBMCs are
incubated with suboptimal concentrations per cell number of beads coupled to a CD4-binding and
beads coupled to a CD8-binding agent, and cells recovered using a magnetic field. Based on the
observed relationship between concentration of the reagent per cell and yield for the beads used,
one or the other of the CD4-binding or CD8-binding reagent is included at a higher concentration
(e.g., higher number of Cd4 or CD8 binding molecules included per cell), thereby producing an
output ratio of CD8+:CD4+ cells following selection, which is greater than or less than 1, such as
1.5:1 or 2:1 or 3:1 or 1:1.5, 1:2, or 1:3. The output ratio may be selected based on the desired ratio
of CD8 vs. CD4 cells in a composition containing engineered cells to be produced following one or
more additional steps, such as activation, transduction, and/or expansion.
sd-660418
Claims (47)
1. A method of enriching for CD4+ and CD8+ T cells, the method comprising: (a) contacting cells of a sample containing primary human T cells with a first immunoaffinity reagent that specifically binds to CD4 and a second immunoaffinity reagent that specifically binds to CD8 in an incubation composition, under conditions whereby the immunoaffinity reagents specifically bind to CD4 and CD8, respectively, on the surface of cells in the sample; and (b) recovering cells bound to the first and/or the second immunoaffinity reagent, thereby generating an enriched composition comprising CD4+ cells and CD8+ cells at a culture- initiating ratio, wherein: the first and/or second immunoaffinity reagent are present in the incubation composition at a sub-optimal yield concentration, whereby the enriched composition contains less than 70% of the total CD4+ cells in the incubation composition and/or less than 70% of the CD8+ cells in the incubation composition, thereby producing a composition enriched for CD4+ and CD8+ T cells.
2. The method of claim 1, further comprising: (a) incubating cells of the enriched composition in a culture-initiating composition in a culture vessel under stimulating conditions, thereby generating stimulated cells, wherein the cells are at or substantially at the culture-initiating ratio; and (b) introducing a genetically engineered antigen receptor into the stimulated cells of (a), thereby generating an output composition comprising CD4 T cells and CD8 T cells expressing the genetically engineered antigen receptor.
3. The method of claim 1 or claim 2, wherein the enriching for CD4+ and CD8+ T cells is performed in a closed system.
4. The method of any one of claims 1-3, wherein the first and second immunoaffinity reagents are present in the incubation composition at a sub-optimal yield concentration, whereby the enriched composition comprises less than 70% of the total CD4+ cells in the incubation composition and less than 70% of the total CD8+ cells in the incubation composition.
5. The method of any one of claims 1-4, wherein: the first immunoaffinity reagent is present in the incubation composition at a sub-optimal yield concentration, whereby the enriched composition comprises less than 60%, less than 50%, less than 40%, less than 30% or less than 20% of the total CD4+ cells in the incubation composition; and/or the second immunoaffinity reagent is present in the incubation composition at a sub-optimal yield concentration, whereby the enriched composition comprises less than 60%, less than 50%, less than 40%, less than 30% or less than 20% of the total CD8+ cells in the incubation composition.
6. The method of claim any one of claims 1-5, wherein the sample comprises at least 1 x 10 CD3+ T cells.
7. The method of any one of claims 1-6, wherein the concentration of one of the first and second immunoaffinity reagents in the incubation composition is greater than that of the other, whereby the greater concentration effects a higher yield in the enriched composition of CD4+ cells or CD8+ cells, respectively, compared to the yield of the other of the CD4+ or CD8+ cells, thereby producing the culture-initiating ratio in the enriched composition.
8. The method of claim 7, wherein: the concentration of one of the first and second immunoaffinity reagents is greater, as compared to the concentration of the other of the first and second immunoaffinity reagents, by at least 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold or 10-fold; and/or the higher yield in the enriched composition is greater by about 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold or 10-fold.
9. The method of any one of claims 1-8, wherein the culture-initiating ratio of CD4 cells to CD8 cells is between at or about 10:1 and at or about 1:10, between at or about 5:1 and at or about 1:5, or between at or about 2:1 and at or about 1:2.
10. The method of any of claims 2 and 4-22, wherein the desired ratio of CD4+ to CD8+ cells is at or about 1:1.
11. The method of any one of claims 1-10, wherein the sample is obtained from a human subject and: + + + the culture-initiating ratio of CD4 cells to CD8 cells is different than the ratio of CD4 cells to CD8 cells in the sample from the subject; and/or the culture-initiating ratio of CD4 cells to CD8 cells is at least 10 %, at least 20 %, at least 30 %, at least 40 %, or at least 50 % greater or less than the ratio of CD4 cells to CD8 cells in the sample from the subject.
12. The method of any one of claims 2-11, wherein greater than 95% or greater than 98% of the cells in the culture-initiating composition are CD4+ cells and CD8+ cells.
13. The method of any one of claims 1-12, wherein each of the immunoaffinity reagents comprises an antibody.
14. The method of claim 13, wherein the antibody is immobilized on the outside surface of a sphere.
15. The method of claim 14, wherein the sphere is a magnetic bead.
16. The method of claim 14 or claim 15, wherein: the antibody comprises one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on the sphere, whereby the antibody is reversibly immobilized to the sphere; and the method further comprises, after contacting cells in the sample to the first and second immunoaffinity reagents, applying a competition reagent to disrupt the bond between the binding partner and binding reagent, thereby recovering the selected cells from the sphere.
17. The method of claim 16, wherein: the binding partner is selected from among biotin, a biotin analog, or a peptide capable of binding to the binding reagent; and the binding reagent is selected from among streptavidin, a streptavidin analog or mutein, avidin, an avidin analog or mutein.
18. The method of claim 17, wherein: the binding partner comprises a peptide comprising the sequence of amino acids set forth in SEQ ID NO:6 and/or the binding reagent is streptavidin mutein comprising the sequence of amino acids set forth in SEQ ID NO: 12, 13, 15 or 16.
19. The method of claim 17 or claim 18, wherein: the binding reagent is a multimer comprising one or more monomers of streptavidin or a streptavidin mutein; and the binding partner is a peptide comprising a sequential arrangement of at least two modules each capable of reversibly binding with at least one monomer of the binding reagent.
20. The method of claim 19, wherein the binding partner comprises the sequence of amino acids set forth in any of SEQ ID NOS: 7-10.
21. The method of any one of claims 16-20, wherein the competition reagent is biotin or a biotin analog.
22. The method of any one of claims 1-21, wherein the first and/or second immunoaffinity reagent comprises an antibody having a dissociation rate constant (k ) for binding -5 -1 between the antibody and cell surface marker of greater than or greater than about 3 x 10 sec .
23. The method of any one of claims 1-22, wherein the first and/or second immunoaffinity reagent comprises an antibody having an affinity with a dissociation constant (K ) -7 -10 in the range of about 10 to about 10 .
24. The method of any one of claims 1-23, wherein one or more steps are carried out in an automated fashion.
25. The method of any one of claims 3-24, wherein the closed system is automated.
26. The method of any one of claims 1-25, comprising: prior to contacting cells of the sample with the first and second immunoaffinity reagents, determining the ratio of CD4+ to CD8+ T cells in the sample; and based on the ratio of CD4+ cells to CD8+ T cells in the sample, choosing the concentration of the first and/or second immunoaffinity reagent to produce the enriched composition comprising CD4+ cells and CD8+ cells at the culture-initiating ratio.
27. The method of any one of claims 1-26, wherein the first and/or second immunoaffinity reagent comprises an antigen-binding fragment (Fab).
28. The method of any one of claims 1-27, wherein the method produces an output composition comprising a ratio of CD4 cells to CD8 cells that is between at or about 2:1 and at or about 1:5.
29. The method of claim 28, wherein the ratio of CD4 cells to CD8 cells in the output composition is 1:1 or is about 1:1.
30. The method of any one of claims 1-29, wherein the sample is obtained from a subject.
31. The method of claim 30, wherein the subject is a subject to whom said genetically engineered T cells or cells for adoptive cell therapy will be administered.
32. The method of claim 30, wherein the subject is a subject other than a subject to whom said genetically engineered T cells or cells for adoptive therapy will be administered.
33. The method of any one of claims 1-32, wherein the sample is blood or a blood- derived sample.
34. The method of any one of claims 1-33, wherein the sample is a white blood cell sample.
35. The method of any one of claims 1-34, wherein the sample is an apheresis sample, a peripheral blood mononuclear cell (PBMC) sample, or a leukapheresis sample.
36. The method of any one of claims 2-35, wherein incubating the composition in a culture vessel under stimulating conditions is performed prior to, during and/or subsequent to introducing a genetically engineered antigen receptor.
37. The method of any one of claim 2-35, wherein incubating the composition in a culture vessel under stimulating conditions is performed prior to, during and subsequent to introducing a genetically engineered antigen receptor.
38. The method of any one of claims 2-37, wherein the stimulating conditions comprise conditions whereby T cells of the composition proliferate.
39. The method of any one of claims 2-38, wherein the stimulating conditions comprise an agent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex.
40. The method of claim 39, wherein the one or more components of the TCR complex comprises the CD3 zeta chain.
41. The method of any one of claims 2-40, wherein the stimulating conditions comprise the presence of an anti-CD3 antibody, and anti-CD28 antibody, anti1BB antibody, and/or a cytokine.
42. The method of claim 41, wherein the anti-CD3 antibody and/or the anti-CD28 antibody is present on the surface of a solid support.
43. The method of claim 41 or claim 42, wherein the cytokine comprises IL-2, IL-15, IL- 7, and/or IL-21.
44. The method of any one of claims 2-43, wherein the genetically engineered antigen receptor comprises a T cell receptor (TCR) or a functional non-TCR antigen receptor.
45. The method of claim 44, wherein the genetically engineered antigen receptor specifically binds to an antigen expressed by cells of a disease or condition to be treated.
46. The method of claim 44 or claim 45, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR).
47. The method of claim 46, wherein the CAR comprises an extracellular antigen- recognition domain and an intracellular signaling domain comprising an ITAM-containing sequence and an intracellular signaling domain of a T cell costimulatory molecule.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201461983415P | 2014-04-23 | 2014-04-23 | |
US61/983,415 | 2014-04-23 | ||
NZ726346A NZ726346B2 (en) | 2014-04-23 | 2015-04-23 | Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ763754A NZ763754A (en) | 2021-09-24 |
NZ763754B2 true NZ763754B2 (en) | 2022-01-06 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2021209195B2 (en) | Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy | |
AU2016341533B2 (en) | Methods for culturing cells and kits and apparatus for same | |
EP3877054B1 (en) | Process for producing genetically engineered t cells | |
WO2016166568A1 (en) | Methods, kits and apparatus for expanding a population of cells | |
JP2024517863A (en) | Methods for stimulating and transducing cells | |
NZ763754B2 (en) | Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy | |
NZ763755B2 (en) | Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy | |
RU2795454C2 (en) | Methods and compositions for obtaining genetically engineered cells | |
NZ726346B2 (en) | Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy |