Compositions and methods for hematopoietic stem cell transplants

Abstract

To provide distinct therapeutic populations of cells that form a pharmaceutical composition useful in hematopoietic stem / progenitor cell transplant.SOLUTION: The present disclosure provides a therapeutic population of cells comprising an enriched population of hematopoietic stem / progenitor cells, memory T cells, regulatory T cells, the population of cells being depleted of conventional naive αβ-T cells. The present disclosure further provides methods of treatment using the therapeutic population of cells. In other embodiments, the present disclosure provides methods of producing a therapeutic population of cells.SELECTED DRAWING: Figure 19

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 471,769, filed March 15, 2017, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Allogeneic hematopoietic stem cell transplantation (HSCT) typically involves the transfer of hematopoietic cells from an immunocompatible healthy donor into a patient after a conditioning regimen. Healthy hematopoietic stem cells (HSCs) can replace a patient's damaged hematopoietic tissue, and specific donor-derived immune cells can have therapeutic effects against cancer, infectious diseases, and immune system disorders. However, currently used methods of allogeneic HSCT involve either heterogeneous cell mixtures containing nontherapeutic contaminating cells or purified but limited cell mixtures that lack the potential therapeutic benefit of a complete graft. In the former scenario, many nontherapeutic cells contaminating the therapeutically important cells are harmless, but even minor populations of specific undesirable cell types can have severe adverse consequences for the recipient. For example, residual tumor cells or teratoma stem-like cells contaminating the transplanted cell population can seed tumors in the patient. In another example, a subset of circulating T cells can cause graft-versus-host disease (GVHD), a severe and often fatal complication of allogeneic HSCT. In such cases, the pathology caused by the contaminating cells overrides the therapeutic benefits of other T cells introduced during transplantation. While allogeneic HSCT is often a curative treatment for the underlying disorder, the medical procedure itself can be harmful to the patient if the contaminating cells in the graft react to their new host environment. Thus, there is a significant unmet need for transplant HSC compositions with a low number of harmful cells and an optimal therapeutic cell mixture. Summary of the Invention

[0003] overview In some embodiments, disclosed herein are pharmaceutical compositions comprising one or more unit doses of a cell graft, each unit dose of the cell graft comprising a therapeutic cell population per kilogram (kg) of body weight of the subject receiving the cell graft. In some embodiments, each unit dose of the therapeutic cell population comprises 3×10 5 More hematopoietic stem / progenitor cells (HSPCs), 3 × 10 5 More memory T cells (Tmem), 5 × 10 5 More regulatory T cells (Tregs) and 3 × 10 5 In some embodiments, the unit dose comprises less than 0.5 x 10 normal naive αβ-T cells. 3 ~2000×10 3 In some embodiments, the HSPCs further comprise invariant natural killer T (iNKT) cells. + and Tmem is CD3 + CD45RA - CD45RO + and Tregs are CD4 + CD25 + CD127 - / lo , CD45RA + or a combination thereof, where normal naive αβ-T cells express CD3 + CD45RA + CD25 - Va24Ja18 - In some embodiments, the iNKT is CD3 + Vα24Jα18 + is.

[0004] In some embodiments, the pharmaceutical compositions disclosed herein comprise a therapeutic cell population enriched for HSPCs, memory T cells (Tmems), and Tregs, wherein normal naive αβ-T cells have been depleted and the therapeutic cell population comprises a ratio of normal naive αβ-T cells to Tregs of less than 1:5. In some embodiments, the pharmaceutical composition further comprises invariant iNKT, wherein the therapeutic cell population comprises a ratio of normal naive αβ-T cells to iNKT of less than 100:1. In some embodiments, the therapeutic cell population comprises a ratio of normal naive αβ-T cells to HSPCs that is less than 1:400; and a ratio of normal naive αβ-T cells to Tmems that is less than 1:800. In some embodiments, the therapeutic cell population comprises iNKT, wherein the therapeutic cell population comprises a ratio of naive normal αβ-T cells to HSPCs of less than 1:400; a ratio of naive normal αβ-T cells to Tmem of less than 1:800; or a ratio of naive normal αβ-T cells to iNKT of less than 100:1. In some embodiments, the therapeutic cell population comprises a ratio of naive normal αβ-T cells to HSPCs of less than 1:400. In some embodiments, the therapeutic cell population comprises a ratio of naive normal αβ-T cells to Tmem of less than 1:3.

[0005] In some aspects, provided herein are methods of treating a disease or disorder comprising administering a therapeutic cell population to a subject in need thereof, wherein the therapeutic cell population is greater than or equal to 1.0 x 10 6 ~50×10 6 HSPCs, concentration 0.1 x 10 cells / kg subject weight 6 ~1000×10 6 Tmem concentration of cells / kg subject weight, 0.1 x 10 6 ~1000×10 6 Tregs at a concentration of 3 × 10 cells / kg subject weight, and 5 Disclosed are methods that include conventional naive αβ-T at a concentration of fewer cells / kg of subject body weight. In some embodiments, the therapeutic cell population is 0.5×10 3 ~2000×10 3 Further included are iNKT cells at a concentration of cells / kg subject weight.

[0006] In some aspects, as used herein, (A) The sample is treated with a binding molecule that specifically binds to CD34 and a CD34 + contacting the cells under conditions that result in an enriched population and a population of CD34-depleted cells; + recovering the enriched population of cells and recovering the population of CD34-depleted cells; and (B) The population of CD34-depleted cells is combined with a binding molecule that specifically binds CD25 and a CD25 + contacting the cells under conditions that result in an enriched population and a population of CD25-depleted cells; + recovering the enriched population of cells and recovering the population of CD25-depleted cells; and (C) The population of CD25-depleted cells is coupled to a binding molecule that specifically binds CD45RA and CD45RA + contacting under conditions to obtain an enriched population of cells and a population of CD45RA-depleted cells, and recovering the population of CD45RA-depleted cells; and (D) The population enriched for CD34+ cells, CD25 + formulating the enriched population of cells and the population of CD45-depleted cells as pharmaceutical compositions suitable for administration to a subject. The present invention discloses a method for producing a pharmaceutical composition comprising processing at least one sample to obtain a therapeutic cell population. [The present invention 1001] 1. A pharmaceutical composition comprising a therapeutic cell population enriched for hematopoietic stem / progenitor cells (HSPCs), memory T cells (Tmem), and regulatory T cells (Tregs), comprising: the cell population is depleted of normal naive αβ-T cells, and the pharmaceutical composition comprises a ratio of normal naive αβ-T cells to Tregs that is less than 1:5; Pharmaceutical compositions. [The present invention 1002] The pharmaceutical composition of this invention 1001, further comprising an enriched population of invariant natural killer T cells (iNKT). [The present invention 1003] The pharmaceutical composition comprising one or more unit doses of a cell transplant, wherein each unit dose of the cell transplant comprises a therapeutic cell population per kilogram (kg) of body weight of the subject receiving the cell transplant, and wherein each unit dose of the therapeutic cell population comprises: 3×10 5 More hematopoietic stem / progenitor cells (HSPCs), 3×10 5 more memory T cells (Tmem), 5×10 5 More regulatory T cells (Tregs), and 3×10 5 Less normal naive αβ-T The pharmaceutical composition of the present invention 1001 or 1002, comprising: [The present invention 1004] Unit dose is 0.5 × 10 3 The pharmaceutical composition of the present invention 1003, further comprising more iNKT cells. [The present invention 1005] Each unit dose of the therapeutic cell population comprises: 1.0×10 6 ~50×10 6 hematopoietic stem / progenitor cells (HSPCs), 0.3×10 6 ~1000×10 6 memory T cells (Tmem), 0.5×10 6 ~1000×10 6 regulatory T cells (Tregs), and 3×10 5 Less normal naive αβ-T The pharmaceutical composition of any one of 1001 to 1004 of the present invention, comprising: [The present invention 1006] The unit dose is 0.5 x 10 3 ~2000×10 3 Any of the aforementioned pharmaceutical compositions of the present invention, further comprising invariant natural killer T (iNKT) cells at a concentration of cells / kg. [The present invention 1007] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the Tregs comprise a population of naive Tregs, a population of memory Tregs, or both. [The present invention 1008] Tmem is a transcription factor that regulates the function of central memory T cells (T CM ) population, effector memory T cells (T EM ), or any combination thereof. [The present invention 1009] Central memory T stem cells (T SCM Any of the aforementioned pharmaceutical compositions of the present invention comprising a population of [The present invention 1010] HSPCs are CD34 + Any of the pharmaceutical compositions of the present invention, [The present invention 1011] HSPCs express CD133 + , CD90 + , CD38 - , CD45RA - , Lin - or any combination thereof. [The present invention 1012] HSPC cKIT + Any of the pharmaceutical compositions of the present invention, [The present invention 1013] HSPCs express CD19 - , TCRα - or a combination thereof. [The present invention 1014] Tmem, CD3 + , CD45RA - , CD45RO + or any combination thereof. [The present invention 1015] Tregs, CD4 + , CD25 + , CD127 - / lo , FoxP3 +or any combination thereof. [The present invention 1016] Naive Tregs express CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA + , CD45RO - or any combination thereof. [The present invention 1017] Memory Tregs are CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA - , CD45RO + or any combination thereof. [The present invention 1018] T SCM However, CD45RA + and CD4 + or CD8 + Any of the pharmaceutical compositions of the present invention, [The present invention 1019] T SCM But CD95 + , CD122 + , CXCR3 + , LFA-1 + or any combination thereof. [The present invention 1020] T CM However, CD45RO + and CD4 + or CD8 + Any of the pharmaceutical compositions of the present invention, [The present invention 1021] T CM However, CD45RA - , CD62L + , CCR7 + or any combination thereof. [The present invention 1022] T EM But CD4 + , CD45RO + , CD45RA - , CD62L - , CCR7 - or any combination thereof. [The present invention 1023] iNKT, CD1d-tet + , 6B11 + or both. [The present invention 1024] iNKT is Vα24Jα18 + Any of the pharmaceutical compositions of the present invention, [The present invention 1025] Normal naive αβ-T cells express CD25 - , CD127 + , or both, as well as TCRα + and CD45RA + Any of the pharmaceutical compositions of the present invention, [The present invention 1026] Normal naive αβ-T cells express TCRα + TCRβ + CD45RA + CD45RO - CD25 - CD95 - IL-2Rβ - CD127 + Any of the pharmaceutical compositions of the present invention, [The present invention 1027] Any of the pharmaceutical compositions of the present invention, wherein the ratio of HSPC to Tmem is 500:1 to 1:1,000. [The present invention 1028] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the ratio of HSPC to Treg is about 100:1 to about 1:30. [The present invention 1029] Any of the pharmaceutical compositions of the present invention, wherein the ratio of HSPCs to naive Tregs is 1:500 to 100:1. [The present invention 1030] Any of the pharmaceutical compositions of the present invention, wherein the ratio of HSPC to memory Treg is 1:500 to 10,000:1. [The present invention 1031] Any of the pharmaceutical compositions of the present invention, wherein the ratio of HSPC to iNKT is 1:2 to 500,000:1. [The present invention 1032] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the ratio of normal naive αβ-T cells to HSPCs is less than 1:3, preferably less than 1:400. [The present invention 1033] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the ratio of normal naive αβ-T cells to Tmem is less than 1:30, preferably less than 1:800. [The present invention 1034] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the ratio of normal naive αβ-T cells to naive Tregs is less than 1:1, preferably less than 1:10. [This invention 1035] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the ratio of normal naive αβ-T cells to memory Tregs is less than 1:1, preferably less than 1:100. [The present invention 1036] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the ratio of normal naive αβ-T cells to iNKT cells is less than 100:1, preferably less than 1:1. [This invention 1037] Any of the pharmaceutical compositions of the present invention, wherein the ratio of Tmem to Treg is 2000:1 to 1:10, preferably 30:1 to 1:1. [The present invention 1038] Any of the pharmaceutical compositions of the present invention, wherein the ratio of Tmem to naive Treg is 3:1 to 0.1:1. [This invention 1039] Any of the pharmaceutical compositions of the present invention, wherein the ratio of Tmem to memory Treg is 27:1 to 0.9:1. [The present invention 1040] Any of the pharmaceutical compositions of the present invention, wherein the ratio of iNKT to Tmem is about 5:1 to about 1:1,000,000. [This invention 1041] Unit dose is 1.0 x 10 6 ~50×10 6 Any of the pharmaceutical compositions of the present invention, comprising HSPCs of the present invention. [The present invention 1042] Unit dose is 0.3 × 10 6 ~1000×10 6 Any of the pharmaceutical compositions of the present invention, comprising memory T cells of the present invention. [This invention 1043] Unit dose is 0.5 × 10 6 ~1000×10 6 Any of the pharmaceutical compositions of the present invention, comprising Treg cells of the above formula (I). [This invention 1044] Unit dose is 0.2 × 10 6 ~500×10 6 Any of the pharmaceutical compositions of the present invention, comprising naive Treg cells. [This invention 1045] Unit dose is 0.5 × 10 6 ~500×10 6 Any of the pharmaceutical compositions of the present invention, comprising memory Treg cells of the above formula (I). [The present invention 1046] Unit dose is 0.5 × 10 3 ~2000×10 3 Any of the pharmaceutical compositions of the present invention, comprising iNKT cells of the present invention. [This invention 1047] Unit dose is 3 x 10 5 Any of the aforementioned pharmaceutical compositions of the present invention, comprising fewer normal naive αβ-T cells. [This invention 1048] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the cell population is obtained by processing one or more tissue harvests. [This invention 1049] The pharmaceutical composition of claim 1048, wherein the one or more tissue harvests are from one or more donors. [The present invention 1050] The pharmaceutical composition of invention 1048 or 1049, wherein the tissue collection is from an HLA-matched sibling donor, an HLA-matched unrelated donor, a partially matched unrelated donor, a haploidentical related donor, an autologous donor, an HLA-mismatched allogeneic donor, a donor pool, or any combination thereof. [This invention 1051] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the HSPCs are provided by a donor who is haplotype-matched to the subject. [This invention 1052] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the Treg, Tmem, iNKT, or any combination thereof, is provided by a donor who is an HLA-matched sibling donor or an HLA-matched unrelated donor. [This invention 1053] Any of the preceding pharmaceutical compositions of the present invention, wherein the cells are formulated for infusion or injection. [This invention 1054] Any of the aforementioned pharmaceutical compositions of the present invention, wherein the cell population constitutes a formulation for administration to a subject. [This invention 1055] 1054. The pharmaceutical composition of claim 10, wherein the formulation comprises Normosol-R and human serum. [The present invention 1056] The pharmaceutical composition of invention 1054 or 1055, wherein human serum is 1% of the total formulation. [This invention 1057] Use of any of the pharmaceutical compositions of the present invention in the treatment of a disease or disorder. [This invention 1058] The use of the present invention 1057, wherein the disease or disorder is leukemia, lymphoma, chronic infection, or autoimmune disease, malignant or non-malignant blood disease, AML, ALL, CML, CLL, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, MDS, lymphoproliferative disorders, type 1 diabetes, inborn errors of metabolism, genetic disorders, sickle cell anemia, beta-thalassemia, multiple sclerosis, solid organ transplantation, Crohn's disease, ulcerative colitis, lupus, hemophagocytic lymphohistiocytosis, glycogen storage disease, mucopolysaccharidosis, or any other disease that would benefit from HSPC transplantation. [This invention 1059] The use of either of inventions 1057 or 1058, wherein each therapeutic cell population is administered to said subject as an individual pharmaceutical composition. [The present invention 1060] The use of any of inventions 1057 or 1058, wherein the therapeutic cell populations are administered to said subject as a single pharmaceutical composition. [This invention 1061] The use of any of claims 1057 to 1060, wherein the cells are isolated from a donor that is an HLA-matched sibling donor, an HLA-matched unrelated donor, a partially matched unrelated donor, a haploidentical related donor, an autologous donor, an HLA-mismatched donor, a donor pool, or any combination thereof. [This invention 1062] The use of any of claims 1057 to 1061, wherein the therapeutic cell population is allogeneic or autologous. [This invention 1063] 1063. The use of any of claims 1057 to 1062, wherein the therapeutic cell population is autologous. [This invention 1064] The use of any of claims 1057 to 1063, wherein the therapeutic cell population is haplotype-matched. [This invention 1065] The use of any of claims 1057-1064, wherein the therapeutic cell population is isolated from mobilized peripheral blood, mobilized apheresis blood products, bone marrow, umbilical cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof. [The present invention 1066] 1065. The use of any of claims 1057 to 1065, wherein the therapeutic cell population is derived from a single tissue harvest. [This invention 1067] 1067. The use of any of claims 1057 to 1066, wherein the therapeutic cell population is derived from one or more tissue harvests. [The present invention 1068] 8. The use of any of claims 1057 to 1067, wherein the therapeutic cell population comprises HSPCs provided by at least a first donor and Tregs and Tmems provided by at least a second donor. [The present invention 1069] The use of the present invention 1068, wherein the therapeutic cell population comprises iNKT cells provided by at least a second donor. [The present invention 1070] The use of invention 1068 or 1069, wherein the haplotype of the first donor is matched to said subject. [This invention 1071] 1070. The use of any of claims 1068 to 1070, wherein the second donor is an HLA-matched sibling donor or an HLA-matched or partially matched unrelated donor. [This invention 1072] The use of any of claims 1057 to 1071, wherein said subject is a human, a non-human primate, a cow, a horse, a sheep, a goat, a pig, a dog, a cat, a mouse, a rabbit, a rat, or a guinea pig. [This invention 1073] Processing at least one sample (a) Enriched hematopoietic stem / progenitor cell (HSPC) populations; (b) enriched populations of regulatory T cells (Tregs); (c) Enriched memory T cell (Tmem) population obtaining the compound; (d) formulating the enriched populations of HSPCs, memory T cells, and Tregs into pharmaceutical compositions suitable for administration to a subject. Including, wherein the populations of (a) to (c) are depleted of normal naive αβ-T cells; A method of making any of the pharmaceutical compositions of the present invention. [This invention 1074] The sample was processed to obtain Lin + The method of claim 1073, further comprising the step of obtaining a cell-depleted population of cells. [This invention 1075] The method of any one of claims 1073 to 1074, wherein obtaining an enriched population of Tregs comprises obtaining an enriched population of naive Tregs, an enriched population of memory Tregs, or both. [This invention 1076] Obtaining an enriched population of Tmem is considered to be an enriched central memory T cell (T CM ), a population of enriched effector memory T cells (T EM 1073-1075, or any combination thereof. [This invention 1077] The method of any of claims 1073 to 1076, further comprising the step of processing said sample to obtain an enriched population of iNKT cells. [This invention 1078] Enriched HSPC populations are at least 50% CD34 + 1073. The method of any of claims 1073 to 1077, comprising HPSCs. [This invention 1079] Lin + The cell-depleted cell population is 1%-30% Lin + cells, preferably less than 1% Lin + 1079. The method of any one of claims 1073 to 1078, comprising a cell. [The present invention 1080] 1079. The method of any of claims 1073 to 1079, wherein the enriched population of Tregs comprises 20% to 99.9% Tregs. [This invention 1081] 1073-1080. The method of any of claims 1073-1080, wherein the enriched population of Tmems comprises 10% to 99.9% Tmems. [This invention 1082] Any of the methods of claims 1073 to 1081, wherein the enriched population of iNKT comprises 10% to 99.9% iNKT. [This invention 1083] Any of the methods of claims 1073 to 1082, wherein formulating the pharmaceutical composition comprises combining an enriched HSPC population, a memory T cell population, a Treg population, an iNKT population, or any combination thereof, into a mixed population of enriched cells. [This invention 1084] 1083. The method of claim 1083, wherein the mixed population of enriched cells comprises a ratio of HSPCs to memory T cells of 500:1 to 1:1,000. [This invention 1085] 1083 or 1084, wherein the mixed population of enriched cells comprises a ratio of HSPCs to naive Tregs of 1:500 to 100:1. [The present invention 1086] 6. The method of any of claims 1083 to 1085, wherein said mixed population of enriched cells comprises a ratio of HSPCs to memory Tregs of 1:500 to 10,000:1. [This invention 1087] 1087. The method of any of claims 1083 to 1086, wherein the mixed population of enriched cells comprises a ratio of HSPC to iNKT of 1:2 to 500,000:1. [This invention 1088] 8. The method of any of claims 1083 to 1087, wherein said mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to HSPCs that is less than 1:3, preferably less than 1:400. [This invention 1089] 9. The method of any of claims 1083 to 1088, wherein said mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to Tmem that is less than 1:30, preferably less than 1:800. [The present invention 1090] 1089. The method of any of claims 1083 to 1089, wherein said mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to naive Tregs that is less than 1:1, preferably less than 1:10. [This invention 1091] 1090. The method of any of claims 1083 to 1090, wherein said mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to memory Tregs that is less than 1:1, preferably less than 1:100. [This invention 1092] 1092. The method of any of claims 1083 to 1091, wherein said mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to iNKT cells that is less than 100:1, preferably less than 1:1. [This invention 1093] 13. The method of any of claims 1083 to 1092, wherein said mixed population of enriched cells comprises a ratio of Tmem to Treg that is between 30:1 and 1:1. [This invention 1094] 1093. The method of any of claims 1083 to 1093, wherein said mixed population of enriched cells comprises a ratio of Tmem to naive Treg that is between 3:1 and 0.1:1. [This invention 1095] 1095. The method of any of claims 1083 to 1094, wherein said mixed population of enriched cells comprises a ratio of Tmem to memory Treg that is between 27:1 and 0.9:1. [This invention 1096] 1096. The method of any of claims 1083 to 1095, wherein said mixed population of enriched cells comprises less than 0.0014% normal naive αβ-T cells. [This invention 1097] 1096. The method of any of claims 1074 to 1096, wherein the Lin+ cells express CD19, CD11c, CD66B, CD14, CD20, or any combination thereof. [This invention 1098] 10. The method of any one of claims 1073 to 1097, wherein the HSPC is CD34+. [This invention 1099] 1098. The method of any of claims 1073 to 1098, wherein the HSPCs are CD19- and TCRα / β-. [The present invention 1100] 109. The method of any of claims 1073 to 1099, wherein the HSPCs are CD133+, CD90+, CD38-, CD45RA-, Lin-, or any combination thereof. [The present invention 1101] Tmem, CD45RA - , CD45RO + or any combination thereof. [The present invention 1102] Tmem is T CM The method of the present invention 1101. [The present invention 1103] T CM However, CD45RO + and CD4 + or CD8 + The method of the present invention 1102. [The present invention 1104] T CMHowever, CD45RA - , CD62L + , CCR7 + 1102 or 1103, or any combination thereof. [This invention 1105] Tmem is T EM The method of the present invention 1101. [The present invention 1106] T EM But CD4 + , CD45RA + , CD45RO - , CD62L - , CCR7 - , or any combination thereof. [This invention 1107] Tregs, CD4 + , CD25 + , CD127 - / lo , FoxP3 + or any combination thereof. [This invention 1108] The method of the present invention 1107, wherein the Tregs are naive Tregs, memory Tregs, or both. [This invention 1109] Naive Tregs express CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA + , CD45RO - or any combination thereof. [The present invention 1110] Memory Tregs are CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA - , CD45RO + or any combination thereof. [The present invention 1111] 11. The method of any of claims 1073 to 1110, wherein the sample comprises mobilized peripheral blood, mobilized apheresis blood products, bone marrow, umbilical cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof. [The present invention 1112] 12. The method of any of claims 1073 to 1111, wherein the sample is prepared for processing with a density gradient, Ficoll, Percoll, red blood cell hypotonic lysis, ammonium chloride-potassium (ACK) buffer, or any combination thereof. [The present invention 1113] The method of any one of claims 1073 to 1111, wherein said sample is obtained by a single tissue sampling. [This invention 1114] 11. The method of any one of claims 1073 to 1111, wherein said sample is obtained by one or more tissue samplings. [This invention 1115] Any of the methods of claims 1073 to 1114, wherein the enriched cell population is obtained by density separation, tetrameric antibody complex-mediated enrichment / depletion, magnetic-activated cell sorting, multi-parameter fluorescence-based molecular phenotyping, or any combination thereof. [The present invention 1116] (A) The sample is treated with a molecule that specifically binds to CD34 and a CD34 + Cell populations and CD34 - The cells were contacted under conditions that allowed a population of CD34 + A population of cells is recovered from the sample and CD34 - recovering a population of cells from the sample; and (B) CD34 - Processing the population of cells to obtain at least one population of enriched therapeutic cells comprising Tregs, Tmems, iNKTs, or any combination thereof. Any of the methods of claims 1073 to 1115, comprising: [This invention 1117] 1116. The method of claim 1116, wherein step B comprises performing precision sorting to obtain an enriched population of therapeutic cells. [This invention 1118] Process B is CD34 - contacting the population of cells with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof. The method of the present invention 1116 or 1117, comprising: [This invention 1119] Process B is (i) CD34 - The population of cells is treated with at least one molecule that specifically binds to CD45RA and CD45RA + Cell populations and CD45RA - The cells are contacted under conditions in which a population of CD45RA - harvesting the population of cells; and (ii) CD45RA to obtain enriched therapeutic cell populations + To perform precise selection from a population of cells The method of the present invention 1116, comprising: [The present invention 1120] Precision selection, CD45RA + contacting the population of cells with a molecule that specifically binds CD4, a molecule that specifically binds CD8, a molecule that specifically binds CD25, a molecule that specifically binds CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof. The method of the present invention 1119, comprising: [This invention 1121] Precision selection, CD45RA - contacting the population of cells with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, or a combination thereof; The method of the present invention 1120 further comprising: [This invention 1122] Process B is (i) CD34- The cell population was analyzed by Lin + at least one binding molecule that specifically binds to the marker; + Populations of cells and Lin - The cells were contacted under conditions in which a population of cells was formed, and Lin - harvesting the population of cells; and (ii) Lin to obtain an enriched population of therapeutic cells - To perform precise selection from a population of cells The method of the present invention 1116, comprising: [This invention 1123] Lin + 1123. The method of claim 1122, wherein the marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof. [This invention 1124] Precision selection, Lin - contacting the population of cells with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, a CD1d-tet molecule, a 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof. The method of the present invention 1122 or 1123, comprising: [This invention 1125] Process B is (i) CD34 - A population of cells was cultured using at least one Lin + at least one binding molecule that specifically binds to the marker; + Populations of cells and Lin - The cells were contacted under conditions in which a population of cells was formed, and Lin - harvesting the population of cells; and (ii) Lin - The population of cells is treated with a binding molecule that specifically binds to CD25 and a CD25 + Cell populations and CD25 - The cells are contacted under conditions in which a population of CD25 +The cell population is harvested, thereby generating a population of cells containing Tregs, and CD25 - harvesting the population of cells; and (iii) CD25 - a binding molecule that specifically binds to CD45RA; and + Cell populations and CD45RA - The cells are contacted under conditions in which a population of CD45RA - Harvesting a population of cells The method of the present invention 1116, comprising: [Invention 1126] Lin + 1126. The method of claim 1125, wherein the marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof. [This invention 1127] Step (ii) is Lin - The cells are treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Population of cells, 6B11 + populations of cells, or combinations thereof, and CD1d-tet - Population of cells, 6B11 - and contacting the cells under conditions in which CD1d-tet + Harvesting a population of cells The method of claim 1125 or 1126, further comprising: [This invention 1128] CD25 + Populations of cells and CD1d-tet + Population of cells, 6B11 + 1128. The method of any of claims 1119 to 1127, wherein the cells or both are simultaneously harvested. [This invention 1129] In order to obtain a population of naive Treg cells, a population of memory Treg cells, a population of iNKT cells, or any combination thereof, the CD25 +The method of any one of claims 1125 to 1128, further comprising performing precision sorting of the population of cells. [The present invention 1130] CD45RA + The method of any of claims 1125 to 1129, wherein the population of cells is collected and further subjected to fine selection to obtain a population of Treg, iNKT or both. [This invention 1131] The method of any of claims 1117 to 1130, wherein the precision selection comprises purification using multi-parameter fluorescence-based molecular phenotyping. [This invention 1132] (A) A first coarse selection is performed on at least a first sample, thereby detecting CD34 + Obtaining an enriched population of cells; (B) A second sample is subjected to a second coarse selection, thereby obtaining Lin - Obtaining a population of cells; (C)Lin - The population of cells was subjected to a third crude selection, which resulted in the CD45RA - Obtaining a population of memory T cells and CD45RA + Obtaining a population of cells; and (D)CD45RA + Perform precision selection on a population of cells to obtain a population of Tregs Any of the methods of claims 1073 to 1115, comprising: [This invention 1133] A second sample was prepared from the CD34 - The method of claim 1132, comprising a population of cells. [This invention 1134] The method of any one of claims 1132 to 1133, wherein the first sample comprises at least one haplotype-matched sample. [This invention 1135] The method of any one of claims 1132 to 1133, wherein the first sample comprises at least two haploidentical samples. [This invention 1136] 1136. The method of any of claims 1132 to 1135, wherein the first sample comprises mobilized peripheral blood, a mobilized apheresis blood product, bone marrow, umbilical cord blood, non-mobilized blood, a non-mobilized apheresis blood product, or any combination thereof. [This invention 1137] Any of the methods of claims 1132 to 1136, wherein the second sample comprises peripheral blood mononuclear cells (PBMCs), mobilized peripheral blood, mobilized apheresis blood products, bone marrow, umbilical cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof. [This invention 1138] Any of the methods of claims 1132 to 1137, wherein the first, second, or third crude sorting comprises density separation, tetrameric antibody complex-mediated enrichment / depletion, magnetic-activated cell sorting, apheresis, leukapheresis, or any combination thereof. [This invention 1139] The method of any of claims 1132 to 1138, wherein the precision selection comprises purification using multi-parameter fluorescence-based molecular phenotyping. [This invention 1140] The method of any of claims 1132 to 1139, wherein precision selection results in a population of naive Treg cells, a population of memory Treg cells, a population of iNKT cells, or any combination thereof. [This invention 1141] Precision selection, CD45RA + The cells are then treated with a binding molecule that specifically binds to CD25 and a CD25 + Cell populations and CD25 - contacting the cells under conditions that result in a population of cells that are CD25 + Harvesting a population of cells, thereby obtaining a population of Tregs Any of the methods of the present invention 1132 to 1140, comprising: [This invention 1142] CD25 + The method of claim 1141, wherein the population of cells is further selected under conditions to obtain a population of naive Treg cells. [This invention 1143] Precision selection, CD45RA+ The cells are treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Population of cells, 6B11 + and contacting the cells under conditions that result in CD1d-tet + Population of cells, 6B11 + harvesting cells, or both Any of the methods of the present invention 1132 to 1142, comprising: [This invention 1144] The method of any of claims 1132 to 1143, wherein the first sample or the second sample is allogeneic, autologous, or a combination thereof. [Invention 1145] The method of any of claims 1132 to 1144, wherein the second sample is derived from an HLA-matched unrelated donor, an HLA-matched sibling donor, or a combination thereof. [Invention 1146] Lin + 1146. The method of any of claims 1132 to 1145, wherein the marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof. [This invention 1147] (A) The sample was + a binding molecule that specifically binds to the marker; + Populations of cells and Lin - The cells are contacted under conditions that result in a population of Lin - harvesting the population of cells; and (B) Lin - The cells were treated with a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25, and a binding molecule that specifically binds to CD34. + A population of cells, CD25 + population of cells, and CD34 - CD25 - contacting the cells under conditions that result in a population of CD34 + Cells and CD25 + Cells were harvested and CD34 - CD25 - harvesting the population of cells; and (C)CD34 - CD25 - The population of cells is treated with a binding molecule that specifically binds to CD45RA and a CD45RA + Cell populations and CD45RA - and contacting the cells under conditions that result in a population of CD45RA - recovering the population of Any of the methods of claims 1073 to 1115, comprising: [Invention 1148] Lin + 1147. The method of claim 1147, wherein the marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof. [This invention 1149] Process B is Lin - The cells are treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Population of cells, 6B11 + and contacting the cells under conditions that result in CD1d-tet + cells, 6B11 + harvesting cells, or a combination thereof; The method of any one of claims 1147 to 1148, further comprising: [This invention 1150] CD34 + cells, CD25 + cells, CD1d-tet + cells, 6B11 + cells, or any combination thereof, thereby detecting CD34 + cells, CD25 + cells, CD1d-tet + cells, 6B11 + 1149. The method of any of claims 1147 to 1149, further comprising the step of obtaining the population of cells, or any combination thereof. [This invention 1151] Precision selection, CD34 + cells, CD25 +cells, CD1d-tet + cells, 6B11 + The cells, or any combination thereof, are treated with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof, and a CD34 + cells, CD4 + CD25 + CD127 - / Lo cells, CD1d-tet + contacting the cells, or combinations thereof, under conditions that result in a highly enriched population of cells; The method of the present invention 1150, comprising: [This invention 1152] (A) The sample is roughly selected and Lin + Populations of cells and Lin - Obtaining a population of cells and - harvesting the population of cells; and (B) Lin - By performing crude sorting of cell populations, we identified populations enriched for HSPCs and Tmem and CD45RA + A population of cells was obtained, and the HSPC and Tmem populations were recovered, and CD45RA + harvesting the population of cells; and (C)CD45RA + A process of obtaining a Treg population by precise selection of a cell population. Any of the methods of claims 1073 to 1115, comprising: [This invention 1153] Precision selection, CD45RA + The population of cells is subjected to a binding molecule that specifically binds CD34, a binding molecule that specifically binds CD4, and a binding molecule that specifically binds CD127, and a population of CD34+ cells and a population of CD4 + CD25 + CD127 - / Lo and contacting the cells under conditions that result in a population of CD34+ cells and a population of CD4 + CD25+ CD127 - / Lo Harvesting a population of cells The method of the present invention 1152 further comprises: [This invention 1154] Precision selection, CD45RA + The cell population is contacted with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, to induce CD1d-tet + Population of cells, 6B11 + harvesting cells, or a combination thereof; The method of the present invention 1152 to 1153 further comprises: [This invention 1155] Any of the methods of 1152 to 1154, wherein the crude sorting comprises density separation, tetrameric antibody complex-mediated enrichment / depletion, magnetic-activated cell sorting, apheresis, leukapheresis, or any combination thereof. [Invention 1156] The method of any of claims 1152 to 1155, wherein precision selection comprises purification using multi-parameter fluorescence-based molecular phenotyping. [This invention 1157] (A) The sample is treated with a binding molecule that specifically binds to CD34 and a CD34 + Cell populations and CD34 - contacting the cells under conditions that result in a population of CD34 + The cell population is harvested and CD34 - harvesting the population of cells; and (B) CD34 - The population of cells is treated with a binding molecule that specifically binds to CD25 and a CD25 + Cell populations and CD25 - contacting the cells under conditions that result in a population of CD25 + The cell population is harvested and CD25 - harvesting the population of cells; and (C)CD25 - The population of cells is treated with a binding molecule that specifically binds to CD45RA and a CD45RA + Cell populations and CD45RA- and contacting the cells under conditions that result in a population of CD45RA - Harvesting the population of cells Any of the methods of claims 1073 to 1115, comprising: [This invention 1158] Process B is (i) CD34 - The population of cells is treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Population of cells, 6B11 + populations of cells, or combinations thereof, and CD1d-tet - Population of cells, 6B11 - and contacting the cells under conditions that result in the formation of a population of CD1d-tet cells, or a combination thereof. + Population of cells, 6B11 + A population of cells, or a combination thereof, is harvested and CD1d-tet - Population of cells, 6B11 - and recovering the population of cells, or both, thereby obtaining a population of iNKT-depleted cells; and (ii) The population of iNKT-depleted cells was treated with a binding molecule that specifically binds to CD25 and a CD25 + Cell populations and CD25 - contacting the cells under conditions that result in a population of CD25 + The cell population is harvested and CD25 - Harvesting a population of cells The method of the present invention 1157 further comprising: [This invention 1159] Process B is CD34 - The population of cells is treated with a binding molecule that specifically binds to CD25, and CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD25 + population of cells, and CD1d-tet + Population of cells, 6B11 + cells, or a combination thereof, and CD34 - CD25 -Under conditions that result in a population of iNKT-depleted cells, CD25 + Populations of cells and CD1d-tet + Population of cells, 6B11 + cells, or a combination thereof, and - CD25 - Harvesting a population of iNKT-depleted cells The method of the present invention 1157, comprising: [The present invention 1160] Process B is (i) CD34 - The population of cells is treated with a binding molecule that specifically binds to CD25, and CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD25 + Populations of cells and CD1d-tet + Population of cells, 6B11 + cells, or a combination thereof and CD34 - CD25 - Under conditions that result in a population of iNKT-depleted cells, CD25 + Populations of cells and CD1d-tet + Population of cells, 6B11 + cells, or a combination thereof, and - CD25 - Recovering a population of iNKT-depleted cells; and (ii) CD25 + Populations of cells and CD1d-tet + Population of cells, 6B11 + The precision selection of the cells, or a combination thereof, is carried out by contacting the cells with a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof, and the CD4 + CD25 + CD127 - / Lo cells, CD1d-tet + cells, 6B11 + Obtaining a population of cells enriched for a cell, or any combination thereof The method of the present invention 1157, comprising: [This invention 1161] Step (i) is CD34 - contacting the population of cells with a tag-containing anti-CD25 antibody and a biotinylated 6B11 monoclonal antibody, and contacting the 6B11-biotin with a conjugated tag, streptavidin; The method of the present invention 1160, comprising: [This invention 1162] 1161. The method of claim 1161, wherein the tag is phycoerythrin (PE). [This invention 1163] The method of any one of claims 1161 to 1162, wherein streptavidin is conjugated to PE / Cy7. [Invention 1164] CD34 - The method of any of claims 1161 to 1163, wherein the cells are then contacted with anti-tag magnetic particles. [Invention 1165] The method of claim 1164, wherein the anti-tag magnetic particles are anti-PE magnetic particles. [Invention 1166] CD25 + Cells and 6B11 + 1166. The method of any of claims 1160 to 1165, wherein the cells are separated using magnetic separation. [This invention 1167] (ii) is CD25 + Cells and 6B11 + The population of cells is subjected to precise sorting with a binding molecule that specifically binds to CD4 and a binding molecule that specifically binds to CD127. + CD25 + CD127 - / Lo Cell-enriched cell populations and 6B11 + CD127 + by contacting the cells under conditions that result in an enriched population of cells, or any combination thereof. Any of the methods of the present invention 1160 to 1166, comprising: [Invention 1168] 1167. The method of claim 1167, wherein the CD4 binding molecule is an anti-CD4 PerCP-labeled antibody, or the CD127 binding molecule is an anti-CD127 APC-labeled antibody, or a combination thereof. [This invention 1169] 1169. The method of any one of claims 1167 to 1168, wherein the precision sorting comprises fluorescence activated cell sorting and detection of PE-labeled CD25, PE / Cy7-labeled 6B11, PerCP-labeled CD4, and APC-labeled CD127. [This invention 1170] CD34 collected in step A + Population of cells, CD25 recovered in step B + population of cells, or both, expressing CD34 + cells, CD25 + The method of claim 1157, wherein the cells, or a combination thereof, are further processed by precision selection comprising contacting the cells with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD127, a binding molecule that specifically binds to CD45RA, or any combination thereof. [This invention 1171] (A) The sample is subjected to a step of isolating a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25 with a binding molecule that specifically binds to CD34. + A population of cells, CD25 + population of cells, and CD34 - CD25 - contacting the cells under conditions that result in a population of CD34 + Cell populations and CD25 + The cell population is harvested and CD34 - CD25 - harvesting the population of cells; and (B) CD34 - CD25 - The population of cells is treated with a binding molecule that specifically binds to CD45RA and a CD45RA + Cell populations and CD45RA - and contacting the cells under conditions that result in a population of CD45RA - Harvesting the population of cells Any of the methods of claims 1073 to 1115, comprising: [This invention 1172] Process A is The sample is treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Population of cells, 6B11 + and contacting the cells under conditions that result in the formation of a population of CD1d-tet cells, or a combination thereof. + Population of cells, 6B11 + harvesting a population of cells, or a combination thereof; The method of the present invention 1171 further comprising: [This invention 1173] The cell population obtained in step A is subjected to precision selection by contacting the cells with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, CD1d-tet, or any combination thereof, to obtain CD34. + cells, CD4 + CD25 + CD127 - / Lo cells, CD1d-tet + Obtaining a population of cells enriched for the cells, or any combination thereof. The method of invention 1171 or 1172 further comprising: [This invention 1174] simultaneously processing said sample to obtain an enriched population of cells containing HSPCs, Tmems, naive Tregs, memory Tregs and containing less than 5% of undesired cell types. Any of the methods of claims 1073 to 1115, comprising: [Invention 1175] 1174. The method of claim 1174, wherein the sample is contacted with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD8, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, a binding molecule that specifically binds to CD45RA, a binding molecule that specifically binds to CD45RO, or any combination thereof. [Invention 1176] The sample is contacted with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet is detected. + Population of cells, 6B11 + Recovering the population of cells, or a combination thereof. The method of the present invention 1175 further comprising: [This invention 1177] contacting the sample with a binding molecule that specifically binds to CD34; + 1177. The method of any of claims 1174 to 1176, wherein the population of cells is harvested, thereby generating a population of HSPCs. [This invention 1178] The sample is contacted with a binding molecule that specifically binds to CD3 and does not bind to a binding molecule that specifically binds to CD45RA, a binding molecule that specifically binds to CD45RO, or a combination thereof, to detect CD3 + CD45RA - CD45RO + The method of any one of claims 1174 to 1177, wherein the population of cells is recovered. [This invention 1179] The sample is contacted with a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, a binding molecule that specifically binds to CD45RA, a binding molecule that specifically binds to CD45RO, or any combination thereof, to obtain CD4 + CD25 + CD127 - / lo CD45RA + CD45RO - A population of cells, CD4 + CD25 + CD127 - / lo CD45RA - CD45RO + The method of any one of claims 1174 to 1178, wherein the population of cells is recovered. [This invention 1180] 1073-1179. The method of any of claims 1073-1179, wherein the therapeutic cell population comprises less than 2% normal naive αβ-T cells. [This invention 1181] 11. The method of any of claims 1073 to 1180, wherein the molecule that specifically binds to CD34, a Lin+ marker, CD25, CD45RA, CDR45RO, CD4, CD8, CD127, CD90, CD133, CD38, CD95, CD122, CXCR3, LFA-1, CD62L, CCR7, or any other cell marker is an antibody or antibody fragment. [This invention 1182] The method of any of claims 1073 to 1181, wherein said antibody or antibody fragment is coupled to a fluorescent dye, a hapten, or a magnetic particle. [This invention 1183] The method of any of claims 1073 to 1182, wherein said cells are freshly isolated. [Brief explanation of the drawings]

[0007] [Figure 1A] FIG. 1A shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 1B] FIG. 1B shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 2A] FIG. 2A shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 2B] FIG. 2B shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 3] FIG. 3 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 4] FIG. 4 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 5] FIG. 5 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 6] FIG. 6 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 7] FIG. 7 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 8]FIG. 8 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 9] FIG. 9 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 10] FIG. 10 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 11] FIG. 11 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 12] FIG. 12 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 13] FIG. 13 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 14] FIG. 14 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 15] FIG. 15 shows a schematic diagram of a method for producing a sculpt graft cell composition. [Figure 16] FIG. 16 shows the experimental design for evaluating the performance of Sculpt cell grafts compared to bone marrow transplant (BMT) and hematopoietic stem cell transplant (HSCT) compositions. [Figure 17] Figures 17A-D show the performance of Sculpt cell grafts compared to bone marrow transplant (BMT) and hematopoietic stem cell transplant (HSCT) compositions. Figure 17A: Kaplan-Meier curves of percent survival over time. Figure 17B: Final outcomes assessed as overall survival, relapse, engraftment failure, and GVHD. Figure 17C: Exemplary photomicrographs of J774 GPF-Luc tumors in spleens excised from HSCT mice. Figure 17D: Representative bioluminescent images comparing the presence of GFP+ cells generated using Xenogen IVIS100 on day +10. [Figure 18] Figure 18 shows Kaplan-Meier curves of percent survival of mice treated with sculpt cell grafts generated using magnetic sorting (MACS) and fluorescence activated sorting (FACS) compared to sculpt cell grafts generated using MACS alone. [Figure 19] FIG. 19 shows Kaplan-Meier curves showing survival rates of mice receiving Sculpt cell grafts versus mice receiving CD34+ cell compositions containing boosted T cells after isolation. [Figure 20] Figure 20 shows Kaplan-Meier curves illustrating mouse survival in an adoptive transfer xenograft model, in which mouse cohorts were administered either human PBMCs or human tissue-derived sculpt cell graft compositions. [Figure 21] Figures 21A-B show enrichment of regulatory T cells and iNKT cells from peripheral blood monocytes (PBMCs) using MACS. Figure 21A shows flow cytometry data showing the % purity and % yield of Tregs in the PBMCs before sorting and in the enriched population. Figure 21B shows flow cytometry data showing the % purity and % yield of iNKT cells in the PBMCs before sorting and in the enriched population. [Figure 22] Figures 22A-B show the enrichment of memory T cells from PBMCs using magnetic sorting. Figure 22A shows the % yield of CD3+CD45RO+ cells and the ratio of naive T:memory T cells obtained using magnetic sorting of PBMCs. Figure 22B shows flow cytometry data showing CD45RO and CD45RA cells in pooled PBMCs and CD45RA-depleted samples. [Figure 23] Figures 23A-B show magnetic separation of CD25+ cells. Figure 23A shows flow cytometry analysis of magnetically separated CD25+ cells, demonstrating efficient separation of CD25+ CD127+ Tregs and characterizing memory and naive Treg cell subpopulations. Figure 23B shows exemplary performance of the magnetic separation strategy on Tregs and naive Tregs from two donors. [Figure 24]Figures 24A-B show the biotinylation titration of the 6B11 antibody. Figure 24A shows the binding of 6B11.1, 6B11.2, and 6B11.3 biotinylated antibodies to samples from three separate donors at increasing concentrations of 6B11 antibody from zero to 1 μg / μL. Figure 24B shows flow cytometry analysis of 6B11 biotin / streptavidin PE-Cy7 staining and CD127 APC staining for samples using a no-6B11 control or 6B11 antibody biotinylated with 100 μM (condition 1), 250 μM (condition 2), or 1 mM (condition 3) biotin. DETAILED DESCRIPTION OF THE INVENTION

[0008] Detailed Description In certain embodiments, the present disclosure provides unique therapeutic cell populations that constitute pharmaceutical compositions useful for hematopoietic stem / progenitor cell transplantation. For example, some embodiments of the present disclosure provide therapeutic cell populations that contain enriched populations of hematopoietic stem / progenitor cells (HSPCs), memory T cells (Tmem), and regulatory T cells (Treg), and that are depleted of normal naive αβ-T cells. In some embodiments, the therapeutic cell population further comprises invariant natural killer T (iNKT) cells. Furthermore, the present disclosure provides methods of treatment using the therapeutic cell populations. In other embodiments, the present disclosure provides methods of producing the therapeutic cell populations.

[0009] Therapeutic cell populations are sculpted to enrich for cells with therapeutic benefit and deplete cell populations that are harmful to the graft recipient (e.g., inducing secondary disease). HSPCs provide short-term benefit by reconstituting blood and immune system function, e.g., restoring red blood cell, platelet, and neutrophil counts. Persistent engraftment of HSPCs results in the reconstitution of adaptive immune function by generating T and B cell populations. Donor T cells as a general population can be both beneficial and detrimental to engraftment. Tmem cells mediate both graft-versus-infection and graft-versus-leukemia effects and are beneficial because they reduce the risk of graft-versus-host disease (GVHD). Naive and memory Treg cells are beneficial because they help suppress graft rejection and suppress GVHD. In contrast, normal naive T cells contribute to GVHD and inappropriate immune reconstitution. Therefore, the reduction or elimination of normal naive T cells in the sculpt grafts disclosed herein enhances the therapeutic benefit by reducing the occurrence of GVHD in the graft recipient.

[0010] definition In this description, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range, as well as fractions thereof (e.g., tenths and hundredths of integers), where appropriate, unless otherwise indicated. Additionally, any numerical range described herein for any physical characteristic, such as polymer subunits, size, or thickness, should be understood to include any integer within the stated range, unless otherwise indicated. As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise indicated. The term "consisting essentially of" limits the scope of a claim to the stated materials or steps, or to those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, the terms "a" and "an" should be understood to refer to "one or more" listed elements. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the terms "including," "having," and "comprising" are used interchangeably and these terms and variations thereof are intended to be construed as open ended.

[0011] As used herein, the term "therapeutic cells" refers to cells selected or administered to a subject based on their ability to provide a therapeutic benefit to the subject. Exemplary therapeutic cells include hematopoietic stem / progenitor cells, memory T cells, regulatory T cells, and invariant natural killer T cells. A therapeutic cell population can contain more than one type of therapeutic cell, such as HSPCs, Tmem, Treg, iNKT, or any combination thereof. A therapeutic cell population may consist essentially of one type of therapeutic cell. In the context of a therapeutic cell population, a percentage (%) of therapeutic cells refers to the percentage of a cell type contained in a therapeutic cell combination or composition in which the total number of therapeutic cells is up to 100%, and a particular therapeutic cell type is a fraction of the total number of therapeutic cells. For example, a population of therapeutic cells containing 30% HSPCs indicates that approximately 30% of the entire therapeutic cell population are HSPCs. A therapeutic cell population may be present within a larger population of cells that have a neutral effect on the subject, but the neutral cells are not included in calculating the total number of therapeutic cells.

[0012] As used herein, the term "hematopoietic stem / progenitor cells" or "HSPCs" refers to hematopoietic stem cells and / or hematopoietic progenitor cells that express high levels of the phenotypic markers CD34, CD133, CD90, or any combination thereof, relative to other types of hematopoietic cells (e.g., the cells are positive for expression of the phenotypic markers as measured by flow cytometry, Western blot, or other methods known in the art). HSPCs may also be negative for expressing markers relative to other types of hematopoietic cells. For example, such markers include CD19, TCRα, TCRβ, CD45RA, Lin, CD38, or any combination thereof. Preferably, HSPCs express CD34 + cells and / or CD19 - TCRα -HSPCs can self-renew or differentiate into (i) myeloid progenitor cells (which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, or dendritic cells); or (ii) lymphoid progenitor cells (which ultimately give rise to T-cells, B-cells, and lymphoid cells called natural killer cells (NK-cells)). For a general discussion of hematopoiesis and HSPC differentiation, see Rowe et al. Cell Stem Cell. 2016.18:707-720.

[0013] As used herein, the term "normal naive αβ-T cells" or "naive T cells" refers to cells that express the phenotypic markers TCRα / β, CD45RA, and have intermediate to high levels of CD127 (CD127 + ) and no or low expression of CD45RO and CD25. In some embodiments, naive Tcon is characterized by expression of phenotypic markers of naive Tcon, such as TCRα, TCRβ, CD3, CD4 or CD8, CD62L, CCR7, CD28, CD127, and CD45RA. In some embodiments, naive Tcon is characterized by expression of phenotypic markers of naive Tcon, such as TCRα, TCRβ, CD3, CD4 or CD8, CD62L, CCR7, CD28, CD127, and CD45RA. + CD25 - CD45RA + and includes polymorphic TCR alpha and polymorphic TCR beta. In some embodiments, naive Tcon are not naive regulatory T cells as defined herein. Naive Tcon cells do not express the Vα24Jα18 TCR found on iNKT cells.

[0014] As used herein, the term "regulatory T cells" or "Treg" refers to a subclass of T cells that have the ability to suppress autoimmune responses and express the phenotypic markers CD4, CD25, and have low or no expression of CD127. Tregs also express FOXP3, but CD127 expression is limited to CD4. + CD25 + It has been demonstrated to be inversely correlated with FOXP3 expression on CD4 + CD25 + CD127- / low The phenotype is considered to be an acceptable surrogate marker for Tregs and a practical substitute for intracellular staining of FOXP3 (Cozzo C, et al. J Immunol. 2003 Dec 1; 171:5678-82; Liu W, et al. J Exp. Med. 2006. 203(7):1701-1711; Seddiki N, et al. J Exp. Med. 2006; 203(7):1693-1700). Tregs may include at least two subclasses, referred to herein as naive Tregs and memory Tregs.

[0015] As used herein, the term "naive Treg" refers to the non-antigen-sensitized regulatory T cell that expresses the phenotypic markers CD4, CD25 and CD45RA as primary cells, and does not express or expresses low levels of CD45RO and CD127.Naive Treg is advantageous because it has higher flexibility in response to antigen than antigen-sensitized Treg.In addition, naive Treg has a longer life span than antigen-sensitized Treg.

[0016] As used herein, the term "memory Tregs" refers to antigen-sensitized regulatory T cells that have the ability to exert a suppressive effect on autoimmunity and that express the phenotypic markers CD4, CD25, and CD45RO, and have low or no expression of CD127 and CD45RA.

[0017] As used herein, the term "memory T cells" or "Tmem" refers to antigen-sensitized T cells that express the phenotypic markers TCRα, TCRβ, CD3, CD4 or CD8, CD95, and IL-2Rβ. Memory T cells have the ability to confer immunity and persist in an inactive state for long periods of time. Memory T cells have the ability to rapidly acquire effector function upon antigen restimulation. The memory T cell population is divided into the subclass of central memory T cells (T CM ) and effector memory T cells (T EM ) and any combination thereof.

[0018] As used herein, "central memory T cells" or "T CM " refers to antigen-primed T cells that express the phenotypic markers CD4 or CD8, CD62L, CD45RO, CCR7, IL-2Rβ, CD28, CD127, and CD95, and that do not express CD45RA or express it at a lower level compared to naive T cells. Central memory T cells are T cells that express CD45RA after antigen restimulation. EM They can differentiate into cells.

[0019] As used herein, "effector memory T cells" or "T EM " refers to antigen-primed T cells that express the phenotypic markers CD4 or CD8, CD45RO, CD127, IL-2Rβ, and CD95, and have low or no expression of CD45RA, CD62L, CCR7, and CD28. Effector memory T cells undergo terminal differentiation and acquire effector function after restimulation with antigen.

[0020] As used herein, "central memory T stem cells" or "T SCM " refers to antigen-sensitized T cells that express the phenotypic markers CD4 or CD8, CD45RA, CD62L, CD95, IL-2Rβ, CCR7, CXCR3, CD122, and LFA-1. T SCM The cells have the ability of memory T cells to rapidly acquire effector function after antigen restimulation, but exhibit enhanced stem cell-like characteristics, e.g., T CM They have a long-term persistence compared to cells. SCM The cells can represent central memory, effector memory and effector T cell subsets.

[0021] As used herein, "invariant natural killer T cells" or "iNKT" comprise a human Vα24Jα18 TCR α chain (referred to herein as "Vα24Jα18 +iNKT cells are a subclass of CD1d-restricted natural killer T (NKT) cells that express a highly conserved αβ-T cell receptor (referred to as "iNKT cells"). iNKT cells can be identified by binding to CD1d multimers, such as those loaded with α-galactosylceramide (GalCer), PBS-57, PBS-44, or other natural or synthetic glycolipids, which can be found as tetramers, dendrimers and other structures, Fc fusions, or any combination thereof. Another method of identification is an antibody or combination of antibodies that specifically recognizes the Vα24Jα18 region. Examples include Vα24 antibodies, Jα18 antibodies, or the monoclonal antibody clone 6B11 that specifically binds to a unique region of the Vα24Jα18 TCR and can be used to identify iNKT cells (Montoya et al. Immunology. 2007. 122(1):1-14). In some embodiments, iNKT cells are identified by binding to CD1d tetrimer-loaded glycolipids. + (CD1d-tet + ), 6B11 + or both. iNKT cells are herein interchangeably referred to as CD1d-tet + , 6B11 + or Vα24Jα18 + Without wishing to be limited to a particular mechanism, it is believed that iNKT cells promote / accelerate the activity of Tregs and HSPCs.

[0022] As referred to herein, "lineage positive" or "Lin + "The cells express phenotypic markers such as CD19, CD11c, CD66B, CD14, CD20, or any combination thereof. As referred to herein, "lineage negative" or "Lin - "The cells do not express or have low expression of the phenotypic markers CD19, CD11c, CD66B, CD14, CD20, or any combination thereof compared to HSPCs, Tregs, Tmem, or iNKT cells. Lin + The cells express phenotypic markers present on mature erythroid cells, granulocytes, macrophages, NK cells, and B and T lymphocytes.

[0023] As used herein, a "sample" refers to a cell source (e.g., a biological tissue) from which a population of cells can be isolated, enriched, or depleted. In some embodiments, the sample is generally unprocessed or minimally processed beforehand. For example, the sample can be mobilized peripheral blood, mobilized apheresis blood products, bone marrow, umbilical cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof. In some embodiments, the sample is prepared by processing using a density gradient, Ficoll, Percoll, hypotonic lysis of red blood cells, ammonium chloride-potassium (ACK) buffer, washing in a pH-balanced isotonic buffer, or any combination thereof, or is minimally processed. In some embodiments, the sample is obtained from a single tissue collection. In some embodiments, the sample is obtained from one or more tissue collections.

[0024] As used herein, "donor" refers to one or more individuals from whom a sample is collected. For example, a donor can refer to a human leukocyte antigen (HLA)-matched sibling, an HLA-matched unrelated donor, a partially matched unrelated donor, a haploidentical related donor, an autologous donor, an HLA-mismatched allogeneic donor, a donor pool, or any combination thereof. In some embodiments, a donor can be a subject. "Donor tissue" refers to tissue collected from a donor. The donor tissue can be a sample. The donor tissue is generally of the same species as the subject.

[0025] As used herein, "subject" or "recipient" refers to one or more individuals who require treatment, therapy, or cell transplantation as disclosed herein. Subjects that can be treated by the present invention are generally humans. However, additional subjects include non-human primates, cows, horses, sheep, goats, pigs, dogs, cats, mice, rabbits, rats, or guinea pigs. The subject can be male or female, and can be of any suitable age, such as infants, juveniles, adolescents, adults, and elderly subjects. During and after treatment, the subject becomes a recipient or transplant recipient.

[0026] As used herein, "tissue harvesting" refers to the process of collecting donor tissue or samples from a donor. Non-limiting examples of tissue harvesting include collecting bone marrow, peripheral blood, umbilical cord blood, etc. from a donor. Tissue harvesting can be performed by any method known in the art.

[0027] As used herein, "enriched," with respect to a cell population or cell type in a mixture, indicates that the cell population has been processed to increase the relative amount or relative ratio of the enriched cell type compared to other cells in the mixture (e.g., the cell type being counted). Thus, depending on the source of the original cell population subjected to the enrichment process, a mixture or composition may include a cell population that is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more (in number or count) "enriched" compared to other cells in the mixture. In some embodiments, the enrichment process may result in a cell population that is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 5,000-fold, 10,000-fold, or more "enriched" relative to other cells in the mixture. For example, in some embodiments, a cell mixture enriched for iNKT cells may contain about 0.03-1% iNKT cells, 0.05%-0.5% iNKT cells, 0.1%-1% iNKT cells, or any combination thereof. Exemplary methods for enriching cell populations include magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS).

[0028] As used herein, "depleted," with respect to a cell population or cell type in a mixture, indicates that the cell population has been processed to reduce the relative amount or relative ratio of the cell type being depleted compared to other cells in the mixture (e.g., the cell type being counted). In some embodiments, subjecting cells to a depletion process can result in a mixture or composition comprising a cell population that is "depleted" (by number or count) to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001%, 0.0000001%, 0.0000001% or less. In some embodiments, subjecting cells to a depletion process can result in a mixture or composition comprising a population that is 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold or less depleted compared to the unprocessed sample. In some embodiments, a depleted cell type is no longer detectable using conventional methods after the processing step that depletes the cell type.

[0029] In certain embodiments, the mixture is enriched for the amount of a particular cell type and depleted for the amount of a different cell type, e.g., CD34 + Cells were enriched, while CD34 - The cells are depleted.

[0030] A cell population "positive" for a marker exhibits uniform staining of the cell population above the level seen in an isotype control. In some embodiments, decreased expression or low expression of one or more markers indicates a decrease in mean fluorescence intensity (MFI) of at least 1 log 10 or a measurement at least 1 log 10 less than a reference control. In some embodiments, increased expression or high expression of one or more markers indicates an increase in MFI of at least 1 log 10 or a measurement at least 1 log 10 greater than an isotype control or reference control. In some embodiments, an increase in MFI of at least 2-fold compared to a reference population indicates that the cells are positive for expression of the marker. For example, a cell population positive for a marker may exhibit an MFI that is 2-4, 4-10, 10-100, and 100-1,000, 1,000-10,000, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000 or more times greater than an isotype control. In some embodiments, a cell population positive for one or more markers indicates that the percentage of cells that exhibit the markers when compared to a reference cell population can be at least 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 cells, 95% of the cells, and 100% of the cells, as well as any percentage between 50% and 100%.

[0031] A cell population "negative" for a marker indicates the absence of significant staining of the cell population with a specific antibody above the isotype control. In some embodiments, a decrease in MFI of at least 2-fold compared to a reference population indicates that the cells are negative for expression of the marker. For example, a cell population negative for a marker may exhibit an MFI that is 2-4, 4-10, 10-100, 100-1,000, 1,000-10,000, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, or more times less than a positive control. In some embodiments, decreased or under-expressed expression of one or more markers indicates that the percentage of cells in a population of cells that exhibit the markers is reduced by at least 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 cells, 95% of the cells, and 100% of the cells, as well as any percentage between 20% and 100%, when compared to a reference cell population.

[0032] As used herein, "percent purity" or "% purity" refers to the number of cells of interest multiplied by 100 and then divided by the number of cell events counted when measured on a flow cytometer, hemocytometer, Coulter counter, microscopy, or other cell counting method (number of cells of interest x 100 / number of cell events).

[0033] As used herein, "total percent yield" or "total yield %" refers to the number of desired cells after a processing step multiplied by 100 and then divided by the number of desired cells in the original population (number of desired cells after processing x 100 / number of desired cells in original population). "Percent yield of a processing step" or "% yield of a processing step" refers to the number of desired cells after a processing step multiplied by 100 and then divided by the number of desired cells in the population before processing (number of desired cells after processing x 100 / number of desired cells in the population before processing).

[0034] As used herein, "coarse sorting" refers to a method of enriching or depleting a population of cells, such that, depending on the source of the original cell population subjected to coarse sorting, the resulting population may contain at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of a particular cell population compared to the starting cell mixture. Methods for performing coarse sorting are well known in the art and may include density separation, apheresis / leukapheresis, tetrameric antibody complex-mediated enrichment / depletion, and magnetic-activated cell sorting (MACS), such as CLINIMACS®, PRODIGY®, or EASYSEP™ / ROBOSEP™.

[0035] As used herein, "fine-grain sorting" refers to a method for enriching or depleting a population of cells, such that the resulting population contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more of a particular cell population compared to the starting cell mixture. Methods for fine-grain sorting are well known in the art and may include multi-parameter fluorescence-based molecular phenotyping, such as fluorescence-activated cell sorting (FACS), and microfluidic-based sorting. Additional methods for fine-grain sorting are described, for example, in U.S. Provisional Patent Application No. 62 / 421,979, which is incorporated herein by reference in its entirety.

[0036] As used herein, the term "binding molecule" can refer to any of a large number of different molecules or aggregates, and the terms are used interchangeably. Proteins, polypeptides, peptides, nucleic acids (nucleotides, oligonucleotides, and polynucleotides), antibodies, saccharides, polysaccharides, lipids, receptors, test compounds (especially those generated by combinatorial chemistry), each or in combination (when bound to each other by themselves or via targeting ligands) can be binding molecules.

[0037] As used herein, "specifically binds to" or "specific for" refers to the ability of a binding molecule (e.g., antibody) or binding domain to bind to a target (molecule or complex). 5 M -1 (This is the off-rate [k off ] for the on-rate [k on ]) or greater, but does not significantly associate or associate with other molecules or components in the sample. Binding molecules or binding domains can be classified as "high affinity" or "low affinity" binding molecules or domains. A "high affinity" binding molecule or domain has an affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 or at least 10 13 M -1 K a A "low affinity" binding molecule or binding domain refers to a binding molecule or binding domain having a binding affinity of 10 7 M -1 Up to 10 6 M -1Up to 10 5 M -1 K up to a Alternatively, affinity is measured in units of M and expressed as the equilibrium dissociation constant (K d ) (e.g., 10 -5 M~10 -13 M).

[0038] As used herein, "sculpted cell graft" or "sculpted graft" refers to a cell population that has been processed from a starting cell population to obtain a specific cell type within a specified range of numbers and to reduce or eliminate undesired cell types to a specified range. The range is typically expressed as the number of a particular type of cell per kg of patient weight, but may also be expressed as the total number within the graft. A sculpted cell graft may comprise a cell mixture containing a non-naturally occurring ratio of target cells to accounting cells or a non-naturally occurring indicated percentage.

[0039] As used herein, a "unit dose" refers to a specified minimum number, specified number, or range of therapeutic cell populations per kilogram (kg) of body weight of the subject receiving the sculpt cell graft. It will be appreciated that the number of unit doses will vary depending on the subject's size. A unit dose may be divided into fractions of unit doses depending on the subject's body weight. In some embodiments, a therapeutic cell population (e.g., HPSC, Tmem, Treg, iNKT, etc.) may be divided into individual containers for administration to a subject.

[0040] therapeutic cell compositions In certain embodiments, the present disclosure provides unique therapeutic cell populations that constitute pharmaceutical compositions useful for hematopoietic stem / progenitor cell transplantation. The therapeutic cell populations may comprise enriched populations of hematopoietic stem / progenitor cells (HSPCs), memory T cells (Tmem), regulatory T cells (Tregs), and are depleted of normal naive αβ-T cells. In some embodiments, Tregs include naive Tregs, memory Tregs, or both. In some embodiments, Tmem include central memory T stem cells (T SCM ), central memory T cells (T CM ) population, effector memory T cells (T EM ), or any combination thereof. In some embodiments, the therapeutic population comprises an enriched population of invariant natural killer T cells (iNKT).

[0041] In some embodiments, the therapeutic composition may comprise HSPCs and Tmem. In some cases, the therapeutic composition may comprise HSPCs, Tmem, and Tregs. In some cases, the therapeutic composition may comprise HSPCs and Tregs. In some cases, the therapeutic composition may comprise HSPCs and iNKT cells. In some cases, the therapeutic composition may comprise HSPCs, Tregs, Tmem, and iNKT cells.

[0042] In certain embodiments of the present disclosure, the HSPCs are CD34 + HSPCs may also or alternatively express CD133 + , CD90 + , CD38 - , CD45RA - , Lin - In some embodiments, the HSPCs may be described as CD19 - , TCRα / β - or a combination thereof. In some embodiments, Tregs are CD25 + , CD4 + and CD127 - / lo In some embodiments, Tregs are CD4 + , CD25+ , CD127 - / lo , FoxP3 + In some embodiments, naive Tregs are CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA + , CD45RO - In some embodiments, memory Tregs are CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA - , CD45RO + In some embodiments, Tmem is CD25, or any combination thereof. - , CD45RA - In some embodiments, Tmem is a CD3 + , CD45RA - , CD45RO + or any combination thereof. In some embodiments, T SCM CD45RA + and CD4 + or CD8 + T SCM Furthermore, CD95 + , CD122 + , CXCR3 + , LFA-1 + or any combination thereof. CM CD45RO + and CD4 + or CD8 + T CM Furthermore, CD45RA - , CD62L + , CCR7 + or any combination thereof. EM CD4 + , CD45RO + , CD45RA- , CD62L - , CCR7 - In some embodiments, the iNKT is CD1d-tet + , 6B11 + In some embodiments, the iNKT is Vα24Jα18 + In some embodiments, the iNKT cells are CD25 + , 6B11 + , CD4 + , Vα24Jα18 + , CD127 dim / - In any embodiment described herein, normal naive αβ-T cells may be CD25 - , CD45RA + In some embodiments, normal naive αβ-T cells are TCRα / β + CD45RA + and CD25 - , CD127 + Or both. Normal naive αβ-T cells also express TCRα + TCRβ + CD45RA + CD45RO - CD25 - CD95 - IL-2Rβ - CD127 + Vα24Jα18 - It can be written as:

[0043] In certain embodiments, the concentration of therapeutic cells is described as a ratio of HSPCs to another cell type. In some embodiments, the ratio of HSPCs to Tmem is 500:1 to 1:1,000, 400:1 to 1:1,000, 300:1 to 1:1,000, 200:1 to 1:1,000, 100:1 to 1:1,000, 50:1 to 1:1,000, 10:1 to 1:1,000, 5:1 to 1:1,000, 4:1 ~1:1,000, 3:1~1:1,000, 2:1~1:1,000, 1:1~1:1,000, 500:1~1:900, 500:1~1:800, 500:1~1:700, 500:1~1:600, 500:1~1:500, 500:1~1:400, 500:1~1:300, 500:1 ~1:200, 500:1~1:100, 500:1~1:50, 500:1~1:20, 500:1~1:10, 500:1~1:9, 500:1~1:8, 500:1~1:7, 500:1~1:6, 500:1~1:5, 500:1~1:4, 500:1~1:3, 500:1~1:2, 50 Ranges include 0:1 to 1:1, 400:1 to 1:900, 300:1 to 1:800, 200:1 to 1:700, 100:1 to 1:600, 50:1 to 1:500, 10:1 to 1:400, 5:1 to 1:300, 4:1 to 1:200, 3:1 to 1:100, 2:1 to 1:50, or 1:1 to 1:20.

[0044] In some embodiments, the ratio of HSPC to Tmem includes ranges of 10:1 to 1:200, 100:1 to 1:2,000, or 1,000:1 to 1:20,000.

[0045] The ratio of HSPCs to Tregs may include ranges of 20:1 to 1:3, 100:1 to 1:30, or 200:1 to 1:300. The ratio of HSPCs to naive Tregs may include ranges of 1:500-100:1, 1:400-100:1, 1:300-100:1, 1:200-100:1, 1:100-100:1, 1:50-100:1, 1:20-100:1, 1:10-100:1, 1:5-100:1, 1:1-100:1, 1:200-50:1, 1:200-20:1, 1:200-10:1, 1:200-5:1, 1:100-1:1, 40:1-1:3, 200:1-1:15, or 400:1-1:150. The ratios of HSPCs to memory Tregs were 1:500–10,000:1, 1:400–10,000:1, 1:300–10,000:1, 1:200–10,000:1, 1:100–10,000:1, 1:50–10,000:1, 1:20–10,000:1, 1:10–10,000:1, 1:5–10,000:1, 1:1–10,000:1, 1:500–5,000:1, and 1:500– The ranges may include 1,000:1, 1:500 to 900:1, 1:500 to 800:1, 1:500 to 700:1, 1:500 to 600:1, 1:500 to 500:1, 1:500 to 400:1, 1:500 to 300:1, 1:500 to 200:1, 1:500 to 100:1, 1:500 to 50:1, 1:500 to 20:1, 1:500 to 10:1, 1:500 to 5:1, or 1:500 to 1:1.

[0046] The ratios of HSPC to iNKT were 1:2–1,000,000:1, 1:2–500,000:1, 1:1–500,000:1, 100:1–1,000,000:1, 100:1–500,000:1, 100:1–100,000:1, 500:1–1,000,000:1, 500:1–500,000:1, and 500 :1 to 100,000:1, 1,000:1 to 100,000:1, 1,000:1 to 1,000,000:1, 1,000:1 to 500,000:1, 1,000:1 to 100,000:1, 10,000:1 to 1:2, 100,000:1 to 1:20, or 1,000,000:1 to 1:200.

[0047] In certain embodiments, the concentration of therapeutic cells is described as a ratio of normal naive αβ-T cells to the therapeutic cell type, such as a ratio of normal naive αβ-T cells to HSPCs of less than 1:3, less than 1:50, less than 1:100, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1,000, less than 1:1,500, less than 1:2,000, less than 1:3,000, less than 1:4,000, less than 1:5,000, less than 1:6,000, less than 1:7,000, less than 1:8,000, less than 1:9,000, less than 1:10 The ratio may be less than 1:1,000, less than 1:50,000, less than 1:100,000, less than 1:200,000, less than 1:300,000, less than 1:400,000, less than 1:500,000, less than 1:600,000, less than 1:700,000, less than 1:800,000, less than 1:900,000, or less than 1:1,000,000.

[0048] The ratio of normal naive αβ-T cells to Tmem can be less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1:50000.

[0049] The ratio of normal naive αβ-T cells to Tregs can be less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1:50000. The ratio of normal naive αβ-T cells to naive Tregs can be less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1:50000. The ratio of normal naive αβ-T cells to memory Tregs can be less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1:50000.

[0050] The normal ratio of naive αβ-T cells to iNKT is less than 100:1, less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:100, less than 1:150, less than 1:200, less than 1:300, less than 1:400, less than 1:5 ... It may be less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, less than 1:50000.

[0051] In some embodiments of the present disclosure, the ratio of Tmem to Treg is independent of the initial cell concentration (e.g., substantially altered from the initial concentration). This provides an advantage by allowing the concentration of Tmem to be controlled (e.g., dose-titrated) independently of the concentration of Treg. The ratio of Tmem to Treg can be 30:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, or 2:1 to 1:1. In certain embodiments, the ratio of Tmem to Treg can be 1:1 to 200:1, 1:10 to 2000:1, or 1:100 to 20,000:1. The ratio of Tmem to naive Tregs can be 5:1 to 1:10, 3:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, or 1:1 to 1:10. The ratio of Tmem to memory Tregs can be 27:1 to 0.9:1, 30:1 to 1:10, 25:1 to 1:10, 20:1 to 1:10, 15:1 to 1:10, 10:1 to 1:10, 30:1 to 1:9, 30:1 to 1:8, 30:1 to 1:7, 30:1 to 1:6, 30:1 to 1:5, 30:1 to 1:4, 30:1 to 1:3, 30:1 to 1:2, or 30:1 to 1:1.

[0052] In some embodiments of the present disclosure, the ratio of Treg to iNKT can be 20,000:1 to 1:5, 200,000:1 to 1:50, or 2,000,000:1 to 1:500.

[0053] In some embodiments of the present disclosure, the ratio of iNKT to Tmem can be 2:1 to 1:100,000, 5:1 to 1:1,000,000, or 10:1 to 1:10,000,000.

[0054] In some embodiments, the number of normal naive aβ-T cells in the composition can be less than about 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 27%, 30%, 32%, or 35% of the number of HSPCs. In some embodiments, the number of normal naive aβ-T cells in the composition can be less than about 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 27%, 30%, 32%, or 35% of the number of HSPCs. In some embodiments, the number of normal naive aβ-T cells in the composition is about 2% to about 7%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 30%, about 2% to about 35%, about 7% to about 10%, about 7% to about 15%, about 7% to about 20%, about 7% to about 25%, about 7% to about 30%, about 7% to about 35%, about It may be 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 35%, about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 25% to about 30%, about 25% to about 35%, or about 30% to about 35%.

[0055] In some embodiments, the number of normal naive aβ-T cells in the composition can be less than about 0.1%, 1%, 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 22%, or 25% of the number of Tmem cells, hi some embodiments, the number of normal naive aβ-T cells in the composition can be up to about 20%. In some embodiments, the number of normal naive aβ-T cells in the composition can be about 0.1% to about 2%, about 0.1% to about 5%, about 0.1% to about 10%, about 0.1% to about 15%, about 0.1% to about 20%, about 2% to about 5%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 10% to about 15%, about 10% to about 20%, or about 15% to about 20% of the number of Tmem cells.

[0056] In some embodiments, the number of normal naive aβ-T cells in the composition can be less than about 0.05%, 0.5%, 1%, 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, or 30% of the number of Treg cells. In some embodiments, the number of normal naive aβ-T cells in the composition can be about 0.05% to about 1%, about 0.05% to about 5%, about 0.05% to about 10%, about 0.05% to about 15%, about 0.05% to about 20%, about 0.05% to about 30%, about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 30%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 10% to about 15%, about 10% to about 20%, about 10% to about 30%, about 15% to about 20%, about 15% to about 30%, or about 20% to about 30% of the number of Treg cells.

[0057] In some embodiments, the number of normal naive aβ-T cells in the composition can be less than about 1%, 10%, 20%, 50%, 70%, 80%, or 90% of the number of iNKT cells. In some embodiments, the number of normal naive aβ-T cells in the composition can be about 1% to about 10%, about 1% to about 20%, about 1% to about 50%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 10% to about 20%, about 10% to about 50%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 20% to about 50%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 70% to about 80%, about 70% to about 90%, or about 80% to about 90% of the number of iNKT cells.

[0058] In certain embodiments, the therapeutic cell population comprises 10%-65% HSPCs. In certain embodiments, the therapeutic cell population comprises 2%-20% Tregs. In certain embodiments, the therapeutic cell population comprises 25%-90% Tmems. In certain embodiments, the therapeutic cell population comprises less than 2% normal naive αβ-T cells. In certain embodiments, the therapeutic cell population comprises: 10%-65% HSPCs; 2%-20% Tregs; 25%-90% Tmems; and less than 2% normal naive αβ-T cells. In some embodiments, the population of Tregs comprises 2%-5%, 2%-10%, 2%-20%, 2%-30%, 2%-40%, 2%-50%, 5%-10%, 5%-20%, 5%-30%, 5%-40%, 5%-50%, 10%-20%, 10%-30%, 10%-40%, 10%-50%, 15%-20%, 15%-30%, 15%-40%, or 15%-50% naive Tregs. In some embodiments, the population of Tregs comprises 75%-95% memory Tregs. In some embodiments, the population of naive T cells comprises 0.1%-10% T SCM In some embodiments, the population of Tmem is between 0% and 99% T CM In some embodiments, the population of Tmem is between 0% and 99% T EMIn certain embodiments, the population further comprises 0.01% to 5%, 0.05% to 5%, 0.1% to 5%, 0.5% to 5%, 1% to 5%, 0.01% to 1.5%, 0.05% to 1.5%, 0.1% to 1.5%, 0.5% to 1.5%, or 1% to 1.5% iNKT.

[0059] In any embodiment disclosed herein, the cell population can be obtained by processing the sample from one or more tissue collections.In some embodiments, the sample or tissue collection is from one donor.The one or more tissue collections can be from one or more donors.For example, the tissue collection can be from HLA-matched sibling donors, HLA-matched unrelated donors, partially matched unrelated donors, haploidentical related donors, autologous donors, completely HLA-mismatched allogeneic donors, donor pools, or any combination thereof.

[0060] In any embodiment disclosed herein, the cell population may be formulated for administration to a subject. In some cases, the cell population may be formulated with an excipient. In some embodiments, the cells are formulated for infusion or injection. The excipient may include Normosol-R and human serum. The human serum may be 0.2% of the total formulation. The human serum may be 0.5% of the total formulation. The human serum may be 1% of the total formulation. The human serum may be 2% of the total formulation. The formulation may also include a pH buffer, such as 0.1 mM to 100 mM phosphate, pH 6.0-9.0, 0.1 mM to 100 mM HEPES, pH 6.0-9.0, 0.1 mM to 100 mM bicarbonate, pH 6.0-9.0, 0.1 mM to 100 mM citrate, pH 6.0-9.0, 0.1 mM to 100 mM acetate, pH 4.0-8.0, or any combination thereof. The formulation may include an electrolyte, such as 5 mM to 400 mM NaCl, 0.5 mM to 50 mM KCl, 0.05 mM to 50 mM CaCl, 0.05 mM to 50 mM MgCl, 0.05 mM to 50 mM LiCl, 0.05 mM to 50 mM MnCl, or any combination thereof. The formulation may include an energy source, such as 0.1 mM to 100 mM glucose, 0.1 mM to 100 mM pyruvate, 0.1 mM to 100 mM fructose, 0.1 mM to 100 mM sucrose, 0.1 mM to 50 mM glycerol, 0.1 mM to 100 mM gluconolactone, 0.1 mM to 100 mM gluconate, or any combination thereof. The formulation may include an antioxidant, for example, 0.05 to 10 mM glutathione (reduced), 0.05 to 10 mM glutathione (oxidized), 0.001 to 10 mM β-mercaptoethanol, 0.001 to 10 mM dithiothreitol, 0.01 to 100 mM ascorbate, 0.001 to 10 mM tris(2-carboxyethyl)phosphine, or any combination thereof.The formulation may contain a stabilizer, for example, 0.01% to 10% human serum albumin, 0.01% to 10% bovine serum albumin, 0.1% to 99% human serum, 0.1% to 99% fetal bovine serum, 0.01% to 10% IgG, 0.1% to 10% immunoglobin, 0.06% to 60% trehalose, or 0.1% to 20% polymer molecules such as polyethylene glycocol (MW 200 to 20,000,000), or any combination thereof.

[0061] In some embodiments disclosed herein, the cell population is formulated for administration to the subject based on the subject's body weight (e.g., number of cells / kg subject body weight). The cell population can be formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a unit dose of a cell graft (e.g., the cell population), and each unit dose of the cell graft comprises a therapeutic cell population per kilogram (kg) of body weight of the subject receiving the cell graft. In some embodiments, the population or unit dose of cells is about 1 x 10 per kg of subject. 6 HSPC ~ approx. 20×10 6 In some embodiments, the population or unit dose of cells comprises at least about 1 x 10 cells per kg of subject. 6 In some embodiments, the population or unit dose of cells comprises up to about 20 x 10 cells per kg of subject. 6 In some embodiments, the population or unit dose comprises about 0.5 x 10 HSPCs. 6 ~50×10 6 , 1.0×10 6 ~20×10 6 or 2.0 x 10 6 ~10×10 6 Contains HSPC cells in the range of cells / kg subject weight.

[0062] In some embodiments, the population or unit dose of cells is about 1 x 10 cells per kg of subject. 6 HSPC ~ approx. 2×10 6 HSPC, approx. 1×10 6 HSPC ~ approx. 5×10 6 HSPC, approx. 1×106 HSPC ~ about 7×10 6 HSPC, about 1×10 6 HSPC ~ about 10×10 6 HSPC, about 1×10 6 HSPC ~ about 15×10 6 HSPC, about 1×10 6 HSPC ~ about 20×10 6 HSPC, about 2×10 6 HSPC ~ about 5×10 6 HSPC, about 2×10 6 HSPC ~ about 7×10 6 HSPC, about 2×10 6 HSPC ~ about 10×10 6 HSPC, about 2×10 6 HSPC ~ about 15×10 6 HSPC, about 2×10 6 HSPC ~ about 20×10 6 HSPC, about 5×10 6 HSPC ~ about 7×10 6 HSPC, about 5×10 6 HSPC ~ about 10×10 6 HSPC, about 5×10 6 HSPC ~ about 15×10 6 HSPC, about 5×10 6 HSPC ~ about 20×10 6 HSPC, about 7×10 6 HSPC ~ about 10×10 6 HSPC, about 7×10 6 HSPC ~ about 15×10 6 HSPC, about 7×10 6 HSPC ~ about 20×10 6 HSPC, about 10×10 6 HSPC ~ about 15×l0 6 HSPC, about 10×10 6 HSPC ~ about 20×10 6 HSPC or about 15×10 6 HSPC ~ about 20×10 6 HSPC is included. In some embodiments, the cell population is about 1×10 per kg of subject 6 HSPC, about 2×10 6 HSPC, about 5×10 6 HSPC, about 7×10 6HSPC, approx. 10×10 6 HSPC, approx. 15×10 6 HSPC or approximately 20 x 10 6 Including HSPCs.

[0063] In some embodiments, the population or unit dose of cells is about 1 x 10 cells per kg of subject. 6 Tmem cells ~ approximately 100 × 10 6 In some embodiments, the population or unit dose of cells comprises at least about 1 x 10 cells per kg of subject. 6 In some embodiments, the population or unit dose of cells comprises up to about 100 x 10 cells per kg of subject. 6 In some embodiments, the population or unit dose of cells comprises about 1 x 10 cells per kg of subject. 6 Tmem cells ~approximately 10 x 10 6 Tmem cells, approximately 1 × 10 6 Tmem cells ~ approximately 20 × 10 6 Tmem cells, approximately 1 × 10 6 Tmem cells ~ approximately 50 × 10 6 Tmem cells, approximately 1 × 10 6 Tmem cells ~approximately 75 × 10 6 Tmem cells, approximately 1 × 10 6 Tmem cells ~ approximately 100 × 10 6 Tmem cells, approximately 10 × 10 6 Tmem cells ~ approximately 20 × 10 6 Tmem cells, approximately 10 × 10 6 Tmem cells ~ approximately 50 × 10 6 Tmem cells, approximately 10 × 10 6 Tmem cells ~approximately 75 × 10 6 Tmem cells, approximately 10 × 10 6 Tmem cells ~approximately 100 x 10 6 Tmem cells, approximately 20 × 10 6 Tmem cells ~ approximately 50 × 10 6 Tmem cells, approximately 20 × 10 6 Tmem cells ~approximately 75 × 10 6 Tmem cells, approximately 20 × 10 6 Tmem cells ~approximately 100 x 10 6 Tmem cells, approximately 50 × 106 Tmem cells ~approximately 75 × 10 6 Tmem cells, approximately 50 × 10 6 Tmem cells ~approximately 100 x 10 6 Tmem cells or approximately 75 × 10 6 Tmem cells ~approximately 100 x 10 6 In some embodiments, the population or unit dose of cells comprises about 1 x 10 cells per kg of subject. 6 Tmem cells, approximately 10 × 10 6 Tmem cells, approximately 20 × 10 6 Tmem cells, approximately 50 × 10 6 Tmem cells, approximately 75 × 10 6 Tmem cells or approximately 100 × 10 6 Contains Tmem cells.

[0064] In some embodiments, the population or unit dose of cells is about 0.5 x 10 cells per kg of subject. 6 Treg cells ~ approx. 2.5×10 6 In some embodiments, the population or unit dose of cells comprises at least about 0.5 x 10 cells per kg of subject. 6 In some embodiments, the population or unit dose of cells comprises up to about 2.5 x 10 cells per kg of subject. 6 In some embodiments, the population or unit dose of cells comprises about 0.5 x 10 cells per kg of subject. 6 Treg cells ~ approx. 1 x 10 6 Treg cells, approximately 0.5 x 10 6 Treg cells ~ approx. 1.5×10 6 Treg cells, approximately 0.5 x 10 6 Treg cells ~ approx. 2 x 10 6 Treg cells, approximately 0.5 x 10 6 Treg cells ~ approx. 2.5×10 6 Treg cells, approximately 1 x 10 6 Treg cells ~ approx. 1.5×10 6 Treg cells, approximately 1 x 10 6 Treg cells ~ approx. 2 x 10 6 Treg cells, approximately 1 x 10 6 Treg cells ~ approx. 2.5×10 6Treg cells, approximately 1.5 x 10 6 Treg cells ~ approx. 2 x 10 6 Treg cells, approximately 1.5 x 10 6 Treg cells ~ approx. 2.5×10 6 Treg cells or approximately 2 × 10 6 Treg cells ~ approx. 2.5×10 6 In some embodiments, the population or unit dose of cells comprises about 0.5 x 10 cells per kg of subject. 6 Treg cells, approximately 1 x 10 6 Treg cells, approximately 1.5 x 10 6 Treg cells, approximately 2 x 10 6 Treg cells or approximately 2.5 × 10 6 In some embodiments, the population or unit dose of cells comprises about 0.1 x 10 Treg cells. 6 ~About 500×10 6 , about 0.2×10 6 ~About 500×10 6 , about 0.3×10 6 ~About 500×10 6 , about 0.4×10 6 ~About 500×10 6 , about 0.5×10 6 ~About 500×10 6 , about 0.6×10 6 ~About 500×10 6 , about 0.7×10 6 ~About 500×10 6 , about 0.8×10 6 ~About 500×10 6 , about 0.9×10 6 ~About 500×10 6 or about 1 x 10 6 ~About 500×10 6 In some embodiments, the population or unit dose of cells comprises naive Tregs in the range of 0.005 x 10 cells / kg subject weight. 6 ~500×10 6 Contains memory Tregs in a range of cells / kg subject weight.

[0065] In some embodiments, the population or unit dose of cells is 0.5 x 10 3 ~2000×10 3Cells / kg subject weight, 0.5 x 10 3 ~1×10 7 cells / kg subject body weight or 1.0 x 10 4 ~2.5×10 6 In some embodiments, the population or unit dose of iNKT cells comprises about 0.01 x 10 cells / kg of subject body weight. 6 iNKT cells ~ approx. 3 x 10 6 In some embodiments, the population or unit dose of cells comprises at least about 0.01 x 10 cells per kg of subject. 6 In some embodiments, the population or unit dose of cells comprises up to about 3 x 10 cells per kg of subject. 6 In some embodiments, the population or unit dose of cells comprises about 0.01 x 10 cells per kg of subject. 6 iNKT cells ~ approx. 0.1×10 6 iNKT cells, approximately 0.01×10 6 iNKT cells ~ approx. 1 x 10 6 iNKT cells, approximately 0.01×10 6 iNKT cells ~ approx. 1.5×10 6 iNKT cells, approximately 0.01×10 6 iNKT cells ~ approx. 2 x 10 6 iNKT cells, approximately 0.01×10 6 iNKT cells ~ approx. 3 x 10 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells ~ approx. 1 x 10 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells ~ approx. 1.5×10 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells ~ approx. 2 x 10 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells ~ approx. 3 x 10 6 iNKT cells, approximately 1 x 10 6 iNKT cells ~ approx. 1.5×10 6 iNKT cells, approximately 1 x 10 6 iNKT cells ~ approx. 2 x 10 6 iNKT cells, approximately 1 x 10 6 iNKT cells ~ approx. 3 x 10 6iNKT cells, approximately 1.5 x 10 6 iNKT cells ~ approx. 2 x 10 6 iNKT cells, approximately 1.5 x 10 6 iNKT cells ~ approx. 3 x 10 6 iNKT cells or approximately 2 × 10 6 iNKT cells ~ approx. 3 x 10 6 In some embodiments, the population or unit dose of cells comprises about 0.01 x 10 cells per kg of subject. 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells, approximately 1 x 10 6 iNKT cells, approximately 1.5 x 10 6 iNKT cells, approximately 2 x 10 6 iNKT cells or approximately 3 × 10 6 Including iNKT cells.

[0066] In some embodiments, the population or unit dose of cells is 1 x 10 6 In some embodiments, the population or unit dose of cells comprises 3×10 normal naive αβ-T cells in the range of less than 3×10 cells / kg subject body weight. 5 In some embodiments, the population or unit dose of cells comprises 7.5 x 10 normal naive αβ-T cells in the range of less than 7.5 x 10 cells / kg subject body weight. 4 5 x 10 cells / kg subject weight or less 4 Less than 1 x 10 cells / kg 4 Less than 0.5 x 10 cells / kg 4 cells / kg or less than 1 x 10 3 cells / kg subject body weight, containing normal naive αβ-T cells in the range of less than 1000 cells / kg subject body weight.

[0067] In some embodiments, each unit dose of therapeutic cell population comprises 1.0 x 10 6 ~50×10 6 of hematopoietic stem / progenitor cells (HSPCs), 0.1 × 10 6 ~1000×10 6 of memory T cells (Tmem), 0.1 × 10 6 ~1000×10 6 of regulatory T cells (Tregs) and 3 × 10 5In some embodiments, each unit dose of therapeutic cell population contains less than 3.0 x 10 normal naive αβ-T. 6 ~50×10 6 of hematopoietic stem / progenitor cells (HSPCs), 0.3 × 10 6 ~1000×10 6 of memory T cells (Tmem), 0.5 × 10 6 ~1000×10 6 of regulatory T cells (Tregs) and 3 × 10 5 In some embodiments, each unit dose of therapeutic cell population contains less than 1.0 x 10 normal naive αβ-T. 6 ~50×10 6 of hematopoietic stem / progenitor cells (HSPCs), 0.3 × 10 6 ~1000×10 6 of memory T cells (Tmem), 0.5 × 10 6 ~1000×10 6 of regulatory T cells (Tregs) and 3 × 10 5 Contains less normal naive αβ-T.

[0068] In any one embodiment disclosed herein, HSPC can be provided by the donor that is haplotype-matched with the subject.In some embodiments, Treg, Tmem, iNKT, or any combination thereof, are provided by the donor that is HLA-matched sibling donor or HLA-matched unrelated donor or partially matched HLA-matched unrelated donor.In some embodiments, HSPC is provided by the donor that is haplotype-matched with the subject, and Treg, Tmem, iNKT, or any combination thereof, are provided by the donor that is HLA-matched sibling donor or HLA-matched unrelated donor.

[0069] Treatment method The present disclosure provides a method of performing cell transplant therapy in a subject having a disease, condition, or disorder, comprising: administering to the subject any of the therapeutic cell transplant compositions described herein. For example, a therapeutic composition comprising cells can be transfused into a subject in need thereof.

[0070] Subjects that can be treated include those suffering from leukemia, lymphoma, chronic infection, or autoimmune diseases, malignant or non-malignant blood diseases, AML, ALL, CML, CLL, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, MDS, lymphoproliferative diseases, type 1 diabetes, congenital metabolic disorders, genetic diseases, severe combined immunodeficiency, sickle cell anemia, β-thalassemia, multiple sclerosis, solid organ transplantation, Crohn's disease, ulcerative colitis, lupus, hemophagocytic lymphohistiocytosis, glycogenosis, breast cancer, other solid tumors, leukodystrophy, mucopolysaccharidosis, or any other disease that would benefit from HSPC transplantation. In some embodiments, the subject suffers from relapsed ALL or AML or primary refractory ALL or AML with less than 10% blasts. In some embodiments, the subject suffers from ≥CR1 or high-risk AML with minimal residual disease positive. In some embodiments, the subject suffers from >=CR1 or high-risk ALL with minimal residual disease positive. In some embodiments, the subject suffers from high-risk CML. In some embodiments, the subject suffers from high-risk myeloproliferative disorders. In some embodiments, the subject suffers from relapsed non-Hodgkin lymphoma responsive to treatment. In some embodiments, the subject suffers from MDS with less than 10% blasts at the time of transplantation. In some specific embodiments, the subject has one or more of the following characteristics: age 18-65, Karnofsky score ≥60 or ECOG ≤2, HCT-comorbidity index ≤4, creatinine <1.5 mg / dL, cardiac ejection fraction >45%, corrected DLCO >60% of predicted value, total bilirubin <3 times the upper limit of normal (ULN) (unless due to Dubin-Johnson syndrome), AST and ALT <3 times ULN, not pregnant or lactating, HIV negative, and no co-existing diseases that can limit life expectancy to <6 months.In certain specific embodiments, the subject has one or more of the following characteristics: patient age 0 - 3, 3 - 6, 6 - 12, 12 - 14, 12 - 18, 18 - 65, 65 - 70, 70 - 75, 75 - 80, 80 - 90 or older, or any range therebetween, Karnofsky score ≥ 60 or ≥ 80, or ECOG ≤ 2, HCT - co - morbidity index ≤ 4, creatinine < 1.5 mg / dL, cardiac ejection fraction > 45%, corrected DLCO > 60% of predicted value, total bilirubin < 3 times the upper limit or < 1.5 times the upper limit of normal value (ULN) (unless due to Gilbert's syndrome), AST and ALT < 3 times ULN or < 1.5 times, not pregnant or lactating, HIV negative, and no co - existing diseases that may limit life expectancy to < 6 months.

[0071] The therapeutic cell compositions described herein can be administered in place of conventional HCT. Exemplary therapeutic cell compositions are compatible with reduced - intensity conditioning (RIC) and myeloablative (MA) regimens. Due to the low risk of GVHD, MA conditioning can be used in place of RIC for some patients, particularly for patients with the underlying malignancy. The therapeutic cell compositions disclosed herein have less GVHD, so the risk of GVHD outweighs the alloreactivity, and alloreactivity can be beneficial in the case of multiple myeloma where autologous transplantation is currently in clinical use. Non - malignant conditions are likely to benefit from increased immune reconstitution, and thus, lower infection rates, high engraftment rates, and persistent chimerism.

[0072] The therapeutic cell compositions described herein are administered to a subject according to known methods or modifications thereof that are apparent to those skilled in the art.

[0073] An "effective amount" or "therapeutically effective amount" refers to the amount of a composition described herein sufficient to aid in the treatment of a disease when administered to a subject (e.g., a human). The amount of a composition that constitutes a "therapeutically effective amount" varies depending on the cell preparation, the disease state and its severity, the mode of administration, and the age of the subject being treated, but can be routinely determined by one of ordinary skill in the art given their knowledge and the present disclosure. When referring to an individual active ingredient or composition administered alone, the therapeutically effective dose refers to that ingredient or composition alone. When referring to a combination, the therapeutically effective dose refers to the combined amount of active ingredients, composition, or both that results in the therapeutic effect, whether administered sequentially, concurrently, or simultaneously.

[0074] In some embodiments, the HSPC cells are CD34 + cells 1.0×10 6 ~50×10 6 Cells / kg subject weight, 1.0 x 10 6 ~20×10 6 cells / kg subject body weight, or 2.0 x 10 6 ~10×10 6 In some embodiments, the HSPC cells are administered at a concentration of about 1 x 10 cells / kg of subject body weight. 6 HSPC ~ approx. 20×10 6 In some embodiments, the HSPC cells are administered at a concentration of at least about 1 x 10 HSPC cells per kg of subject. 6 In some embodiments, the HSPC cells are administered at a concentration of up to about 20 x 10 HSPC cells per kg of subject. 6 In some embodiments, the HSPC cells are administered at a concentration of about 1 x 10 HSPC cells per kg of subject. 6 HSPC ~ approx. 2×10 6 HSPC, approx. 1×10 6 HSPC ~ approx. 5×10 6 HSPC, approx. 1×10 6 HSPC ~ approx. 7×10 6 HSPC, approx. 1×10 6 HSPC~approx. 10×10 6 HSPC, approx. 1×10 6 HSPC ~ approx. 15×10 6 HSPC, approx. 1×106 HSPC ~ approx. 20×10 6 HSPC, approx. 2×10 6 HSPC ~ approx. 5×10 6 HSPC, approx. 2×10 6 HSPC ~ approx. 7×10 6 HSPC, approx. 2×10 6 HSPC~approx. 10×10 6 HSPC, approx. 2×10 6 HSPC ~ approx. 15×10 6 HSPC, approx. 2×10 6 HSPC ~ approx. 20×10 6 HSPC, approx. 5×10 6 HSPC ~ approx. 7×10 6 HSPC, approx. 5×10 6 HSPC~approx. 10×10 6 HSPC, approx. 5×10 6 HSPC ~ approx. 15×10 6 HSPC, approx. 5×10 6 HSPC ~ approx. 20×10 6 HSPC, approx. 7×10 6 HSPC~approx. 10×10 6 HSPC, approx. 7×10 6 HSPC ~ approx. 15×10 6 HSPC, approx. 7×10 6 HSPC ~ approx. 20×10 6 HSPC, approx. 10×10 6 HSPC ~ approx. 15×10 6 HSPC, approx. 10×10 6 HSPC ~ approx. 20×10 6 HSPC or approximately 15 x 10 6 HSPC ~ approx. 20×10 6 In some embodiments, the HSPC cells are administered at a concentration of about 1 x 10 HSPC cells per kg of subject. 6 HSPC, approx. 2×10 6 HSPC, approx. 5×10 6 HSPC, approx. 7×10 6 HSPC, approx. 10×10 6 HSPC, approx. 15×10 6 HSPC or approximately 20 x 10 6 HSPCs are administered at a concentration of

[0075] In some embodiments, Tmem is 0.1 x 10 6 ~1000×10 6 Cells / kg subject weight, 1.0 x 10 6 ~250×10 6 cells / kg subject weight, or 2.9 x 10 6 ~10.1×10 6 Cells / kg subject weight, 10.1 x 10 6 ~30.1×10 6 Cells / kg subject weight, 30.1 x 10 6 ~101×10 6 Cells / kg subject weight, 1.0 x 10 6 ~100×10 6 In some embodiments, Tmem cells are administered at a concentration of about 1 x 10 cells / kg of subject body weight. 6 Tmem cells ~approximately 100 x 10 6 In some embodiments, the Tmem cells are administered at a concentration of at least about 1 x 10 Tmem cells per kg of subject. 6 In some embodiments, Tmem cells are administered at a concentration of up to about 100 x 10 Tmem cells per kg of subject. 6 In some embodiments, the Tmem cells are administered at a concentration of about 1 x 10 Tmem cells per kg of subject. 6 Tmem cells ~approximately 10 x 10 6 Tmem cells, approximately 1 × 10 6 Tmem cells ~ approximately 20 × 10 6 Tmem cells, approximately 1 × 10 6 Tmem cells ~ approximately 50 × 10 6 Tmem cells, approximately 1 × 10 6 Tmem cells ~approximately 75 × 10 6 Tmem cells, approximately 1 × 10 6 Tmem cells ~approximately 100 x 10 6 Tmem cells, approximately 10 × 10 6 Tmem cells ~ approximately 20 × 10 6 Tmem cells, approximately 10 × 10 6 Tmem cells ~ approximately 50 × 10 6 Tmem cells, approximately 10 × 10 6 Tmem cells ~approximately 75 × 10 6 Tmem cells, approximately 10 × 10 6Tmem cells ~approximately 100 x 10 6 Tmem cells, approximately 20 × 10 6 Tmem cells ~ approximately 50 × 10 6 Tmem cells, approximately 20 × 10 6 Tmem cells ~approximately 75 × 10 6 Tmem cells, approximately 20 × 10 6 Tmem cells ~approximately 100 x 10 6 Tmem cells, approximately 50 × 10 6 Tmem cells ~approximately 75 × 10 6 Tmem cells, approximately 50 × 10 6 Tmem cells ~approximately 100 x 10 6 Tmem cells or approximately 75 × 10 6 Tmem cells ~approximately 100 x 10 6 In some embodiments, the Tmem cells are administered at a concentration of about 1 x 10 Tmem cells per kg of subject. 6 Tmem cells, approximately 10 × 10 6 Tmem cells, approximately 20 × 10 6 Tmem cells, approximately 50 × 10 6 Tmem cells, approximately 75 × 10 6 Tmem cells or approximately 100 × 10 6 It is administered at a concentration of Tmem cells.

[0076] In some embodiments, Tregs are 0.1 x 10 6 ~1000×10 6 Cells / kg subject weight, 0.1 x 10 6 ~5×10 6 cells / kg subject body weight, or 0.5 x 10 6 ~2.5×10 6 In some embodiments, Tregs are administered at a concentration of about 0.5 x 10 cells / kg of subject body weight. 6 Treg cells ~ approx. 2.5×10 6 In some embodiments, the Tregs are administered at a concentration of at least about 0.5 x 10 Tregs per kg of subject. 6 In some embodiments, Tregs are administered at a concentration of up to about 2.5 x 10 Tregs per kg of subject. 6In some embodiments, Tregs are administered at a concentration of about 0.5 x 10 Tregs per kg of subject. 6 Treg cells ~ approx. 1 x 10 6 Treg cells, approximately 0.5 x 10 6 Treg cells ~ approx. 1.5×10 6 Treg cells, approximately 0.5 x 10 6 Treg cells ~ approx. 2 x 10 6 Treg cells, approximately 0.5 x 10 6 Treg cells ~ approx. 2.5×10 6 Treg cells, approximately 1 x 10 6 Treg cells ~ approx. 1.5×10 6 Treg cells, approximately 1 x 10 6 Treg cells ~ approx. 2 x 10 6 Treg cells, approximately 1 x 10 6 Treg cells ~ approx. 2.5×10 6 Treg cells, approximately 1.5 x 10 6 Treg cells ~ approx. 2 x 10 6 Treg cells, approximately 1.5 x 10 6 Treg cells ~ approx. 2.5×10 6 Treg cells or approximately 2 × 10 6 Treg cells ~ approx. 2.5×10 6 In some embodiments, Tregs are administered at a concentration of about 0.5 x 10 Tregs per kg of subject. 6 Treg cells, approximately 1 x 10 6 Treg cells, approximately 1.5 x 10 6 Treg cells, approximately 2 x 10 6 Treg cells or approximately 2.5 × 10 6 It is administered at a concentration of Treg cells.

[0077] In some embodiments, naive Tregs are about 0.1 x 10 6 ~About 500×10 6 , about 0.2×10 6 ~About 500×10 6 , about 0.3×10 6 ~About 500×10 6 , about 0.4×10 6 ~About 500×10 6 , about 0.5×10 6 ~About 500×106 , about 0.6×10 6 ~About 500×10 6 , about 0.7×10 6 ~About 500×10 6 , about 0.8×10 6 ~About 500×10 6 , about 0.9×10 6 ~About 500×10 6 or about 1 x 10 6 ~About 500×10 6 Administered at a concentration of cells / kg subject weight

[0078] In some embodiments, memory Tregs are 0.005×10 6 ~500×10 6 The cells are administered at a concentration of 100 cells / kg subject weight.

[0079] In some embodiments, the iNKT cells are 0.5 x 10 2 ~2000×10 3 Cells / kg subject weight, 0.5 x 10 2 ~1×10 4 Cells / kg subject weight, 0.5 x 10 3 ~1×10 5 Cells / kg subject weight, 0.5 x 10 4 ~1×10 6 Cells / kg subject weight, 0.5 x 10 5 ~1×10 7 Cells / kg subject weight 0.5 x 10 2 ~1×10 7 cells / kg subject body weight or 1.0 x 10 4 ~2.5×10 6 In some embodiments, iNKT cells are administered at a concentration of about 0.01 x 10 cells / kg of subject body weight. 6 iNKT cells ~ approx. 3 x 10 6 In some embodiments, the iNKT cells are administered at a concentration of at least about 0.01 x 10 iNKT cells per kg of subject. 6 In some embodiments, the iNKT cells are administered at a concentration of up to about 3 x 10 iNKT cells per kg of subject. 6In some embodiments, the iNKT cells are administered at a concentration of about 0.01 x 10 iNKT cells per kg of subject. 6 iNKT cells ~ approx. 0.1×10 6 iNKT cells, approximately 0.01×10 6 iNKT cells ~ approx. 1 x 10 6 iNKT cells, approximately 0.01×10 6 iNKT cells ~ approx. 1.5×10 6 iNKT cells, approximately 0.01×10 6 iNKT cells ~ approx. 2 x 10 6 iNKT cells, approximately 0.01×10 6 iNKT cells ~ approx. 3 x 10 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells ~ approx. 1 x 10 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells ~ approx. 1.5×10 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells ~ approx. 2 x 10 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells ~ approx. 3 x 10 6 iNKT cells, approximately 1 x 10 6 iNKT cells ~ approx. 1.5×10 6 iNKT cells, approximately 1 x 10 6 iNKT cells ~ approx. 2 x 10 6 iNKT cells, approximately 1 x 10 6 iNKT cells ~ approx. 3 x 10 6 iNKT cells, approximately 1.5 x 10 6 iNKT cells ~ approx. 2 x 10 6 iNKT cells, approximately 1.5 x 10 6 iNKT cells ~ approx. 3 x 10 6 iNKT cells or approximately 2 × 10 6 iNKT cells ~ approx. 3 x 10 6 In some embodiments, the iNKT cells are administered at a concentration of about 0.01 x 10 iNKT cells per kg of subject. 6 iNKT cells, approximately 0.1 x 10 6 iNKT cells, approximately 1 x 10 6 iNKT cells, approximately 1.5 x 10 6 iNKT cells, approximately 2 x 10 6 iNKT cells or approximately 3 × 106 It is administered at a concentration of iNKT cells.

[0080] In some embodiments, the cells administered are greater than 3×10 5 In some embodiments, the administered cells comprise less than 2×10 normal naive αβ-T cells / kg subject body weight. 5 1 x 10 cells / kg subject weight or less 5 7.5 x 10 cells / kg subject, less than body weight 4 cells / kg, less than 5 x 10 4 Less than 1 x 10 cells / kg 4 Less than 0.5 x 10 cells / kg 4 cells / kg or less than 1 x 10 3 Contains fewer normal naive αβ-T cells than cells / kg subject body weight.

[0081] As further discussed in this example, the purity of a therapeutic cell composition can be important to the clinical outcome of treatment. For example, processing a cell fraction that may contain naive Tcon contaminating cells along with therapeutic cells using methods that result in low purity can impair therapeutic efficacy by increasing levels of GVHD, acute GVHD grade 3-4, steroid-resistant acute GVHD grade 3-4, chronic GVHD, graft failure, graft rejection, severe infection, organ failure, VOD / SOS, and relapse compared to therapeutic cell compositions prepared using high-fidelity sorting techniques (e.g., FACS).

[0082] Without wishing to be bound by theory, it is believed that the therapeutic cell compositions disclosed herein provide superior therapeutic benefits over myeloablative transplantation because the Treg, Tmem, and iNKT cells derived from HSPCs provide earlier immune system rescue compared to conventional myeloablative HSPC transplantation. Following conventional myeloablative HSPC transplantation, a subject's immune system may begin to recover significantly over a period of up to one year, providing protective immunity. In comparison, the introduction of Treg, Tmem, or iNKT cells along with HSPCs into the therapeutic composition of the present disclosure is believed to replenish the subject's destroyed immune cells. These replenished cells begin to provide significant immune function immediately after administration. Therefore, the therapeutic cell compositions of the present disclosure provide superior therapeutic benefits over conventional HSPC transplantation. It is also believed that regulatory T cells may assist in engraftment and suppression of GVHD by suppressing alloreactive T cells in solid tissues. It is also believed that iNKT cells provide a positive feedback signal to Tregs to promote a suppressive environment, particularly within solid tissues. HSPCs are also believed to reconstitute the blood and immune system of myeloablative patients, e.g., NK cells, which are further believed to confer a graft-versus-leukemia effect. Memory T cells are also believed to confer anti-infective and anti-leukemia effects for a limited period (less than 5 years). Due to their limited lifespan, these cells are unable to sustain a long-term graft-versus-host disease response.

[0083] In some embodiments, therapeutic cell populations are administered to a subject as separate pharmaceutical compositions. For example, HSPC, Tmem, Treg, or iNKT enriched cell populations can be administered sequentially. In some embodiments, therapeutic cell populations are administered in multiple doses. In some cases, a dose of the cell population can comprise HSPCs. In some cases, a dose of the cell population can comprise Treg cells. In some cases, a dose of the cell population can comprise Tmem cells. In some cases, a dose of the cell population can comprise iNKT cells. In some embodiments, therapeutic cell populations are administered simultaneously to a subject as a single pharmaceutical composition. The doses can be administered as a course of treatment. A course of treatment can include administering a dose to a subject at 1-, 2-, 3-, 4-, 5-, 6-, or 7-day intervals. A course of treatment can also include administering a dose to a subject at 1-, 2-, 3-, or 4-week intervals. A course of treatment may involve administering doses to a subject at 1-month, 2-month, 3-month or 4-month intervals.

[0084] In some embodiments, the therapeutic compositions described herein can be administered as separate cell populations. For example, the first dose of the therapeutic composition can be administered as a population of HSPCs, a population of Treg cells, or a population of Tmem cells, either alone or in any combination. Subsequent doses can then contain any of the above cell types not present in the first dose. The doses can also include iNKT cells. For example, the first dose can include HSPCs, while the second dose includes Treg and Tmem cells. Other combinations of doses can also be administered to the subject.

[0085] In some embodiments, the complete therapeutic composition may comprise multiple doses. The complete therapeutic compositions described herein may be administered in at least one dose. The complete therapeutic compositions described herein may be administered in up to 30 doses. In some cases, the complete therapeutic compositions described herein may be administered in about 2 doses, 5 doses, 10 doses, 15 doses, 20 doses, 25 doses, or 30 doses. In some cases, a dose of the complete therapeutic composition may comprise HSPCs, Treg cells, or Tmem cells. In some cases, each dose of the complete therapeutic composition may comprise a different cell population.

[0086] In some embodiments, the unit dose is at least 3 x 10 per kg of subject 5 In some embodiments, the unit dose may comprise at least 3 x 10 HSPCs per kg of subject. 5 In some embodiments, the unit dose may comprise at least 3 x 10 Treg cells per kg of subject. 5 In some embodiments, the unit dose may comprise at least 3 x 10 Tmem cells per kg of subject. 5 In some embodiments, the unit dose may comprise 3 x 10 iNKT cells per kg of subject. 5 May contain fewer Tcon cells.

[0087] In some embodiments, the complete therapeutic composition can be administered in about 1 day. In some cases, the complete therapeutic composition can be administered for up to 30 days. For example, a first dose of a therapeutic composition described herein can be administered in 1 day, and a second dose can be administered 10 days later. In some cases, the complete therapeutic composition can be administered in about 1 day, 2 days, 5 days, 10 days, 15 days, 20 days, or 30 days.

[0088] In some embodiments, the cells are isolated from a donor that is an HLA-matched sibling donor, an HLA-matched unrelated donor, a partially matched unrelated donor, a haploidentical related donor, an autologous donor, an HLA-mismatched donor, a donor pool, or any combination thereof. In some embodiments, the therapeutic cell population is allogeneic. In some embodiments, the therapeutic cell population is autologous. In some embodiments, the therapeutic cell population is haploidentical. In some embodiments, the therapeutic cell population is isolated from mobilized peripheral blood, mobilized apheresis blood products, bone marrow, umbilical cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof.

[0089] In some embodiments, the therapeutic cell population is obtained by a single tissue collection. In some embodiments, the therapeutic cell population is obtained by one or more tissue collections. In some embodiments, the therapeutic cell population comprises HSPCs provided by at least a first donor and Tregs and Tmems provided by at least a second donor. In some embodiments, the therapeutic cell population comprises iNKT cells provided by the at least a second donor. In some specific embodiments, the haplotype of the first donor is matched with that of the subject. In some specific embodiments, the second donor is an HLA-matched sibling donor or an HLA-matched or partially matched unrelated donor.

[0090] In some embodiments, the subject has been pretreated with radiation, chemotherapy, recombinant protein, antibody, or toxin-conjugated antibody, or any combination thereof, prior to treatment. In some embodiments, the subject is pretreated for cell transplant therapy by first treating the subject with myeloablative therapy. Exemplary myeloablative therapies include chemotherapy or radiation therapy. Myeloablative therapy is thought to provide therapeutic benefit by debulking the tumor. Cancer cells are generally more sensitive to chemotherapy / radiotherapy than many normal cells. However, if tumor-infiltrating cells survive the course of chemotherapy / radiotherapy, the subject is at risk of recurrence. Therefore, high levels of chemotherapy can help improve the elimination of tumor-infiltrating populations; however, this concentration can cause toxicity to normal cells. While some sensitive normal cells are not essential, hematopoietic stem cells are killed to a lethal extent by high levels of chemotherapy. Myeloablative regimens eradicate a sufficient number of HSCs, and the patient would usually die without a transplant. When HSPCs are infused into a myeloablative subject, the donor cells can rescue the subject and reconstitute the subject's blood and immune system for life. In some embodiments, the myeloablative therapy comprises the administration of busulfan, cyclophosphamide, TBI, fludarabine, etoposide, or any combination thereof. In some embodiments, the myeloablative therapy comprises the administration of an anti-cKIT antibody. In some embodiments, the myeloablative therapy comprises the administration of an antibody-drug conjugate. The antibody-drug conjugate can be, for example, an anti-CD45-saporin or anti-cKit-saporin therapeutic antibody. In some embodiments, the myeloablative therapy is a reduced-intensity conditioning therapy.

[0091] In some specific embodiments, the therapeutic cell population is administered to the subject as a combination therapy including an immunosuppressant. Exemplary immunosuppressants include sirolimus, tacrolimus, cyclosporine, mycophenolate, antithymocyte globulin, corticosteroids, calcineurin inhibitors, antimetabolites such as methotrexate, post-transplant cyclophosphamide, or any combination thereof. In some embodiments, the subject is pretreated with sirolimus or tacrolimus alone as prophylaxis against GVHD. In some embodiments, the therapeutic cell population is administered to the subject before the immunosuppressant. In some embodiments, the therapeutic cell population is administered to the subject after the immunosuppressant. In some embodiments, the therapeutic cell population is administered to the subject simultaneously with the immunosuppressant. In some embodiments, the therapeutic cell population is administered to the subject without the immunosuppressant. In some embodiments, the patient receiving the therapeutic cell population receives an immunosuppressant within 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, or less than 1 week.

[0092] In some embodiments, subjects receiving Bu / Flu and / or Bu / Cy are initiated on day +3 with tacrolimus as an intravenous infusion at an initial dose of 0.03 mg / kg / day, with a target of 4-8 ng / ml. In some embodiments, subjects receiving Cy / TBI (also TBI / VP-16 or TBI / VP-16 / Cy) are initiated on day +3 with sirolimus at an initial dose of 6 mg loading dose followed by 2 mg daily, with a target of 3-8 ng / ml. If the subject becomes intolerant to their specific GVHD prophylaxis or there is another reason for a change as determined by the treating physician, prophylaxis may be changed at the treating physician's discretion, with tacrolimus, sirolimus, or mycophenolate mofetil being recommended. If GVHD occurs, the appropriate treatment schedule and dose are initiated. Recipients who develop acute GVHD are treated at the treating physician's discretion.

[0093] Selection and sorting of cell populations Before the formulation or administration of therapeutic cells, the cell source(s) for enriching or depleting therapeutic cells are collected from a donor (e.g., peripheral blood mononuclear cells, bone marrow, umbilical cord blood).Methods for enriching or depleting specific subset cell populations in a cell mixture are well known in the art.For example, cell populations can be enriched or depleted by density separation, rosetting tetrameric antibody complex-mediated enrichment / depletion, magnetic activated cell sorting (MACS), multiparameter fluorescence-based molecular phenotyping, such as fluorescence activated cell sorting (FACS), or any combination thereof.Additional methods for enriching or depleting cell populations are described, for example, in U.S. Provisional Patent Application No. 62 / 421,979; U.S. Patent Application Publication No. 2014 / 0011690; and U.S. Patent Application Publication No. 2016 / 0245805, which are incorporated herein by reference in their entirety. Collectively, these methods of enriching or depleting a cell population may be generally referred to herein as "sorting" the cell population, or contacting the cells "under conditions" such that an enriched (+) or depleted (-) cell population is formed or obtained.

[0094] Accordingly, embodiments of the present disclosure include methods for making pharmaceutical compositions comprising processing at least one sample to obtain: (a) an enriched population of hematopoietic stem / progenitor cells (HSPCs); (b) an enriched population of regulatory T cells (Tregs); (c) an enriched population of memory T cells (Tmems); and (d) formulating the enriched populations of HSPCs, memory T cells, and Tregs as a pharmaceutical composition suitable for administration to a subject, wherein the populations of (a)-(c) are depleted of normal, naive αβ-T cells. In some embodiments, the methods may further comprise processing the sample to obtain an enriched population of iNKT cells.

[0095] In some embodiments, the method further comprises processing the sample to obtain Lin +The method further comprises obtaining a population of cells depleted of cells. The method may comprise obtaining an enriched population of naive Tregs, an enriched population of memory Tregs, or both. In some embodiments, obtaining the enriched population of Tmems comprises obtaining enriched central memory T stem cells (T SCM ) population, enriched central memory T cells (T CM ), a population of enriched effector memory T cells (T EM ), or any combination thereof. In some embodiments, the method may further comprise processing the sample to obtain an enriched population of iNKT cells.

[0096] In some embodiments, the population of enriched HSPCs is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more CD34 + In some embodiments, the Lin + The cell-depleted cell population is 1% to 30% Lin + cells, preferably less than 1% Lin + In some embodiments, the enriched population of Tregs comprises 20% to 99.9% Tregs. In some embodiments, the enriched population of Tmems comprises 10% to 99.9% Tmems. In some embodiments, the enriched population of iNKTs comprises 10% to 99.9% iNKTs.

[0097] In some embodiments, formulating the pharmaceutical composition comprises combining an enriched HSPC population, a memory T cell population, a Treg population, an iNKT population, or any combination thereof, into a mixed population of enriched cells.

[0098] In some embodiments, the mixed population of enriched cells is 500:1 to 1:1,000, 400:1 to 1:1,000, 300:1 to 1:1,000, 200:1 to 1:1,000, 100:1 to 1:1,000, 50:1 to 1:1,000, 10:1 to 1:1,000, 5:1 to 1:1,000, 4:1 to 1:1, 000, 3:1~1:1,000, 2:1~1:1,000, 1:1~1:1,000, 500:1~1:900, 500:1~1:800, 500:1~1:700, 500:1~1:600, 500:1~1:500, 500:1~1:400, 500:1~1:300, 500:1~1:200, 5 00:1~1:100, 500:1~1:50, 500:1~1:20, 500:1~1:10, 500:1~1:9, 500:1~1:8, 500:1~1:7, 500:1~1:6, 500:1~1:5, 500:1~1:4, 500:1~1:3, 500:1~1:2, 500:1~1:1, 400 In some embodiments, the enriched mixed population of cells comprises a ratio of HSPCs to Tmem that includes a range of 10:1 to 1:200, 100:1 to 1:2,000, or 1,000:1 to 1:20,000.

[0099] In some embodiments, the mixed population of enriched cells comprises a ratio of HSPCs to Tregs that can include ranges of 20:1 to 1:3, 100:1 to 1:30, or 200:1 to 1:300. The ratio of HSPCs to naive Tregs may include ranges of 1:500-100:1, 1:400-100:1, 1:300-100:1, 1:200-100:1, 1:100-100:1, 1:50-100:1, 1:20-100:1, 1:10-100:1, 1:5-100:1, 1:1-100:1, 1:200-50:1, 1:200-20:1, 1:200-10:1, 1:200-5:1, 1:100-1:1, 40:1-1:3, 200:1-1:15, or 400:1-1:150.

[0100] In some embodiments, the mixed population of enriched cells is 1:500-10,000:1, 1:400-10,000:1, 1:300-10,000:1, 1:200-10,000:1, 1:100-10,000:1, 1:50-10,000:1, 1:20-10,000:1, 1:10-10,000:1, 1:5-10,000:1, 1:1-10,000:1, 1:500-5,000:1, 1:500-1,000:1 , 1:500-900:1, 1:500-800:1, 1:500-700:1, 1:500-600:1, 1:500-500:1, 1:500-400:1, 1:500-300:1, 1:500-200:1, 1:500-100:1, 1:500-50:1, 1:500-20:1, 1:500-10:1, 1:500-5:1, or 1:500-1:1 ratio of HSPCs to memory Tregs.

[0101] In some embodiments, the mixed population of enriched cells is 1:2 to 1,000,000:1, 1:2 to 500,000:1, 1:1 to 500,000:1, 100:1 to 1,000,000:1, 100:1 to 500,000:1, 100:1 to 100,000:1, 500:1 to 1,000,000:1, 500:1 to 500,000:1, 500:1 to The ratio of HSPCs to iNKTs may include ranges of 100,000:1, 1,000:1 to 100,000:1, 1,000:1 to 1,000,000:1, 1,000:1 to 500,000:1, 1,000:1 to 100,000:1, 10,000:1 to 1:2, 100,000:1 to 1:20, or 1,000,000:1 to 1:200.

[0102] In some embodiments, the mixed population of enriched cells is less than 1:3, less than 1:50, less than 1:100, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1,000, less than 1:1,500, less than 1:2,000, less than 1:3,000, less than 1:4,000, less than 1:5,000, less than 1:6,000, less than 1:7,000, less than 1:8,000, less than 1:9,000, less than 1:10,000, less than 1:50 These include ratios of normal naive αβ-T cells to HSPCs of less than 1:100,000, less than 1:200,000, less than 1:300,000, less than 1:400,000, less than 1:500,000, less than 1:600,000, less than 1:700,000, less than 1:800,000, less than 1:900,000, or less than 1:1,000,000.

[0103] In some embodiments, the mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to Tmem that can be less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1:50000.

[0104] The ratio of normal naive αβ-T cells to Tregs can be less than 1:1, less than 1:10, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1:50000. The ratio of normal naive αβ-T cells to naive Tregs can be less than 1:1, less than 1:10, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1:50000. The ratio of normal naive αβ-T cells to memory Tregs can be less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, or less than 1:50000.The normal ratio of naive αβ-T cells to iNKT is less than 100:1, less than 1:1, less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:7, less than 1:8, less than 1:9, less than 1:10, less than 1:15, less than 1:20, less than 1:30, less than 1:200, less than 1:300, less than 1:400, less than 1:500, less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:100, less than 1:150, less than 1:200, less than 1:300, less than 1:400, less than 1:5 ... It may be less than 1:600, less than 1:700, less than 1:800, less than 1:900, less than 1:1000, less than 1:5000, less than 1:10000, less than 1:15000, less than 1:20000, less than 1:25000, less than 1:30000, less than 1:35000, less than 1:40000, less than 1:45000, less than 1:50000.

[0105] In some embodiments of the present disclosure, the ratio of Tmem to Treg is independent of the initial cell concentration (e.g., substantially altered from the initial concentration). This provides an advantage by allowing the concentration of Tmem to be controlled (e.g., dose-titrated) independently of the concentration of Treg. The ratio of Tmem to Treg can be 30:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, or 2:1 to 1:1. In certain embodiments, the ratio of Tmem to Treg can be 1:1 to 200:1, 1:10 to 2000:1, or 1:100 to 20,000:1. The ratio of Tmem to naive Tregs can be 5:1 to 1:10, 3:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, or 1:1 to 1:10. The ratio of Tmem to memory Tregs can be 27:1 to 0.9:1, 30:1 to 1:10, 25:1 to 1:10, 20:1 to 1:10, 15:1 to 1:10, 10:1 to 1:10, 30:1 to 1:9, 30:1 to 1:8, 30:1 to 1:7, 30:1 to 1:6, 30:1 to 1:5, 30:1 to 1:4, 30:1 to 1:3, 30:1 to 1:2, or 30:1 to 1:1.

[0106] In some embodiments of the present disclosure, the ratio of Treg to iNKT can be 20,000:1 to 1:5, 200,000:1 to 1:50, or 2,000,000:1 to 1:500.

[0107] In some embodiments of the present disclosure, the ratio of iNKT to Tmem can be 2:1 to 1:100,000, 5:1 to 1:1,000,000, or 10:1 to 1:10,000,000.

[0108] In some embodiments, the HSPCs are CD34 + HSPCs also express CD133 + , CD90 + , CD38 - , CD45RA - , Lin - In some embodiments, the HSPCs may be described as CD19 - , TCRα / β - or a combination thereof. + In some embodiments, the Tregs express CD4 + , CD25 + , CD127 - / lo , FoxP3 + In some embodiments, naive Tregs are CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA + , CD45RO - In some embodiments, memory Tregs are CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA - , CD45RO + In some embodiments, Tmem is CD3 + , CD45RA - , CD45RO + or any combination thereof. In some embodiments, T SCM CD45RA +and CD4 + or CD8 + T SCM Furthermore, CD95 + , CD122 + , CXCR3 + , LFA-1 + or any combination thereof. CM CD45RO + and CD4 + or CD8 + T CM Furthermore, CD45RA - , CD62L + , CCR7 + or any combination thereof. EM CD4 + , CD45RO + , CD45RA - , CD62L - , CCR7 - In some embodiments, the iNKT is CD1d-tet + , 6B11 + , Va24Ja18 + In any embodiment described herein, normal naive αβ-T cells are TCRα / β + CD45RA + and CD25 - , CD127 + Or both. Normal naive αβ-T cells also express TCRα + TCRβ + CD45RA + CD45RO - CD25 - CD95 - IL-2Rβ - CD127 + It can be written as:

[0109] In some embodiments, the biological sample can be sorted to isolate a population of interest, such as HSPC, Treg cells, Tmem cells, or a combination thereof. In some cases, the biological sample can be contacted with a molecule that specifically binds to CD34 to isolate HSPC. The sample can be contacted with a molecule that specifically binds to CD34. + is enriched for CD34 + Cell populations and CD34 - A cell population can be obtained. In some cases, CD34 - The cell population can be contacted with a molecule that specifically binds to CD25. + CD34 cell population - CD25 cell population sorting + Cells are enriched, thereby CD34 - CD25 - Cell populations and CD34 - CD25 + A cell population can be obtained. - CD25 + The cell population can be further sorted to obtain a Treg cell population. - CD25 + The cells can be contacted with a molecule that specifically binds CD4 and a molecule that specifically binds CD127. The cells can then be contacted with a molecule that specifically binds CD34 - CD25 + CD4 + CD127 dim / - In some cases, CD34 - CD25 - The cell population can be further sorted to enrich for Tmem cells. The cells can be contacted with a molecule that specifically binds to CD45RA. The cell sample can be enriched for CD34 - CD25 - CD45RA - It can be sorted to obtain a Tmem cell population. + The normal naive αβ-T cells can be discarded due to depletion of normal naive αβ-T cells.

[0110] In some embodiments, biological samples can be sorted to isolate a population of interest, such as HSPCs, Treg cells, Tmem cells, iNKT cells, or a combination thereof. In some cases, biological samples can be contacted with molecules that specifically bind to CD34 to isolate HSPCs. The sample can be contacted with molecules that specifically bind to CD34. + is enriched for CD34 + Cell populations and CD34 - A cell population can be obtained. In some cases, CD34 - The cell population can be contacted with a molecule that specifically binds CD25 and a molecule that specifically binds 6B11. + CD34 cell population - CD25 cell population sorting + and 6B11 + Cells are enriched, thereby CD34 - CD25 - 6B11 - Cell populations and CD34 - CD25 + 6B11 + A cell population can be obtained. - CD25 + 6B11 + The cell population can be further sorted to obtain Treg and iNKT cell populations. - CD25 + 6B11 + The cells can be contacted with a molecule that specifically binds CD4 and a molecule that specifically binds CD127. - CD25 + 6B11 + CD4 + CD127 dim / - In some cases, CD34 - CD25 - 6B11 - The cell population can be further sorted to enrich for Tmem cells. The cells can be contacted with a molecule that specifically binds to CD45RA. The cell sample can be enriched for CD34 - CD25 - 6B11 - CD45RA- It can be sorted to obtain a Tmem cell population. + The normal naive αβ-T cells can be discarded due to the depletion of normal naive αβ-T cells. In some embodiments, the method of making a therapeutic composition described herein can include separately sorting different cell populations. For example, HSPCs, Treg cells, Tmem cells, and / or iNKT cells can be separately sorted. The separately sorted populations can be mixed to form a therapeutic composition. In some cases, the separate cell populations can be isolated from different donors. For example, HSPCs can be isolated from donor 1, and Treg and Tmem cells can be sorted from donor 2. Alternatively, all cell populations can be isolated from the same donor. In some embodiments, the sample comprises mobilized peripheral blood, mobilized apheresis blood products, bone marrow, umbilical cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof. In some embodiments, the sample comprises cultured cells derived from PBMCs. In some embodiments, the sample comprises cultured cells derived from induced pluripotent stem cells (iPSCs). In some embodiments, the sample is prepared for density gradient, Ficoll, Percoll, hypotonic red blood cell lysis, ammonium chloride-potassium (ACK) buffer, or any combination thereof. In some embodiments, the sample is obtained by a single tissue collection. In some embodiments, the sample is obtained by one or more tissue collections.

[0111] Selection scheme example 1 In certain embodiments, a method for making a pharmaceutical composition includes: A. combining a sample with a molecule that specifically binds CD34 and a CD34 + Cell populations and CD34 - The cells were contacted under conditions that allowed a population of CD34 + The population of cells is recovered from the sample and CD34 - B. recovering said population of cells from said sample; and B. CD34 -and processing the population of cells to obtain at least one population of enriched therapeutic cells comprising Tregs, Tmems, iNKTs, or any combination thereof (see Figures 1A and 1B). In some embodiments, step B includes performing fine sorting to obtain the population of enriched therapeutic cells (see Figure 1A). For example, step B includes performing fine sorting to obtain the population of enriched therapeutic cells comprising CD34 - This may include contacting the population of cells with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof.

[0112] In certain embodiments, step B comprises: (i) detecting CD34 - and isolating the population of cells from at least one molecule that specifically binds to CD45RA. + Cell populations and CD45RA - The cells are contacted under conditions in which a population of CD45RA - (ii) recovering said population of cells; and (iii) detecting a CD45RA α-glucan-containing compound to obtain said population of enriched therapeutic cells. + and performing fine selection of cells from said population. Fine selection includes selecting cells that express CD45RA + The fine selection may include contacting the population of cells with a molecule that specifically binds CD4, a molecule that specifically binds CD8, a molecule that specifically binds CD25, a molecule that specifically binds CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof. - The method can further include contacting the population of cells with a molecule that specifically binds CD45RA, a molecule that specifically binds CD45RO, or a combination thereof.

[0113] In certain embodiments, step B comprises: (i) detecting CD34 - The population of cells was analyzed by Lin + at least one binding molecule that specifically binds to the marker;+ Populations of cells and Lin - The cells were contacted under conditions in which a population of cells was formed, and Lin - (ii) recovering said population of cells; and (iii) obtaining said population of enriched therapeutic cells. - In some embodiments, the fine selection comprises performing fine selection of cells from the population. - contacting the population of cells with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, a CD1d-tet molecule, a 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof.

[0114] In certain embodiments, step B comprises: (i) detecting CD34 - The population of cells is subjected to at least one Lin + at least one binding molecule that specifically binds to the marker; + Populations of cells and Lin - The cells were contacted under conditions in which a population of cells was formed, and Lin - (ii) recovering said population of cells; and (iii) - the population of cells, a binding molecule that specifically binds to CD25, and a CD25 + Cell populations and CD25 - contacting the cells under conditions to form a population of CD25 cells; + The cell population is harvested, thereby generating a population of cells containing Tregs, and CD25 - recovering said population of cells; and (iii) CD25 - the population of cells, a binding molecule that specifically binds to CD45RA, and + Cell populations and CD45RA - The cells are contacted under conditions in which a population of CD45RA - In some embodiments, step (ii) comprises recovering the population of cells (see FIG. 1A). -The cells are treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Population of cells, 6B11 + Populations of cells, or combinations thereof, and CD1d-tet - Population of cells, 6B11 - and contacting the cells under conditions in which CD1d-tet + In some embodiments, the method further comprises recovering the population of cells. + This population of cells and CD1d-tet + The population of cells, 6B11 + In some embodiments, the method comprises recovering the CD25 Treg cells in step (ii) to obtain a population of naive Treg cells, a population of memory Treg cells, a population of iNKT cells, or any combination thereof. + In some embodiments, the method further comprises performing fine sorting of the population of cells. + The population of cells is collected and further subjected to fine selection to obtain Treg, iNKT or both populations.

[0115] Selection scheme example 2 In certain embodiments, a method for producing a pharmaceutical composition includes: (A) performing a first coarse selection on at least a first sample, thereby detecting CD34 + (B) performing a second crude selection on the second sample, thereby obtaining an enriched population of cells; - Obtaining a population of cells; (C) Lin - The population of cells is subjected to a third coarse selection, whereby CD45RA - Obtain a population of memory T cells and identify CD45RA + (D) obtaining a population of CD45RA cells; and + This involves performing fine selection on the population of cells, thereby obtaining a population of Tregs (see Figures 2 and 3).

[0116] In some embodiments, the second sample comprises the CD34- The first sample comprises a population of cells (see FIG. 3). In some embodiments, the first sample comprises at least one haploidentical sample (see FIG. 2A). In some embodiments, the first sample comprises at least two haploidentical samples (see FIG. 2B). In some embodiments, the first sample comprises mobilized peripheral blood, mobilized apheresis blood products, bone marrow, cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof. In some embodiments, the second sample comprises peripheral blood mononuclear cells (PBMCs), mobilized peripheral blood, mobilized apheresis blood products, bone marrow, cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof. In some embodiments, the first sample or the second sample is allogeneic, autologous, or a combination thereof. In some embodiments, the second sample is derived from an HLA-matched unrelated donor, an HLA-matched sibling donor, or a combination thereof (see FIGS. 2A and B).

[0117] In some embodiments, the first, second, or third crude sorting comprises density separation, tetrameric antibody complex-mediated enrichment / depletion, magnetic-activated cell sorting, apheresis, leukapheresis, or any combination thereof.

[0118] In some embodiments, fine selection may involve purification using multi-parameter fluorescence-based molecular phenotyping. Fine selection may yield a population of naive Treg cells, a population of memory Treg cells, a population of iNKT cells, or any combination thereof. Fine selection may involve the selection of CD45RA + a binding molecule that specifically binds to CD25 on a cell; and + Cell populations and CD25 - contacting the cells under conditions that result in a population of CD25 + In some embodiments, the method may include harvesting the population of cells, thereby obtaining a population of Tregs. + The population of cells is further selected under conditions that result in a population of naive Treg cells. In some embodiments, fine selection is performed to identify the CD45RA +The cells are treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Population of cells, 6B11 + and contacting the cells under conditions that result in CD1d-tet + The population of cells, 6B11 + and recovering the cells or both.

[0119] Selection scheme example 3 In certain embodiments, a method for making a pharmaceutical composition comprises: (A) transferring a sample to Lin + a binding molecule that specifically binds to the marker; + Populations of cells and Lin - The cells are contacted under conditions that result in a population of Lin - (B) recovering said population of cells; - The cells were treated with a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25, and a binding molecule that specifically binds to CD34. + A population of cells, CD25 + Cell populations and CD34 - CD25 - contacting the cells under conditions to obtain a population of CD34 cells; + Cells and CD25 + Cells were harvested and CD34 - CD25 - recovering said population of cells; and (C) CD34 - CD25 - the population of cells, a binding molecule that specifically binds to CD45RA, and + Cell populations and CD45RA - and contacting the cells under conditions that result in a population of CD45RA - This involves recovering a population of (see Figure 4).

[0120] In some embodiments, step B comprises: - The cells are treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Population of cells, 6B11 +and contacting the cells under conditions that result in the formation of CD1d-tet cells or a combination thereof. + cells, 6B11 + In some embodiments, the method further comprises recovering the CD34 cells, or a combination thereof (see FIG. 4). + cells, CD25 + cells, CD1d-tet + cells, 6B11 + cells, or any combination thereof, thereby detecting CD34 + cells, CD25 + cells, CD1d-tet + cells, 6B11 + In some embodiments, the method further comprises obtaining a population of CD34 cells, or any combination thereof (see FIG. 4). + cells, CD25 + cells, CD1d-tet + cells, 6B11 + The cells, or any combination thereof, are treated with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof, and a CD34 + cells, CD4 + CD25 + CD127 - / Lo cells, CD1d-tet + The method includes contacting the cells, or a combination thereof, under conditions that result in a highly enriched population of cells (see Figure 4).

[0121] Selection scheme example 4 In certain embodiments, the method for making a pharmaceutical composition comprises: (A) performing a rough screening of a sample to identify Lin + Populations of cells and Lin - Obtain a population of cells and - (B) harvesting said population of cells; - By performing crude sorting of the population of cells, we obtained a population enriched for HSPCs and Tmem and CD45RA+ A population of cells is obtained, and the population of HSPCs and Tmem is recovered, and CD45RA + recovering said population of cells; and (C) CD45RA + and performing fine sorting of the population of cells to obtain a population of Tregs (see Figure 5). In some embodiments, fine sorting comprises selecting CD45RA + The population of cells is intercalated with a binding molecule that specifically binds CD34, a binding molecule that specifically binds CD4, and a binding molecule that specifically binds CD127, and a population of CD34+ cells and a population of CD4 + CD25 + CD127 - / Lo and contacting the cells under conditions that result in a population of CD34+ cells and a population of CD4 + CD25 + CD127 - / Lo In some embodiments, the fine selection further comprises recovering the population of cells. + The cell population is contacted with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, to induce CD1d-tet + Population of cells, 6B11 + In some embodiments, the crude sorting comprises density separation, tetrameric antibody complex-mediated enrichment / depletion, magnetic-activated cell sorting, apheresis, leukapheresis, or any combination thereof.

[0122] Selection scheme example 5 In certain embodiments, the method for making the pharmaceutical composition comprises: (A) combining a sample with a binding molecule that specifically binds CD34 and a CD34 + Cell populations and CD34 - contacting the cells under conditions that result in a population of CD34 + The population of cells is harvested and CD34 - (B) recovering said population of cells; - the population of cells, a binding molecule that specifically binds to CD25, and a CD25 + Cell populations and CD25 -contacting the cells under conditions that result in a population of CD25 + The population of cells is harvested and CD25 - (C) recovering said population of cells; and - the population of cells, a binding molecule that specifically binds to CD45RA, and + Cell populations and CD45RA - and contacting the cells under conditions that result in a population of CD45RA - This involves harvesting a population of cells (see Figures 6, 10, 11, 12).

[0123] In some embodiments, step B comprises: (i) detecting CD34 - The population of cells is treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Population of cells, 6B11 + Populations of cells, or combinations thereof, and CD1d-tet - Population of cells, 6B11 - and contacting the cells under conditions that result in the formation of a population of CD1d-tet cells, or a combination thereof. + Population of cells, 6B11 + and recovering a population of iNKT-depleted cells, or a combination thereof, thereby obtaining a population of iNKT-depleted cells, and - Population of cells, 6B11 - (ii) recovering the population of iNKT-depleted cells, or both, thereby obtaining a population of iNKT-depleted cells; and (ii) recovering the population of iNKT-depleted cells by combining a binding molecule that specifically binds CD25 with a binding molecule that specifically binds CD25. + Cell populations and CD25 - contacting the cells under conditions that result in a population of CD25 + The population of cells is harvested and CD25 - Further comprising harvesting the population of cells (see Figure 6).

[0124] In some embodiments, step B comprises: -and combining the population of cells with a binding molecule that specifically binds to CD25, and CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, and CD25 + Populations of cells and CD1d-tet + Population of cells, 6B11 + cells or a combination thereof and CD34 - CD25 - Under conditions that result in a population of iNKT-depleted cells, CD25 + This population of cells and CD1d-tet + The population of cells, 6B11 + Cells or a combination thereof are harvested and CD34 - CD25 - and recovering the population of iNKT-depleted cells (see Figure 10).

[0125] In some embodiments, step B comprises: (i) detecting CD34 - and combining the population of cells with a binding molecule that specifically binds to CD25, and CD1d-tet, 6B11 monoclonal antibody or functional fragment thereof, or a combination thereof, and CD25 + Populations of cells and CD1d-tet + Population of cells, 6B11 + cells or a combination thereof and CD34 - CD25 - Under conditions that result in a population of iNKT-depleted cells, CD25 + This population of cells and CD1d-tet + The population of cells, 6B11 + Cells or a combination thereof are harvested and CD34 - CD25 - recovering said population of iNKT-depleted cells; and (ii) detecting CD25 + This population of cells and CD1d-tet + The population of cells, 6B11 +The cells or a combination thereof are finely selected by contacting the cells with a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof, to select CD4. + CD25 + CD127 - / Lo cells, CD1d-tet + cells, 6B11 + The method includes obtaining a population of cells enriched for the following:

[0126] In some embodiments, step B comprises: (i) detecting CD34 - The population of cells is contacted with an anti-CD25 antibody containing a tag (e.g., fluorescent phycoerythrin) and a biotinylated 6B11 monoclonal antibody (6B11-biotin). The 6B11-biotin is then contacted with streptavidin conjugated to the same tag as the anti-CD25 antibody. In some embodiments, the streptavidin is conjugated to phycoerythrin / Cy7 (SAv-PE / Cy7). The cells are then contacted with anti-tag magnetic particles. In some embodiments, the magnetic particles are anti-PE magnetic particles (e.g., magnetic beads). The CD25 + The CD25 and 6B11-binding cells were separated using MACS and analyzed by immunoblotting. + Cells and 6B11 + Cell populations and CD34 - CD25 - In some embodiments, step B comprises: (ii) generating a population of iNKT-depleted cells; + Cells and 6B11 + The population of cells is subjected to fine selection by isolating the cells with a binding molecule that specifically binds to CD4 and a binding molecule that specifically binds to CD127. + CD25 + CD127 - / Lo Cell-enriched cell populations and 6B11 + CD127 +The method further includes contacting the cells under conditions that result in an enriched population of cells, or any combination thereof. In some embodiments, the CD4 binding molecule is an anti-CD4 PerCP-labeled antibody. In some embodiments, the CD127 binding molecule is an anti-CD127 APC-labeled antibody. Precision sorting can include FACS, in which the CD25-PE, 6B11-biotin-SAv-PE / Cy7, CD4-PerCP, and CD127-APC are detected.

[0127] In some embodiments, the CD34 recovered in step A + The population of cells, CD25 recovered in step B + The populations of cells or both are selected from the group consisting of the CD34 + cells, the CD25 + The cells or a combination thereof are further processed by precision selection, including contacting the cells or a combination thereof with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD127, a binding molecule that specifically binds to CD45RA, or any combination thereof (see Figure 12).

[0128] Selection scheme example 6 In certain embodiments, a method for making a pharmaceutical composition includes: A. separating a sample into a binding molecule that specifically binds CD34 and a binding molecule that specifically binds CD25, and a binding molecule that specifically binds CD34. + A population of cells, CD25 + Cell populations and CD34 - CD25 - contacting the cells under conditions to obtain a population of CD34 cells; + Cell populations and CD25 + The population of cells is harvested and CD34 - CD25 - B. recovering said population of cells; and B. CD34 - CD25 - the population of cells, a binding molecule that specifically binds to CD45RA, and + Cell populations and CD45RA - and contacting the cells under conditions that result in a population of CD45RA -and recovering the population of cells (see FIG. 7). In some embodiments, step A comprises treating the sample with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet. + Population of cells, 6B11 + and contacting the cells under conditions that result in a population of cells or a combination thereof, and + The population of cells, 6B11 + and recovering the population of cells or a combination thereof (see FIG. 7). In some embodiments, the method further comprises fine-sorting the cell population obtained in step A by contacting the cells with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, CD1d-tet, or any combination thereof, to recover CD34. + cells, CD4 + CD25 + CD127 - / Lo cells, CD1d-tet + The method further includes obtaining a population of cells enriched for the cells, or any combination thereof (see Figure 8).

[0129] Selection scheme example 7 In certain embodiments, a method for producing a pharmaceutical composition comprises simultaneously processing a sample to obtain an enriched population of cells containing HSPCs, Tmems, naive Tregs, and memory Tregs, and containing less than 5% of undesired cell types (see FIG. 13). In some embodiments, the sample is contacted with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD8, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, a binding molecule that specifically binds to CD45RA, a binding molecule that specifically binds to CD45RO, or any combination thereof. In some embodiments, the method comprises contacting the sample with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and detecting CD1d-tet. + Population of cells, 6B11 +In some embodiments, the sample is contacted with a binding molecule that specifically binds to CD34, and the CD34 population or combination thereof is recovered. + The population of cells is collected, thereby producing a population of HSPCs. In some embodiments, the sample is contacted with a binding molecule that specifically binds to CD3 and does not bind to a binding molecule that specifically binds to CD45RA, a binding molecule that specifically binds to CD45RO, or a combination thereof, to collect CD3. + CD45RA - CD45RO + In some embodiments, the sample is contacted with a binding molecule that specifically binds CD4, a binding molecule that specifically binds CD25, a binding molecule that specifically binds CD127, a binding molecule that specifically binds CD45RA, a binding molecule that specifically binds CD45RO, or any combination thereof, to identify CD45. + CD25 + CD127 - / lo CD45RA + CD45RO - A population of cells, CD4 + CD25 + CD127 - / lo CD45RA - CD45RO + The population of cells is harvested.

[0130] In any of the foregoing embodiments, the therapeutic cell population contains fewer than 2%, fewer than 1%, fewer than 0.5%, fewer than 0.1%, fewer than 0.01%, or fewer than 0.001% normal naive αβ-T cells.

[0131] In any of the foregoing embodiments, Lin + The markers can be CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

[0132] In any of the foregoing embodiments, the molecule that specifically binds to CD34, a Lin+ marker, CD25, CD45RA, CDR45RO, CD4, CD8, CD127, CD90, CD133, CD38, CD95, CD122, CXCR3, LFA-1, CD62L, CCR7 or any other cell marker is an antibody or antibody fragment.

[0133] The term "antibody," as used herein, is a broad term and is used in its ordinary sense, including, but not limited to, naturally occurring and non-naturally occurring antibodies, such as single-chain antibodies, chimeric, bifunctional, and humanized antibodies, and antigen-binding fragments thereof. It will be recognized that the choice of epitope or region of the molecule to which the antibody is raised will determine its specificity, for example, for various forms (if present) or the entire molecule (e.g., all or substantially all of the molecule).

[0134] Methods for producing antibodies are well established. Those skilled in the art will recognize that numerous procedures are available for producing antibodies, such as those described in, for example, "Antibodies, A Laboratory Manual," Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, NY. Those skilled in the art will also recognize that antibody-mimicking binding fragments or Fab fragments can be prepared from genetic information by a variety of procedures (Antibody Engineering: A Practical Approach (Borrebaeck, C., ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920 (1992)). Additionally, monoclonal and polyclonal antibodies against molecules, such as proteins and markers, are commercially available (R and D Systems, Minneapolis, Minn.; HyTest Ltd., Turk, Finland; Abcam Inc., Cambridge, Mass., USA; Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald Industries International, Inc., Concord, Mass., USA; BiosPacific, Emeryville, Calif.).

[0135] In some embodiments, the antibody is a polyclonal antibody.In other embodiments, the antibody is a monoclonal antibody.In various embodiments, the antibody of the present disclosure is compatible with the downstream use of the cell population extracted according to the present method.For example, the antibody of the present disclosure can be a non-immunogenic humanized antibody.In some embodiments, the antibody of the present disclosure comprises an epitope tag that is useful for immobilizing the antibody before or after sample extraction, thereby depleting the antibody from the extracted cell population.

[0136] In some embodiments, the antibody is a biotinylated 6B11 antibody. 6B11 is an antibody that binds to the invariant TCR Va24Ja18. In some embodiments, the 6B11 antibody is biotinylated at pH 5.5-6.0, 6.0-6.5, 6.5-7, 7.5-8, 8-8.5, 8.5-9, 9-9.5, or 9.5-10. In some embodiments, the 6B11 antibody is biotinylated in a phosphate, borate, or HEPES buffer. In some embodiments, the buffer is an amine-free buffer. The biotinylation reaction can be performed at approximately 0 mM NaCl, 50 mM NaCl, 100 mM NaCl, 150 mM NaCl, 300 mM NaCl, or 500 mM NaCl, or intermediate values ​​thereof. Modification sites on proteins (e.g., antibodies) include free amines, free thiols, sugar chains, artificially introduced azides, and artificially introduced alkynes. Biotin concentrations can vary from 10 μM to 50 μM, 50 μM to 100 μM, 100 μM to 250 μM, 250 μM to 500 μM, 500 μM to 1 mM, 1 mM to 2 mM, 2 mM to 5 mM, and 5 mM to 10 mM. Reaction times can vary from 10 to 30 minutes, 30 minutes to 1 hour, 1 hour to 1.5 hours, 1.5 hours to 2.5 hours, 2.5 hours to 6 hours, 6 hours to 10 hours, and 10 hours to 18 hours. The biotinylated 6B11 antibody disclosed herein can be synthesized using any of the methods disclosed herein. + It can be used to select, purify or isolate a population of cells (iNKT cells).

[0137] A pair of a capture binding partner and a detection binding partner, e.g., a pair of a capture antibody and a detection antibody, can be used in various embodiments of the present disclosure. Thus, in some embodiments, a selection / purification protocol is typically used that uses two types of binding partners, e.g., two types of antibodies. One binding partner is a capture partner, usually immobilized on a particle, and the other binding partner is a detection binding partner, typically conjugated to a detectable label. Such antibody pairs are available from several commercial sources, e.g., BiosPacific, Emeryville, Calif. Antibody pairs can also be designed and prepared by methods well known in the art. In a specific embodiment, the antibody is biotinylated or biotin-labeled.

[0138] In some embodiments, a second imaging element is present that nonspecifically binds to all members of the initial cell population. This signal can then be read and the amount of fluorescence between cavities can be normalized. One example is an antibody that binds to a protein that is ubiquitously expressed on the cell surface of the starting cell population.

[0139] In some embodiments, the antibody or antibody fragment is coupled to or labeled with a fluorescent dye, hapten, or magnetic particle.

[0140] Several strategies are known in the art that can be used to label binding partners to enable their detection or identification in particle mixtures. The labels can be attached by any known means, for example, by methods that utilize nonspecific or specific interactions. Also, labeling can be done directly or via the binding partner.

[0141] The emission, e.g., fluorescence, from the moiety should be sufficient to permit detection using a detector as described herein. Generally, the compositions and methods of the present disclosure use highly fluorescent moieties, e.g., moieties capable of emitting electromagnetic radiation when stimulated with an electromagnetic radiation source at the moiety's excitation wavelength. Several moieties are suitable for the compositions and methods of the present disclosure.

[0142] Labels that can be activated by energy other than electromagnetic radiation are also useful in the present disclosure. Such labels can be activated, for example, by electricity, heat, or chemical reaction (e.g., chemiluminescent labels). Also, several enzyme-activated labels are known to those skilled in the art.

[0143] Typically, the fluorescence of the moiety will have sufficient quantum efficiency combined with a lack of photobleaching that the moiety is detectable above background levels in the detectors of the present disclosure, with the necessary balance for the desired detection limit, accuracy, and precision of the assay.

[0144] Furthermore, the moiety has properties consistent with its use in the assay of first choice. In some embodiments, the assay is an immunoassay, in which case the fluorescent moiety is attached to an antibody; the moiety must have properties such that it does not aggregate with other antibodies or proteins, or aggregate in excess of what is consistent with the required accuracy and precision of the assay. In some embodiments, the fluorescent moiety stains molecules that have a combination of 1) a high extinction coefficient; 2) a high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with labeling of a molecule of interest (e.g., a protein) so that the molecule can be analyzed using the analytical devices and systems of the present disclosure (e.g., without causing precipitation of the protein of interest or the protein to which the moiety is attached).

[0145] A fluorescent moiety may comprise a single entity (such as a quantum dot or fluorescent molecule) or multiple entities (e.g., multiple fluorescent molecules). "Moiety," as the term is used herein, refers to a group of fluorescent entities, such as multiple fluorophores, it being recognized that each individual entity may be separately bound to a binding partner, or the entities may be bound together so long as they provide sufficient fluorescence for them to be detected as a group.

[0146] In some embodiments, the fluorescent dye molecule comprises at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring comprises a chemically reactive group or a conjugated substance, such as an Alexa Fluor molecule.

[0147] In some embodiments, the label comprises a first type and a second type of label, e.g., two different ALEXA FLUOR® dyes (Invitrogen), where the first type and second type of dye molecules have different emission spectra.

[0148] A non-exhaustive list of useful fluorescent entities for use in the fluorescent moiety includes: ALEXA FLUOR® 488, ALEXA FLUOR® 532, ALEXA FLUOR® 555, ALEXA FLUOR® 647, ALEXA FLUOR® 700, ALEXA FLUOR® 750, fluorescein, B-phycoerythrin, allophycocyanin, PBXL-3, Atto 590, and Qdot 605.

[0149] The label can be bound to the particle or binding partner by any method known in the art, such as absorption, covalent binding, biotin / streptavidin or other binding pairs. The label can also be bound via a linker. In some embodiments, the label is cleaved by the analyte, thereby releasing the label from the particle. Alternatively, the analyte can prevent the cleavage of the linker.

[0150] The isolated cell population may be used immediately or may be cultured in vitro after isolation using methods well known in the art. The cell population may be cultured in a medium such as RPMI-1640, DMEM, X-Vivo 10, X-Vivo 15, or modified media and combinations thereof. The medium may contain 1-20% human serum. The medium may contain cytokines or molecules that activate receptors for, for example, rapamycin, SCF, SDF-1, TPO, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-15, IL-17, IL-18, IL-23, IL-33, TGF-b, IFNg, IFNa, and combinations thereof. The cell population can be stimulated with an agonist, e.g., a particle (microparticle, nanoparticle, protein or cell), comprising a multivalent display of a binding molecule, e.g., anti-CD3, anti-CD28, CD64, CD86, anti-IL-21R, CD137, or combinations thereof.

[0151] Also, cells can be frozen, or frozen before or after separation. When cells are stored for a long period of time, it is preferable to do so at around -80°C or in liquid nitrogen to ensure that the cells can be used again after thawing. For this purpose, cells are usually stored in a medium containing DMSO and / or FCS / HS together, glucose, etc. Immediately after thawing the cells, they can either be used directly for therapeutic purposes or in vitro experiments, or can be expanded and / or differentiated using growth factors, antigens, cells, etc. [Example]

[0152] Example 1 1. Preparation of Sculpt Cell Grafts Cell enrichment and depletion are performed in multiple steps to formulate the final product for infusion. Each selection step is described below along with a brief description of the procedure. A schematic of this method is shown in Figure 14.

[0153] Process A: CD34 +Cell enrichment was performed using immunomagnetic cell selection with the CLINIMACS Cell Selection System (Miltenyi Biotec, Bergish-Gladbach, Germany). Briefly, when two apheresis blood products were collected, the pooled donor apheresis blood products were washed to remove excess plasma and platelets. Based on the manufacturer's guidelines, cell counts and CD34 counts were performed using CLINIMACS buffer supplemented with 0.5% HSA (working buffer) and IVIg to reduce nonspecific binding of Miltenyi microbead reagent to cells. + The volume was adjusted to that indicated for cell counting. The cell product was labeled with CLINIMACS CD34 reagent using the manufacturer's guidelines for product content. Unbound reagent was reduced by dilution with working buffer and removal by centrifugation. The labeled cell product was connected to a CLINIMACS device, which controlled cell loading into a disposable tubing set for immunomagnetic selection of labeled cells. Retained cells were released by removing the magnetic field and entered the positive fraction bag attached to the CLINIMACS tubing set. CD34 + The enriched cells were resuspended in Normosol-R supplemented with 2% HSA as the designated infusion medium. The negative fraction (flow-through of CD34 selection) was retained for further cell processing.

[0154] Step B: Regulatory T cells and iNKT cells were enriched from the negative fraction of Step A (flow-through from CD34 selection) by immunomagnetic selection using the CLINIMACS system. The fraction volume was adjusted, and cells were labeled with PE / Cy7-conjugated CD1d (glycolipid-loaded) tetramer reagent and Miltenyi Biotec's anti-CD25 PE reagent. After washing to remove unbound reagent from the cell suspension, cells were labeled with anti-PE microbead reagent, washed to remove unbound beads, and loaded into the CLINIMACS using an LS tubing set. The labeled cells were retained on a magnetized column. Upon removal of the magnetic field, CD25 +and CD1d (glycolipid-loaded) tetramer + The cells were released from the column into a positive fraction bag attached to a tubing set. + and CD1d (glycolipid-loaded) tetramer + The flow-through containing depleted cells (negative fraction) was retained for the subsequent cell selection procedure (step C below). + Cells and CD1d (glycolipid-loaded) tetramers + Cell enrichment was an intermediate step for further enrichment in step D below.

[0155] Step C: Cells from the CD25 / CD1d-enriched negative fraction were separated into naive and memory subsets by immunomagnetic selection using CD45RA expression as an indicator of donor memory cells. Cells were prepared and labeled with CLINIMACS CD45RA reagent. After washing to remove unbound reagent from the cell suspension, the cells were loaded stepwise onto a CLINIMACS equipped with a depletion tubing set. The labeled cells were retained on a magnetized column and removed from the column into a target cell bag attached to the tubing set. CD45RA + The flow-through fraction containing the cell-depleted cells was added to the final product to ensure inclusion of donor memory cells.

[0156] Step D: Treg and iNKT cells were further purified by cell sorting using a BD FACS ARIA. + and CD1d (glycolipid-loaded) tetramer + CD25 recovered by cell enrichment + The cells were adjusted for labeling with PerCP-conjugated mouse anti-human CD4 and APC-conjugated mouse anti-human CD127 monoclonal antibody reagents, as well as additional CD25 PE (Miltenyi Biotec) and CD1d (glycolipid-loaded) tetramer PE-Cy7. After washing to remove unbound reagents, the cells were incubated with CD4-PerCP +x CD25 PE+,CD127- APC dim / neg cells (Treg) and CD127+ x CD1d (glycolipid-loaded) tetramer-PE / Cy7 + Selection was performed using a gate set for a single lymphoid cell type containing the molecular phenotype (iNKT).

[0157] The cell subsets recovered by the cell selection procedure and intended for transfusion were resuspended in Normosol-R, pH 7.2 (Hospira, Lake Forest, IL) supplemented with 0.5% HSA (Albumin-Human, 25%, Grifols, Clayton, NC). The cell fraction was placed in one blood transfer bag at 1 × 10 8 The cells were pooled at a maximum density of 100 cells / ml and placed in a continuously monitored secure refrigerator at 2-8°C.

[0158] Example 2 Creation of sculpted cell grafts using anti-iNKT antibodies Cell enrichment and depletion are performed in multiple steps to formulate the final product for infusion. Each selection step is described below along with a brief description of the procedure. A schematic of this method is shown in Figure 15.

[0159] Process A: CD34 + Cell enrichment was performed using immunomagnetic cell selection with the CLINIMACS Cell Selection System (Miltenyi Biotec, Bergish-Gladbach, Germany). Briefly, donor apheresis blood products were washed to remove excess plasma and platelets. If two donor apheresis blood products were collected, they were pooled. Based on the manufacturer's guidelines, CD34 cell counts were determined using CLINIMACS buffer supplemented with 0.5% HSA (working buffer) and IVIg to reduce nonspecific binding of Miltenyi microbead reagent to cells. +The volume was adjusted to that indicated for cell counting. The cell product was labeled with CLINIMACS CD34 reagent using the manufacturer's guidelines for product content. Unbound reagent was reduced by dilution with working buffer and removal by centrifugation. The labeled cell product was connected to a CLINIMACS device, which controlled cell loading into a disposable tubing set for immunomagnetic selection of labeled cells. Retained cells were released by removing the magnetic field and entered the positive fraction bag attached to the CLINIMACS tubing set. CD34 + The enriched cells were resuspended in Normosol-R supplemented with 2% HSA as the designated infusion medium. The negative fraction (flow-through of CD34 selection) was retained for further cell processing.

[0160] Step B: Regulatory T cells and iNKT cells were enriched from the negative fraction of Step A (flow-through from CD34 selection) by immunomagnetic selection using the CLINIMACS system. The fraction volume was adjusted, and cells were labeled with anti-CD25-PE (Miltenyi Biotec) and biotin-conjugated 6B11 monoclonal antibody (anti-iNKT). After washing to remove unbound reagent from the cell suspension, cells were labeled with PE / Cy7-conjugated streptavidin and washed again. Cells were then labeled with anti-PE microbead reagent (Miltenyi Biotec), washed to remove unbound beads, and loaded into the CLINIMACS using the CD133 enrichment program with an LS TS tubing set. The labeled cells were retained on the magnetized column. Upon removal of the magnetic field, CD25 + and 6B11 + The cells were released from the column into a target fraction bag attached to a tubing set. + and 6B11 + The flow-through containing the depleted cells (non-target fraction) was retained for the subsequent cell selection procedure (step D below). + Cells and 6B11 + Cell enrichment is an intermediate step for further enrichment in step C below.

[0161] Step C: Treg and iNKT cells were further purified by fluorescence-activated cell sorting. + and 6B11 + CD25 recovered by cell enrichment + The cells were adjusted for labeling with PerCP-conjugated mouse anti-human CD4 (Miltenyi Biotec) and APC-conjugated mouse anti-human CD127 monoclonal antibody reagents (Miltenyi Biotec), as well as additional CD25 PE (Miltenyi Biotec) and streptavidin PE-Cy7. After washing to remove unbound reagents, the cells were incubated with CD4-PerCP + x CD25 PE+,CD127- APC dim / neg cells (Treg) and CD127+ x 6B11-PE / Cy7 + A single lymphocyte cell line containing the molecular phenotype (iNKT) was selected using a set gate.

[0162] Step D: Cells from the CD25 / 6B11-negative fraction were separated into naive and memory subsets by immunomagnetic selection using CD45RA expression as an indicator of donor memory cells. Cells were prepared and labeled with CLINIMACS CD45RA reagent. After washing to remove unbound reagent from the cell suspension, the cells were loaded stepwise onto a CLINIMACS equipped with a depletion tubing set. The labeled cells were retained on a magnetized column and removed from the column into a target cell bag attached to the tubing set. CD45RA + The flow-through fraction containing the cell-depleted cells was added to the final product to ensure inclusion of donor memory cells.

[0163] The cell subsets recovered by the cell selection procedure and intended for transfusion were resuspended in Normosol-R, pH 7.2 (Hospira, Lake Forest, IL) supplemented with 0.5% HSA (Albumin-Human, 25%, Grifols, Clayton, NC). The cell fraction was placed in one blood transfer bag at 1 × 10 8 Combine at a maximum density of cells / ml and place in a continuously monitored secure refrigerator at 2-8°C.

[0164] Table 1. Reagents and cell numbers of interest for exemplary sculpt graft compositions TIFF2025175045000002.tif94158

[0165] Example 3 Sculpted cell grafts versus non-T cell-depleted and T cell-depleted allografts in a murine model of myeloablative hematopoietic cell transplantation (alloMA-HCT) A mouse model of myeloablative and cell graft transplantation was used to evaluate the performance of the exemplary Sculpt cell grafts described herein compared with known cell grafts. Figure 16 shows a diagram of the experimental procedure. To model myeloablative patients, 8-12 week-old BALB / c recipient mice were weighed and then irradiated on day -2 with a 400 / 400 rad split dose at 160 KeV from a Rad-Source RS2000 irradiator through a 0.5 mm Cu filter. Mice were switched to an antibiotic-containing diet for 6 weeks. To model residual disease, 2,500-10,000 J774 GFP-Luc cells were injected intravenously via the tail vein on day -1. The J774 cell line is a myeloid cell line originally derived from BALB / c mice and is syngeneic to the BALB / c recipient mice. J774 cells were engineered to express GFP and luciferase transgenes. Three different cell compositions, summarized in Table 2, were used to model allogeneic hematopoietic cell transplantation (HCT). Cell compositions were prepared from hematopoietic tissue of C57Bl / 6 mice as described below and intravenously infused into BALB / c mice via the retro-orbital plexus on day 0. Experimental cohorts typically consisted of 5–9 mice. Data from seven independent experiments were pooled and presented in Figure 17.

[0166] Table 2: Cell compositions administered to BALB / c mice TIFF2025175045000003.tif104154

[0167] Mice were checked daily for distress. Body condition (BC) scores and body weights were measured twice weekly. Mice with a BC of ≦1.5 were euthanized. Mice that died at a weight <70% of their original weight were recorded as having GVHD. Mice that died at a weight >70% of their original weight within 30 days after transplantation were recorded as having engraftment failure. All dead mice were dissected and examined for tumor growth in the liver and spleen using a fluorescent microscope. The presence of GFP+ tumor nodules was recorded as recurrence (see Figure 17C). A subset of mice was subjected to bioluminescence imaging on a Xenogen IVIS100 on day +10 (see Figure 17D).

[0168] Mice receiving the Sculpt cell graft composition outperformed all other cohorts (see Figures 17A and B). 94% of the mice survived to day +175 without evidence of relapse. 3% of the mice died of GVHD and 3% died of engraftment failure. The improved performance compared to BMT and HSPC compositions indicates that the GvL effect is maintained, but the rates of GVHD and engraftment failure are significantly reduced.

[0169] In comparison, all mice receiving the BMT cell composition died of GVHD before day +30. Bioluminescence imaging on day +10 showed no evidence of recurrence in these mice. Furthermore, postmortem analysis revealed no tumor nodule growth in the hematopoietic system. This result indicates that T cells in the graft were sufficient to combat cancer cells and demonstrates robust GVL by the graft despite the background of uniformly lethal GVHD. Note that in human patients, the source of the allogeneic graft is closely HLA-haplotype-matched to the recipient, whereas BALB / c and C57Bl / 6 mice are completely mismatched. Therefore, the GVHD response observed in the clinic is significantly attenuated compared to the GVHD response observed here.

[0170] Mice receiving the HSCT cell composition had mixed outcomes. 51% of mice survived long-term until day +175. 9% of HSCT recipients died of engraftment failure, as indicated by body weight >70% and death before day +30. 3% of mice died of GVHD, as indicated by body weight <70% at the last measurement before death. 36% of mice had evidence of relapse at the time of death. The high relapse rate in this cohort is attributed to prolonged immunosuppression after HSCT, as T cell counts remain low in the early weeks after transplant. In comparison, human HSCT recipients also have low T cell counts and a high relapse rate in the first year after transplant. Thus, the HSCT transplant model reflects the lack of GvL due to T cell depletion in human patients. CD34 + Higher relapse rates are observed in human patients treated with allogeneic transplants compared to T cell non-replete allografts.

[0171] Example 4 Comparison of sculpted cell grafts generated by MACS / FACS versus MACS alone in a mouse model of alloMA-HCT In this experiment, data from the Sculpt cell graft compositions presented above were compared with another cohort of mice. GVL and GVHD were assessed in animals treated with Sculpt cell grafts generated by MACS alone or a combination of MACS and FACS (see Table 3). These mice were prepared and followed as described above. Cohorts consisted of 5–7 mice, and data from three independent experiments were pooled.

[0172] Table 3. Sculpted cell grafts generated by MACS alone or a combination of MACS and FACS. TIFF2025175045000004.tif126154

[0173] MACS / FACS-Sculpt cell-transplanted mice were prepared using a combination of MACS and FACS as described in Example 3. FACS purity was typically greater than 95%. In comparison, the purity of MACS-only enriched samples was typically 20% to 80%. The allogeneic lymphocytes from MACS-only mice were not further purified by FACS, severely compromising the therapeutic benefit of this composition. Only 54% of MACS-only mice survived to day +175, whereas the survival rate of MACS / FACS-Sculpt cell-transplanted mice was 94% (see Figure 18). All fatal cases showed body weights <70% of their initial weight, indicating GVHD as the cause of death. None of these mice showed evidence of relapse at postmortem. The higher level of lethal GVHD in MACS-only compared to MACS / FACS-based production demonstrates the importance of purity of such cell populations in the context of graft-modifying procedures for myeloablative hematopoietic cell transplantation (MA-HCT). Without wishing to be bound by theory, it is believed that the predominant GVHD in mice treated with Sculpt cell grafts with MACS alone is the result of contaminating Tcon cells.

[0174] Example 5 Comparison of Sculpt Cell Grafts versus BMT with T Cell Booster Infusion in a Murine Model of AlloMA-HCT The sculpt cell graft composition was compared with a cohort administered a BMT composition with various amounts of T cells added to the cKIT+ HSPC fraction (HSPC + T cell boost). By way of background, T cell boost is an experimental clinical approach to bone marrow transplantation. As an example, T cell depletion (e.g., typically CD34+ cell CLINIMACS isolation) is performed at a clinical center, and then a portion of the T cells are boosted or transplanted into the graft at a later date. In the boost procedure, T cells are collected from CD34 - Note that these are bulk T cells because they are obtained from a fraction and not further processed. T cells are typically reintroduced at levels 10-1000 times lower than those naturally present in the BMT graft.

[0175] In this example, each booster cohort received 1 x 10 spleen-derived 5 , 2 × 10 5 , 4×10 5 , or 2 × 10 6 The cKIT+ HSPC fraction containing the boosted T cells was administered. In comparison, the SCULPT cell grafts received an average of 2.5 × 10 5 However, some SCULPT cell graft experiments contained 5 × 10 T cells. 6 as many Sculpt T cells as possible, and 1 × 10 5 A small number of Sculpt T cells were used.

[0176] In the context of adoptive transfer of fully mismatched C57Bl / 6 lymphocytes into BALB / c mice, 1 × 10 5 Even 1×10 T cells were sufficient to induce fatal GVHD in 100% of recipients (Figure 19). 5 ~4×10 5Note that mice receiving booster T cells had a prolonged time to death, but none survived long-term. In contrast, 94% (30 / 32) of SCULPT cell graft-treated mice demonstrated long-term survival up to day +175. Postmortem examination showed no evidence of relapse in any of the mice. Thus, the T cell component of the SCULPT cell graft composition mitigates the GVHD response while conferring a GVL therapeutic benefit. Similar results are not observed with the simple booster infusion of small numbers of T cells into cKIT+ magnetically enriched bone marrow cells.

[0177] Example 6 Time delay to onset of GVHD with human-derived SCLAPT cell grafts versus unprocessed PBMCs in a xenograft model of allo-HCT Leukocyte concentrates from TrimaAccel® LRS chambers were collected from four human donors after plateletpheresis. Red blood cells were removed by density gradient (Ficoll) and ACK lysis. The resulting PBMCs were washed and 2.5 x 10 PBMCs from each donor were collected. 8 Cells were processed into the sculpt cell graft lymphocyte fraction. More specifically, Tregs and iNKTs were first processed using MACS (i.e., crude sorting). The sample was stained with anti-CD25 PE and biotin-conjugated anti-iNKT antibody 6B11, then washed, stained with streptavidin PE-Cy7, then washed again, and stained with anti-PE microbeads. The sample was then positively enriched using the Possel_S program on Miltenyi's AutoMACS. The positively enriched fraction was further stained with anti-CD127 APC and anti-CD4 PerCP and analyzed on a BD FACSAria II to a purity of >98% using the following parameters: Tregs:CD4 + CD25 + CD127 - and iNKT:CD127 + 6B11 + Further purification was carried out according to

[0178] The negative fraction of the AutoMACS column was further stained with anti-CD45RA microbeads, and the CD45RA+ fraction was depleted of naive T cells using the Deplete_S program. The remaining cells were CD45RA-CD45RO+, representing memory T cells.

[0179] The sculpt cell graft was formulated based on the final yield of each subtype, which differed between donors. PBMC cohorts were combined to contain equal numbers of CD3+ cells in the sculpt cell graft composition from matched donors. + T cells were blended to match the number of memory T cells, and the final blend ranges are shown in Table 4.

[0180] Table 4: Lymphocyte Composition TIFF2025175045000005.tif34153

[0181] For xenograft experiments, immunodeficient (NSG) mice were used because a functional mouse immune system would rapidly reject incoming human tissue based on species-specific differences. NSG mice lack B, T, and NK cells, making them suitable hosts for xenograft experiments. To further prepare the host for lymphocyte engraftment, mice were administered a nonlethal dose of radiation. NSG mice were irradiated with 250 rads at 160 keV from a Rad-Source RS2000 irradiator through a 0.5 mm Cu filter and switched to an antibiotic-containing diet. On the same day, five mice per donor cohort were injected with formulated cells and followed as described above. In this context, human PBMCs are known to induce robust GVHD responses. Results show that 70% of mice receiving lymphocytes from the Sculpt cell graft survived to day +90, compared with only 15% of mice receiving an equivalent dose of CD3+ cells in PBMCs (see Figure 20). GVHD was recorded as the cause of death in all deaths. The xenogeneic response of human lymphocytes to mouse tissue is robust; however, Sculpt cell grafts not only delay the onset of lethal GVHD (which may have clinical value), but also result in sustained survival in a significant proportion of mice.

[0182] Example 7 Isolation of regulatory T cells and iNKT The following experiment demonstrates a method for generating Treg and iNKT cell fractions for use in sculpt cell graft compositions and treatments. Peripheral blood monocytes (PBMCs) were isolated from three donors using buffy coat LRS chambers as described in Example 6. PBMCs were pooled and then resuspended to 2 x 10 8 Tregs were stained with anti-CD25 PE, and iNKT cells were stained with biotin-conjugated 6B11 antibody (anti-iNKT) for 30 minutes at room temperature, washed, and then stained with streptavidin PE-Cy7 for 10 minutes at room temperature. Excess antibody was removed by washing the cells with buffer, and then 2 × 10 cells were stained with anti-PE microbeads. 8Cells were stained at 0.05% CI, 0.01- ...

[0183] The performance of Treg and iNKT cell isolation from PBMCs was evaluated by calculating the purity and yield of Treg and iNKT cells in the enriched fraction compared to unenriched cells. In a representative experiment using anti-PE beads, the purity of Tregs increased from approximately 5% to 60% (Figure 21A and Table 5). The purity of NKT cells increased from 0.02% to 0.04% with the tested anti-PE beads (Figure 21B and Table 6).

[0184] Table 5. Percent purity and yield of Treg cells TIFF2025175045000006.tif40128

[0185] Table 6. Percent purity and yield of iNKT cells TIFF2025175045000007.tif40128

[0186] This data demonstrates that by carefully titrating the magnetic selection reagent, highly pure enriched cells can be obtained without adversely compromising yield. It also demonstrates that the negative fraction can be reprocessed under similar or identical conditions to the crude PBMC product to recover more of the desired cells. This unexpected finding enables a tailor-made method for post-CLINIMACS Treg / iNKT fractions.

[0187] Example 8 Isolation of memory T cells PBMCs were isolated from three donors by buffy coat LRS chambers as described in Example 6. PBMCs were pooled and then collected at 1 x 10 9 2 x 10 cells 8 The cells were resuspended in half the manufacturer's recommended amount of CD45RA microbeads (1.0 μL of anti-CD45RA / 6.6 × 10 cells / mL). 6 0.5 μL of anti-CD45RA / 6.6 × 10 cells instead of PBMCs 6 Cells were stained with CD3, CD45RA, and CD45RO (PBMCs). Cells were washed and separated using CLINIMACS Depletion 2.1 (flow rate of 6 mL / min). Both positive and negative fractions were collected and analyzed. Fractions of known volume were removed by comparison with the measured volume of the sample fraction for each sample, stained with fluorescently conjugated monoclonal antibodies against CD3, CD45RA, and CD45RO, washed, and counted using reference beads on a Beckman Coulter CYTOFLEX flow cytometer to calculate purity and yield (Figure 22).

[0188] The performance of naive (Tn) and memory T cell isolation was evaluated by comparing the CD3 + CD45RA + and CD3 + CD45RO + Cell purity and yield were evaluated by calculation and compared with the cells before enrichment. Using half the manufacturer's recommended amount of CD45RA microbeads, the Tmem to Tn ratio increased from 2.25:1 in the starting PBMC sample to 50298:1 in the processed sample (Table 7). Furthermore, the Tmem yield after this processing was 27.5%. This study demonstrates the feasibility of producing sculpt cell grafts using a smaller amount of CD45RA microbeads.

[0189] Table 7. Depleted CD45RA + Performance of memory T cell enrichment using magnetic sorting for cells TIFF2025175045000008.tif30128

[0190] Example 9 Isolation of regulatory T cells and naive regulatory T cells The following experiment demonstrates the characteristics of Treg populations resulting from the exemplary methods disclosed herein. PBMCs were isolated from two donors using buffy coat LRS chambers as described in Example 6. The PBMCs were pooled and then resuspended at 2 x 10 8 Cells were stained at a concentration of 1000 cells / ml. Tregs were sorted using anti-CD25 magnetic microbeads. Cells were sorted using a CLINIMACS LS TS tubing set using the manufacturer's CD133 depletion protocol (loading rate: 10 mL / min, reloaded three times). Treg-enriched cells were collected in the positive fraction, and the remaining cells were collected in the negative fraction.

[0191] The Treg-enriched fraction was stained with anti-CD25 PE, CD4PerCP, CD127APC, CD45RA-FITC, and CD45RO-APC-Cy7 according to the manufacturer's instructions. Excess antibody was removed by washing the cells with buffer. The Treg-enriched fraction was counted using reference beads on a Beckman Coulter Cytoflex system and used to calculate the percent purity and yield. The performance of Treg isolation from PBMCs was evaluated by calculating the purity and yield of Treg cells in the enriched fraction compared to the unenriched cells.

[0192] Figure 23A shows that the Treg-positive fraction was 88.5% CD4 + CD25 + cells, 81.0% of which were CD127 + Furthermore, Figure 23A shows that CD4 + CD25 + CD127 + The cell population (Treg) was 67.1% memory Treg (CD45RO + ) and 17.8% of naive Tregs (CD45RA + Figure 23B shows a summary of the purity and yield of total Tregs and naive Tregs in the Treg-positive fraction.

[0193] This data demonstrates that the methods described herein result in a population of Tregs that contains significant numbers of naive Tregs that would normally be lost during the CD45RA depletion step.

[0194] Example 9 Biotinylation of 6B11 6B11 is an antibody that binds to the invariant TCR Va24Ja18. However, the potency and specificity of this reagent are such that when conjugated to a secondary fluorophore or other molecular handle, performance is usually poor. However, using it and varying the biotinylation conditions can yield unexpectedly good performance.

[0195] The 6B11 hybridoma was modified with three different concentrations of EZ-LINK Sulfo-NHS-Biotin (ThermoFisher) in PBS at pH 7.5 for 2 hours, followed by desalting. The biotinylated antibodies and conditions were as follows: 6B11.1 = 100 μM (condition 1), 6B11.2 = 250 μM (condition 2), and 6B11.3 = 1 mM (condition 3).

[0196] iNKT cells were analyzed using 6B11.1 (condition 1), 6B11.2 (condition 2), or 6B11.3 (condition 3) antibodies, and binding to cell samples from three different donors (donors 73, 74, and 75) was assessed at increasing 6B11 antibody concentrations (0 μg / μL, 0.015 μg / μL, 0.032 μg / μL, 0.062 μg / μL, 0.0125 μg / μL, 0.25 μg / μL, 0.5 μg / μL, and 1 μg / μL). Figure 24A shows that the 6B11.2 biotinylated antibody had superior performance.

[0197] iNKT cells were analyzed using 6B11.1 (condition 1), 6B11.2 (condition 2), or 6B11.3 (condition 3) antibodies in combination with CD127 and streptavidin PE-Cy7. Figure 24B shows that condition 2 demonstrated the most pronounced iNKT cell separation (4.85%). The scatter plots shown were pre-gated on CD3+ single-cell lymphocytes.

[0198] Further embodiments can be obtained by combining the various embodiments described above.The U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications mentioned herein, such as but not limited to, U.S. Provisional Patent Application No. 62 / 421,979 filed on November 14, 2016 and U.S. Provisional Patent Application No. 62 / 471,769 filed on March 15, 2017, are all incorporated herein by reference in their entirety, except where the incorporation of a reference or a portion thereof contradicts the present disclosure.The various aspects of the present embodiment can be modified, if necessary, to obtain further embodiments using the ideas of the various patents, applications and publications.

[0199] These and other changes can be made to the present embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

Claims

1. 1. A pharmaceutical composition comprising a therapeutic cell population enriched for hematopoietic stem / progenitor cells (HSPCs), memory T cells (Tmem), and regulatory T cells (Tregs), comprising: the cell population is depleted of normal naive αβ-T cells, and the pharmaceutical composition comprises a ratio of normal naive αβ-T cells to Tregs that is less than 1:5; Pharmaceutical compositions.

2. 10. The pharmaceutical composition of claim 1, further comprising an enriched population of invariant natural killer T cells (iNKT).

3. The pharmaceutical composition comprising one or more unit doses of a cell transplant, wherein each unit dose of the cell transplant comprises a therapeutic cell population per kilogram (kg) of body weight of the subject receiving the cell transplant, and wherein each unit dose of the therapeutic cell population comprises: 3×10 5 More hematopoietic stem / progenitor cells (HSPCs), 3×10 5 more memory T cells (Tmem), 5×10 5 More regulatory T cells (Tregs), and 3×10 5 Less normal naive αβ-T 3. The pharmaceutical composition of claim 1 or 2, comprising:

4. Unit dose is 0.5 × 10 3 The pharmaceutical composition of claim 3, further comprising more iNKT cells.

5. Each unit dose of the therapeutic cell population comprises: 1.0×10 6 ~50×10 6 Hematopoietic stem / progenitor cells (HSPCs), 0.3×10 6 ~1000×10 6 memory T cells (Tmem), 0.5×10 6 ~1000×10 6 regulatory T cells (Tregs), and 3×10 5 Less normal naive αβ-T 5. The pharmaceutical composition of any one of claims 1 to 4, comprising:

6. The unit dose is 0.5 x 10 3 ~2000×10 3 10. The pharmaceutical composition of any of the preceding claims, further comprising invariant natural killer T (iNKT) cells at a concentration of cells / kg.

7. The pharmaceutical composition of any one of the preceding claims, wherein the Tregs comprise a population of naive Tregs, a population of memory Tregs, or both.

8. Tmem is a transcription factor that regulates the function of central memory T cells (T CM ) population, effector memory T cells (T EM ) or any combination thereof.

9. Central memory T stem cells (T SCM 10. The pharmaceutical composition of claim 1, comprising a population of:

10. HSPCs are CD34 + 2. The pharmaceutical composition of claim 1, wherein

11. HSPCs express CD133 + , CD90 + , CD38 - , CD45RA - , Lin - or any combination thereof.

12. HSPC cKIT + 2. The pharmaceutical composition of claim 1, wherein

13. HSPCs express CD19 - , TCRα - or a combination thereof.

14. Tmem, CD3 + , CD45RA - , CD45RO + or any combination thereof.

15. Tregs, CD4 + , CD25 + , CD127 - / lo , FoxP3 + or any combination thereof.

16. Naive Tregs express CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA + , CD45RO - or any combination thereof.

17. Memory Tregs are CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA - , CD45RO + or any combination thereof.

18. T SCM However, CD45RA + and CD4 + or CD8 + 2. The pharmaceutical composition of claim 1, wherein

19. T SCM But CD95 + , CD122 + , CXCR3 + , LFA-1 + 19. The pharmaceutical composition of claim 18, wherein the compound is selected from the group consisting of:

20. T CM However, CD45RO + and CD4 + or CD8 + 2. The pharmaceutical composition of claim 1, wherein

21. T CM However, CD45RA - , CD62L + , CCR7 + 21. The pharmaceutical composition of claim 20, wherein the compound is selected from the group consisting of:

22. T EM But CD4 + , CD45RO + , CD45RA - , CD62L - , CCR7 - or any combination thereof.

23. iNKT, CD1d-tet + , 6B11 + 10. The pharmaceutical composition of claim 9, wherein the hydroxybenzoate is hydroxybenzoate or hydroxybenzoate.

24. iNKT is Vα24Jα18 + 2. The pharmaceutical composition of claim 1, wherein

25. Normal naive αβ-T cells express CD25 - , CD127 + , or both, as well as TCRα + and CD45RA + 2. The pharmaceutical composition of claim 1, wherein

26. Normal naive αβ-T cells express TCRα + TCRβ + CD45RA + CD45RO - CD25 - CD95 - IL-2Rβ - CD127 + 2. The pharmaceutical composition of claim 1, wherein

27. 2. The pharmaceutical composition of claim 1, wherein the ratio of HSPC to Tmem is 500:1 to 1:1,000.

28. 2. The pharmaceutical composition of claim 1, wherein the ratio of HSPCs to Tregs is from about 100:1 to about 1:

30.

29. The pharmaceutical composition of any of the preceding claims, wherein the ratio of HSPCs to naive Tregs is 1:500 to 100:

1.

30. 2. The pharmaceutical composition of any one of the preceding claims, wherein the ratio of HSPCs to memory Tregs is 1:500 to 10,000:

1.

31. 2. The pharmaceutical composition of claim 1, wherein the ratio of HSPC to iNKT is 1:2 to 500,000:

1.

32. 10. The pharmaceutical composition of any one of the preceding claims, wherein the ratio of normal naive αβ-T cells to HSPCs is less than 1:3, preferably less than 1:

400.

33. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the ratio of normal naive αβ-T cells to Tmem is less than 1:30, preferably less than 1:

800.

34. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the ratio of normal naive αβ-T cells to naive Tregs is less than 1:1, preferably less than 1:

10.

35. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the ratio of normal naive αβ-T cells to memory Tregs is less than 1:1, preferably less than 1:

100.

36. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the ratio of normal naive αβ-T cells to iNKT is less than 100:1, preferably less than 1:

1.

37. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the ratio of Tmem to Treg is between 2000:1 and 1:10, preferably between 30:1 and 1:

1.

38. The pharmaceutical composition of any one of the preceding claims, wherein the ratio of Tmem to naive Treg is between 3:1 and 0.1:

1.

39. The pharmaceutical composition of any of the preceding claims, wherein the ratio of Tmem to memory Treg is between 27:1 and 0.9:

1.

40. The pharmaceutical composition of any one of the preceding claims, wherein the ratio of iNKT to Tmem is from about 5:1 to about 1:1,000,000.

41. Unit dose is 1.0 x 10 6 ~50×10 6 6. The pharmaceutical composition of claim 1, comprising HSPCs of:

42. Unit dose is 0.3 × 10 6 ~1000×10 6 4. The pharmaceutical composition of claim 1, comprising memory T cells of

43. Unit dose is 0.5 × 10 6 ~1000×10 6 2. The pharmaceutical composition of claim 1, comprising Treg cells.

44. Unit dose is 0.2 × 10 6 ~500×10 6 10. The pharmaceutical composition of claim 1, comprising naive Treg cells.

45. Unit dose is 0.5 × 10 6 ~500×10 6 4. The pharmaceutical composition of any one of the preceding claims, comprising memory Treg cells.

46. Unit dose is 0.5 × 10 3 ~2000×10 3 3. The pharmaceutical composition of claim 1, comprising iNKT cells.

47. Unit dose is 3 x 10 5 10. The pharmaceutical composition of any one of the preceding claims, comprising fewer normal naive αβ-T cells.

48. 10. The pharmaceutical composition of any one of the preceding claims, wherein the cell population is obtained by processing one or more tissue harvests.

49. 49. The pharmaceutical composition of claim 48, wherein the one or more tissue harvests are from one or more donors.

50. 50. The pharmaceutical composition of claim 48 or 49, wherein the tissue collection is from an HLA-matched sibling donor, an HLA-matched unrelated donor, a partially matched unrelated donor, a haploidentical related donor, an autologous donor, an HLA-mismatched allogeneic donor, a donor pool, or any combination thereof.

51. The pharmaceutical composition of any one of the preceding claims, wherein the HSPCs are provided by a donor who is haplotype-matched to the subject.

52. The pharmaceutical composition of any one of the preceding claims, wherein the Treg, Tmem, iNKT, or any combination thereof is provided by a donor that is an HLA-matched sibling donor or an HLA-matched unrelated donor.

53. 10. The pharmaceutical composition of any one of the preceding claims, wherein the cells are formulated for infusion or injection.

54. 10. The pharmaceutical composition of any one of the preceding claims, wherein the cell population constitutes a formulation for administration to a subject.

55. 55. The pharmaceutical composition of claim 54, wherein the formulation comprises Normosol-R and human serum.

56. 56. The pharmaceutical composition of claim 54 or 55, wherein the human serum is 1% of the total formulation.

57. 10. Use of a pharmaceutical composition according to any one of the preceding claims in the treatment of a disease or disorder.

58. 58. The use of claim 57, wherein the disease or disorder is leukemia, lymphoma, chronic infection, or autoimmune disease, malignant or non-malignant blood disease, AML, ALL, CML, CLL, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, MDS, lymphoproliferative disease, type 1 diabetes, inborn error of metabolism, genetic disease, sickle cell anemia, beta-thalassemia, multiple sclerosis, solid organ transplant, Crohn's disease, ulcerative colitis, lupus, hemophagocytic lymphohistiocytosis, glycogen storage disease, mucopolysaccharidosis, or any other disease that would benefit from HSPC transplantation.

59. 59. The use of any one of claims 57 or 58, wherein each therapeutic cell population is administered to said subject as an individual pharmaceutical composition.

60. 59. The use of any one of claims 57 or 58, wherein the therapeutic cell population is administered to said subject as a single pharmaceutical composition.

61. 61. The use of any one of claims 57 to 60, wherein the cells are isolated from a donor that is an HLA-matched sibling donor, an HLA-matched unrelated donor, a partially matched unrelated donor, a haploidentical related donor, an autologous donor, an HLA-mismatched donor, a donor pool, or any combination thereof.

62. 62. The use of any one of claims 57 to 61, wherein the therapeutic cell population is allogeneic or autologous.

63. 63. The use of any one of claims 57 to 62, wherein the therapeutic cell population is autologous.

64. 64. The use of any one of claims 57 to 63, wherein the therapeutic cell population is haplotype-matched.

65. 65. The use of any one of claims 57-64, wherein the therapeutic cell population is isolated from mobilized peripheral blood, mobilized apheresis blood products, bone marrow, umbilical cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof.

66. 66. The use of any one of claims 57 to 65, wherein the therapeutic cell population is derived from a single tissue harvest.

67. 67. The use of any one of claims 57 to 66, wherein the therapeutic cell population is derived from one or more tissue harvests.

68. 68. The use of any one of claims 57 to 67, wherein the therapeutic cell population comprises HSPCs provided by at least a first donor and Tregs and Tmems provided by at least a second donor.

69. 69. The use of claim 68, wherein the therapeutic cell population comprises iNKT cells provided by at least a second donor.

70. 70. The use of claim 68 or 69, wherein the haplotype of the first donor is matched to the subject.

71. 71. The use of any one of claims 68 to 70, wherein the second donor is an HLA-matched sibling donor or an HLA-matched or partially matched unrelated donor.

72. 72. The use of any one of claims 57 to 71, wherein the subject is a human, non-human primate, cow, horse, sheep, goat, pig, dog, cat, mouse, rabbit, rat, or guinea pig.

73. Processing at least one sample (a) Enriched hematopoietic stem / progenitor cell (HSPC) populations; (b) enriched populations of regulatory T cells (Tregs); (c) Enriched memory T cell (Tmem) population obtaining the compound; and (d) formulating the enriched populations of HSPCs, memory T cells, and Tregs into pharmaceutical compositions suitable for administration to a subject. Including, wherein the populations of (a) to (c) are depleted of normal naive αβ-T cells; A method of making the pharmaceutical composition of any preceding claim.

74. The sample was processed to obtain Lin + 74. The method of claim 73, further comprising obtaining a cell-depleted population of cells.

75. 75. The method of claim 73 or 74, wherein obtaining an enriched population of Tregs comprises obtaining an enriched population of naive Tregs, an enriched population of memory Tregs, or both.

76. Obtaining an enriched population of Tmem is considered to be an enriched central memory T cell (T CM ) population, enriched effector memory T cells (T EM 76. The method of any one of claims 73-75, comprising obtaining a population of:

77. The method of any one of claims 73 to 76, further comprising processing the sample to obtain an enriched population of iNKT cells.

78. Enriched HSPC populations are at least 50% CD34 + 78. The method of any one of claims 73-77, comprising HPSCs.

79. Lin + The cell-depleted cell population was 1% to 30% Lin + cells, preferably less than 1% Lin + 79. The method of any one of claims 73 to 78, comprising a cell.

80. The method of any one of claims 73 to 79, wherein the enriched population of Tregs comprises between 20% and 99.9% Tregs.

81. The method of any one of claims 73 to 80, wherein the enriched population of Tmems comprises between 10% and 99.9% Tmems.

82. The method of any one of claims 73-81, wherein the enriched population of iNKT comprises between 10% and 99.9% iNKT.

83. 83. The method of any one of claims 73-82, wherein formulating said pharmaceutical composition comprises combining an enriched HSPC population, a memory T cell population, a Treg population, an iNKT population, or any combination thereof, into a mixed population of enriched cells.

84. 84. The method of claim 83, wherein said mixed population of enriched cells comprises a ratio of HSPCs to memory T cells of between 500:1 and 1:1,000.

85. 85. The method of claim 83 or 84, wherein the mixed population of enriched cells comprises a ratio of HSPCs to naive Tregs of 1:500 to 100:

1.

86. 86. The method of any one of claims 83-85, wherein said mixed population of enriched cells comprises a ratio of HSPCs to memory Tregs of 1:500 to 10,000:

1.

87. The method of any one of claims 83-86, wherein said mixed population of enriched cells comprises a ratio of HSPCs to iNKTs of 1:2 to 500,000:

1.

88. 88. The method of any one of claims 83 to 87, wherein said mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to HSPCs that is less than 1:3, preferably less than 1:

400.

89. 89. The method of any one of claims 83 to 88, wherein the mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to Tmem that is less than 1:30, preferably less than 1:

800.

90. 90. The method of any one of claims 83 to 89, wherein the mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to naive Tregs of less than 1:1, preferably less than 1:

10.

91. 91. The method of any one of claims 83 to 90, wherein the mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to memory Tregs that is less than 1:1, preferably less than 1:

100.

92. 92. The method of any one of claims 83 to 91, wherein the mixed population of enriched cells comprises a ratio of normal naive αβ-T cells to iNKT cells of less than 100:1, preferably less than 1:

1.

93. The method of any one of claims 83 to 92, wherein the mixed population of enriched cells comprises a ratio of Tmem to Treg that is between 30:1 and 1:

1.

94. The method of any one of claims 83 to 93, wherein the mixed population of enriched cells comprises a ratio of Tmem to naive Treg that is between 3:1 and 0.1:

1.

95. The method of any one of claims 83-94, wherein the mixed population of enriched cells comprises a ratio of Tmem to memory Treg that is between 27:1 and 0.9:

1.

96. 96. The method of any one of claims 83-95, wherein said mixed population of enriched cells comprises less than 0.0014% normal naive αβ-T cells.

97. The method of any one of claims 74 to 96, wherein the Lin+ cells express CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

98. The method of any one of claims 73 to 97, wherein the HSPCs are CD34+.

99. The method of any one of claims 73 to 98, wherein the HSPCs are CD19- and TCRα / β-.

100. 100. The method of any one of claims 73 to 99, wherein the HSPCs are CD133+, CD90+, CD38-, CD45RA-, Lin-, or any combination thereof.

101. Tmem, CD45RA - , CD45RO + or any combination thereof.

102. Tmem is T CM 102. The method of claim 101, wherein:

103. T CM However, CD45RO + and CD4 + or CD8 + 103. The method of claim 102, wherein:

104. T CM However, CD45RA - , CD62L + , CCR7 + or any combination thereof.

105. Tmem is T EM 102. The method of claim 101, wherein:

106. T EM But CD4 + , CD45RA + , CD45RO - , CD62L - , CCR7 - or any combination thereof.

107. Tregs, CD4 + , CD25 + , CD127 - / lo , FoxP3 + or any combination thereof.

108. The method of claim 107, wherein the Tregs are naive Tregs, memory Tregs, or both.

109. Naive Tregs express CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA + , CD45RO - or any combination thereof.

110. Memory Tregs are CD4 + , CD25 + , CD127 - / lo , FoxP3 + , CD45RA - , CD45RO + or any combination thereof.

111. 111. The method of any one of claims 73-110, wherein the sample comprises mobilized peripheral blood, a mobilized apheresis blood product, bone marrow, umbilical cord blood, non-mobilized blood, a non-mobilized apheresis blood product, or any combination thereof.

112. 112. The method of any one of claims 73-111, wherein the sample is prepared for processing with a density gradient, Ficoll, Percoll, red blood cell hypotonic lysis, ammonium chloride-potassium (ACK) buffer, or any combination thereof.

113. The method of any one of claims 73 to 111, wherein the sample is obtained from a single tissue collection.

114. The method of any one of claims 73 to 111, wherein the sample is obtained by one or more tissue samplings.

115. The method of any one of claims 73-114, wherein the enriched cell population is obtained by density separation, tetrameric antibody complex-mediated enrichment / depletion, magnetic-activated cell sorting, multi-parameter fluorescence-based molecular phenotyping, or any combination thereof.

116. (A) The sample is treated with a molecule that specifically binds to CD34 and a CD34 + Cell populations and CD34 - The cells were contacted under conditions that allowed a population of CD34 + A population of cells is recovered from the sample and CD34 - recovering a population of cells from the sample; and (B) CD34 - Processing the population of cells to obtain at least one population of enriched therapeutic cells comprising Tregs, Tmems, iNKTs, or any combination thereof.

116. The method of any one of claims 73 to 115, comprising:

117. 117. The method of claim 116, wherein step B comprises performing precision sorting to obtain an enriched population of therapeutic cells.

118. Process B is CD34 - contacting the population of cells with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof.

118. The method of claim 116 or 117, comprising:

119. Process B is (i) CD34 - The population of cells is treated with at least one molecule that specifically binds to CD45RA and CD45RA + Populations of cells and CD45RA - The cells are contacted under conditions in which a population of CD45RA - harvesting the population of cells; and (ii) CD45RA to obtain enriched therapeutic cell populations + To perform precise selection from a population of cells 117. The method of claim 116, comprising:

120. Precision selection, CD45RA + contacting the population of cells with a molecule that specifically binds CD4, a molecule that specifically binds CD8, a molecule that specifically binds CD25, a molecule that specifically binds CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof.

120. The method of claim 119, comprising:

121. Precision selection, CD45RA - contacting the population of cells with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, or a combination thereof; 121. The method of claim 120, further comprising:

122. Process B is (i) CD34 - The cell population was analyzed by Lin + at least one binding molecule that specifically binds to the marker; + Populations of cells and Lin - The cells were contacted under conditions in which a population of cells was formed, and Lin - harvesting the population of cells; and (ii) Lin to obtain enriched therapeutic cell populations - To perform precise selection from a population of cells 117. The method of claim 116, comprising:

123. Lin + The method of claim 122, wherein the marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

124. Precision selection, Lin - contacting the population of cells with a molecule that specifically binds to CD45RA, a molecule that specifically binds to CD45RO, a molecule that specifically binds to CD4, a molecule that specifically binds to CD8, a molecule that specifically binds to CD25, a molecule that specifically binds to CD127, a CD1d-tet molecule, a 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof.

124. The method of claim 122 or 123, comprising:

125. Process B is (i) CD34 - A population of cells was cultured using at least one Lin + at least one binding molecule that specifically binds to the marker; + Populations of cells and Lin - The cells were contacted under conditions in which a population of cells was formed, and Lin - harvesting the population of cells; and (ii) Lin - The population of cells is treated with a binding molecule that specifically binds to CD25 and a CD25 + Cell populations and CD25 - The cells are contacted under conditions in which a population of CD25 + The cell population is harvested, thereby generating a population of cells containing Tregs, and CD25 - harvesting the population of cells; and (iii) CD25 - a binding molecule that specifically binds to CD45RA; and + Populations of cells and CD45RA - The cells are contacted under conditions in which a population of CD45RA - Harvesting a population of cells 117. The method of claim 116, comprising:

126. Lin + The method of claim 125, wherein the marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

127. Step (ii) is Lin - The cells are treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Cell population, 6B11 + populations of cells, or combinations thereof, and CD1d-tet - Cell population, 6B11 - and contacting the cells under conditions in which CD1d-tet + Harvesting a population of cells 127. The method of claim 125 or 126, further comprising:

128. CD25 + Populations of cells and CD1d-tet + Cell population, 6B11 + The method of any one of claims 119-127, wherein the cells or both are simultaneously harvested.

129. In step (ii), CD25 T cells are isolated to obtain a population of naive Treg cells, a population of memory Treg cells, a population of iNKT cells, or any combination thereof. + The method of any one of claims 125-128, further comprising performing precision sorting of the population of cells.

130. CD45RA + The method of any one of claims 125-129, wherein the population of cells is collected and further subjected to precision sorting to obtain a population of Treg, iNKT or both.

131. 131. The method of any one of claims 117-130, wherein precision selection comprises purification using multi-parameter fluorescence-based molecular phenotyping.

132. (A) A first coarse selection is performed on at least a first sample, thereby detecting CD34 + Obtaining an enriched population of cells; (B) A second coarse selection is performed on the second sample, thereby determining the Lin - Obtaining a population of cells; (C) Lin - The population of cells was subjected to a third crude selection, which resulted in the CD45RA - Obtaining a population of memory T cells and CD45RA + Obtaining a population of cells; and (D) CD45RA + Perform precision selection on a population of cells to obtain a population of Tregs 116. The method of any one of claims 73 to 115, comprising:

133. A second sample was prepared from the CD34 - 133. The method of claim 132, comprising a population of cells.

134. 134. The method of claim 132 or 133, wherein the first sample comprises at least one haplotype-matched sample.

135. 134. The method of claim 132 or 133, wherein the first sample comprises at least two haplotype-matched samples.

136. 136. The method of any one of claims 132-135, wherein the first sample comprises mobilized peripheral blood, a mobilized apheresis blood product, bone marrow, umbilical cord blood, non-mobilized blood, a non-mobilized apheresis blood product, or any combination thereof.

137. 137. The method of any one of claims 132-136, wherein the second sample comprises peripheral blood mononuclear cells (PBMCs), mobilized peripheral blood, mobilized apheresis blood products, bone marrow, umbilical cord blood, non-mobilized blood, non-mobilized apheresis blood products, or any combination thereof.

138. The method of any one of claims 132-137, wherein the first, second, or third crude sorting comprises density separation, tetrameric antibody complex-mediated enrichment / depletion, magnetic-activated cell sorting, apheresis, leukapheresis, or any combination thereof.

139. 139. The method of any one of claims 132-138, wherein precision selection comprises purification using multi-parameter fluorescence-based molecular phenotyping.

140. The method of any one of claims 132-139, wherein the precision sorting results in a population of naive Treg cells, a population of memory Treg cells, a population of iNKT cells, or any combination thereof.

141. Precision selection, CD45RA + The cells are then treated with a binding molecule that specifically binds to CD25 and a CD25 + Cell populations and CD25 - contacting the cells under conditions that result in a population of cells that are CD25 + Harvesting a population of cells, thereby obtaining a population of Tregs 141. The method of any one of claims 132-140, comprising:

142. CD25 + 142. The method of claim 141, wherein the population of cells is further selected under conditions to obtain a population of naive Treg cells.

143. Precision selection, CD45RA + The cells are treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Cell population, 6B11 + and contacting the cells under conditions that result in CD1d-tet + Cell population, 6B11 + harvesting cells, or both 143. The method of any one of claims 132-142, comprising:

144. 144. The method of any one of claims 132-143, wherein the first sample or the second sample is allogeneic, autologous, or a combination thereof.

145. The method of any one of claims 132-144, wherein the second sample is from an HLA-matched unrelated donor, an HLA-matched sibling donor, or a combination thereof.

146. Lin + The method of any one of claims 132-145, wherein the marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

147. (A) The sample was + a binding molecule that specifically binds to the marker; + Populations of cells and Lin - The cells are contacted under conditions that result in a population of Lin - harvesting the population of cells; and (B) Lin - The cells were treated with a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25, and a binding molecule that specifically binds to CD34. + A population of cells, CD25 + population of cells, and CD34 - CD25 - contacting the cells under conditions that result in a population of CD34 + Cells and CD25 + Cells were harvested and CD34 - CD25 - harvesting the population of cells; and (C) CD34 - CD25 - The population of cells is treated with a binding molecule that specifically binds to CD45RA and a CD45RA + Populations of cells and CD45RA - and contacting the cells under conditions that result in a population of CD45RA - recovering the population of 116. The method of any one of claims 73 to 115, comprising:

148. Lin + The method of claim 147, wherein the marker is CD19, CD11c, CD66B, CD14, CD20, or any combination thereof.

149. Process B is Lin - The cells are treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Cell population, 6B11 + and contacting the cells under conditions that result in CD1d-tet + cells, 6B11 + harvesting cells, or a combination thereof; 149. The method of claim 147 or 148, further comprising:

150. CD34 + cells, CD25 + cells, CD1d-tet + cells, 6B11 + cells, or any combination thereof, thereby detecting CD34 + cells, CD25 + cells, CD1d-tet + cells, 6B11 + 150. The method of any one of claims 147-149, further comprising obtaining the population of cells, or any combination thereof.

151. Precision selection, CD34 + cells, CD25 + cells, CD1d-tet + cells, 6B11 + The cells, or any combination thereof, are treated with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof, and a CD34 + cells, CD4 + CD25 + CD127 - / Lo cells, CD1d-tet + contacting the cells, or combinations thereof, under conditions that result in a highly enriched population of cells; 151. The method of claim 150, comprising:

152. (A) The sample is roughly selected and Lin + Populations of cells and Lin - Obtaining a population of cells and - harvesting the population of cells; and (B) Lin - By performing crude sorting of cell populations, we identified populations enriched for HSPCs and Tmem and CD45RA + A population of cells was obtained, and the HSPC and Tmem populations were recovered, and CD45RA + harvesting the population of cells; and (C) CD45RA + A process of obtaining a Treg population by precise selection of a cell population.

116. The method of any one of claims 73 to 115, comprising:

153. Precision selection, CD45RA + The population of cells is subjected to a binding molecule that specifically binds CD34, a binding molecule that specifically binds CD4, and a binding molecule that specifically binds CD127, and a population of CD34+ cells and a population of CD4 + CD25 + CD127 - / Lo and contacting the cells under conditions that result in a population of CD34+ cells and a population of CD4 + CD25 + CD127 - / Lo Harvesting a population of cells 153. The method of claim 152, further comprising:

154. Precision selection, CD45RA + The cell population is contacted with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, to induce CD1d-tet + Cell population, 6B11 + harvesting cells, or a combination thereof; 154. The method of claims 152-153, further comprising:

155. The method of any one of claims 152-154, wherein the crude sorting comprises density separation, tetrameric antibody complex-mediated enrichment / depletion, magnetic-activated cell sorting, apheresis, leukapheresis, or any combination thereof.

156. 156. The method of any one of claims 152-155, wherein precision selection comprises purification using multi-parameter fluorescence-based molecular phenotyping.

157. (A) The sample is treated with a binding molecule that specifically binds to CD34 and a CD34 + Cell populations and CD34 - contacting the cells under conditions that result in a population of CD34 + The cell population is harvested and CD34 - harvesting the population of cells; and (B) CD34 - The population of cells is treated with a binding molecule that specifically binds to CD25 and a CD25 + Cell populations and CD25 - contacting the cells under conditions that result in a population of CD25 + The cell population is harvested and CD25 - harvesting the population of cells; and (C) CD25 - The population of cells is treated with a binding molecule that specifically binds to CD45RA and a CD45RA + Populations of cells and CD45RA - and contacting the cells under conditions that result in a population of CD45RA - Harvesting the population of cells 116. The method of any one of claims 73 to 115, comprising:

158. Process B is (i) CD34 - The population of cells is treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Cell population, 6B11 + populations of cells, or combinations thereof, and CD1d-tet - Cell population, 6B11 - and contacting the cells under conditions that result in the formation of a population of CD1d-tet cells, or a combination thereof. + Cell population, 6B11 + A population of cells, or a combination thereof, is harvested and CD1d-tet - Cell population, 6B11 - and recovering the population of cells, or both, thereby obtaining a population of iNKT-depleted cells; and (ii) The population of iNKT-depleted cells was incubated with a binding molecule that specifically binds to CD25 and a CD25 + Cell populations and CD25 - contacting the cells under conditions that result in a population of CD25 + The cell population is harvested and CD25 - Harvesting a population of cells 158. The method of claim 157, further comprising:

159. Process B is CD34 - The population of cells is treated with a binding molecule that specifically binds to CD25, and CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD25 + population of cells, and CD1d-tet + Cell population, 6B11 + cells, or a combination thereof, and CD34 - CD25 - Under conditions that result in a population of iNKT-depleted cells, CD25 + Populations of cells and CD1d-tet + Cell population, 6B11 + cells, or a combination thereof, and - CD25 - Harvesting a population of iNKT-depleted cells 158. The method of claim 157, comprising:

160. Process B is (i) CD34 - The population of cells is treated with a binding molecule that specifically binds to CD25, and CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD25 + Populations of cells and CD1d-tet + Cell population, 6B11 + cells, or a combination thereof and CD34 - CD25 - Under conditions that result in a population of iNKT-depleted cells, CD25 + Populations of cells and CD1d-tet + Cell population, 6B11 + cells, or a combination thereof, and - CD25 - Recovering a population of iNKT-depleted cells; and (ii) CD25 + Populations of cells and CD1d-tet + Cell population, 6B11 + The precision selection of the cells, or a combination thereof, is carried out by contacting the cells with a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or any combination thereof, and the CD4 + CD25 + CD127 - / Lo cells, CD1d-tet + cells, 6B11 + Obtaining a population of cells enriched for a cell, or any combination thereof 158. The method of claim 157, comprising:

161. Step (i) is CD34 - contacting the population of cells with a tag-containing anti-CD25 antibody and a biotinylated 6B11 monoclonal antibody, and contacting the 6B11-biotin with a conjugated tag, streptavidin; 161. The method of claim 160, comprising:

162. 162. The method of claim 161, wherein the tag is phycoerythrin (PE).

163. 163. The method of claim 161 or 162, wherein the streptavidin is conjugated to PE / Cy7.

164. CD34 - The method of any one of claims 161 to 163, wherein the cells are then contacted with anti-tag magnetic particles.

165. 165. The method of claim 164, wherein the anti-tag magnetic particles are anti-PE magnetic particles.

166. CD25 + Cells and 6B11 + 166. The method of any one of claims 160-165, wherein the cells are separated using magnetic separation.

167. (ii) is CD25 + Cells and 6B11 + The population of cells is subjected to precise sorting with a binding molecule that specifically binds to CD4 and a binding molecule that specifically binds to CD127. + CD25 + CD127 - / Lo Cell-enriched cell populations and 6B11 + CD127 + by contacting the cells under conditions that result in an enriched population of cells, or any combination thereof.

167. The method of any one of claims 160-166, comprising:

168. The method of claim 167, wherein the CD4 binding molecule is an anti-CD4 PerCP-labeled antibody, the CD127 binding molecule is an anti-CD127 APC-labeled antibody, or a combination thereof.

169. The method of claim 167 or 168, wherein the precision sorting comprises fluorescence-activated cell sorting and detection of PE-labeled CD25, PE / Cy7-labeled 6B11, PerCP-labeled CD4, and APC-labeled CD127.

170. CD34 collected in step A + Population of cells, CD25 recovered in step B + population of cells, or both, expressing CD34 + cells, CD25 + The method of claim 157, wherein the cells, or a combination thereof, are further processed by precision sorting comprising contacting the cells with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD127, a binding molecule that specifically binds to CD45RA, or any combination thereof.

171. (A) The sample is subjected to a step of incubating the sample with a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25, and a step of incubating the sample with a binding molecule that specifically binds to CD34 and a binding molecule that specifically binds to CD25. + A population of cells, CD25 + population of cells, and CD34 - CD25 - contacting the cells under conditions that result in a population of CD34 + Cell populations and CD25 + The cell population is harvested and CD34 - CD25 - harvesting the population of cells; and (B) CD34 - CD25 - The population of cells is treated with a binding molecule that specifically binds to CD45RA and a CD45RA + Populations of cells and CD45RA - and contacting the cells under conditions that result in a population of CD45RA - Harvesting the population of cells 116. The method of any one of claims 73 to 115, comprising:

172. Process A is The sample is treated with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet + Cell population, 6B11 + and contacting the cells under conditions that result in the formation of a population of CD1d-tet cells, or a combination thereof. + Cell population, 6B11 + harvesting a population of cells, or a combination thereof; 172. The method of claim 171, further comprising:

173. The cell population obtained in step A is subjected to precision selection by contacting the cells with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, CD1d-tet, or any combination thereof, to obtain CD34. + cells, CD4 + CD25 + CD127 - / Lo cells, CD1d-tet + Obtaining a population of cells enriched for the cells, or any combination thereof.

173. The method of claim 171 or 172, further comprising:

174. simultaneously processing said sample to obtain an enriched population of cells containing HSPCs, Tmems, naive Tregs, memory Tregs and containing less than 5% of undesired cell types.

116. The method of any one of claims 73 to 115, comprising:

175. 175. The method of claim 174, wherein the sample is contacted with a binding molecule that specifically binds to CD34, a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD8, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, a binding molecule that specifically binds to CD45RA, a binding molecule that specifically binds to CD45RO, or any combination thereof.

176. The sample is contacted with CD1d-tet, 6B11 monoclonal antibody or a functional fragment thereof, or a combination thereof, and CD1d-tet is detected. + Cell population, 6B11 + Recovering the population of cells, or a combination thereof.

176. The method of claim 175, further comprising:

177. contacting the sample with a binding molecule that specifically binds to CD34; + The method of any one of claims 174-176, wherein the population of cells is harvested, thereby producing a population of HSPCs.

178. The sample is contacted with a binding molecule that specifically binds to CD3 and does not bind to a binding molecule that specifically binds to CD45RA, a binding molecule that specifically binds to CD45RO, or a combination thereof, to detect CD3 + CD45RA - CD45RO + The method of any one of claims 174-177, wherein the population of cells is harvested.

179. The sample is contacted with a binding molecule that specifically binds to CD4, a binding molecule that specifically binds to CD25, a binding molecule that specifically binds to CD127, a binding molecule that specifically binds to CD45RA, a binding molecule that specifically binds to CD45RO, or any combination thereof, to obtain CD4 + CD25 + CD127 - / lo CD45RA + CD45RO - A population of cells, CD4 + CD25 + CD127 - / lo CD45RA - CD45RO + The method of any one of claims 174-178, wherein the population of cells is harvested.

180. The method of any one of claims 73-179, wherein the therapeutic cell population comprises less than 2% normal naive αβ-T cells.

181. 181. The method of any one of claims 73-180, wherein the molecule that specifically binds to CD34, a Lin+ marker, CD25, CD45RA, CDR45RO, CD4, CD8, CD127, CD90, CD133, CD38, CD95, CD122, CXCR3, LFA-1, CD62L, CCR7, or any other cell marker is an antibody or antibody fragment.

182. 182. The method of any one of claims 73-181, wherein said antibody or antibody fragment is coupled to a fluorescent dye, a hapten, or a magnetic particle.

183. The method of any one of claims 73-182, wherein the cells are freshly isolated.

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