Methods for allogeneic hematopoietic stem cell transplantation

Abstract

Various embodiments of the present disclosure provide therapeutic compositions and associated methods for improved hematopoietic stem cell transplantations, including methods to enhance protection from graft versus host disease while maintaining effective immune responses such as graft versus tumor immune responses.

Description

Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT METHODS FOR ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 755,142 filed February 6, 2025, which is hereby incorporated by reference in its entirety for all purposes.BACKGROUND

[0002] Patients with hematologic malignancies such as leukemia and lymphoma beyond first remission or with refractory relapse are rarely cured with standard chemotherapy. Myeloablative allogeneic hematopoietic cell transplantation (alloHCT) is associated with improved survival in these patients. Myeloablative alloHCT is a procedure in which the patient undergoes chemotherapy or radialion to ablate or destroy tissue in the bone causing the malignancy. They then receive hematopoietic cells, including hematopoietic stem and progenitor cells (HSPC) from a donor’s blood. However, alloHCT has a major drawback in that it often results in graft versus host disease (GVHD). GVHD is a condition in which the transplanted donor peripheral blood stem cells view the patient’s body as foreign, and the donated cells attack the patient’s tissue (e.g., skin, GI tissue, liver tissue, and lung tissue) resulting in a number of complications, many of which can be serious and result in morbidity and mortality. Thus, there is a need for improved methods and therapies for hematopoietic cell transplantation which have a reduced incidence and severity of GVHD including, reduced morbidity and mortality.BRIEF SUMMARY

[0003] Certain aspects of the present disclosure relate to a multi-component cellular therapy product comprising: a) a first population of isolated CD45+cells comprising CD34+hematopoietic stem and progenitor cells (HSPCs); b) a second population of isolated CD45+cells comprising isolated fresh regulatory T cells (Tregs); c) a third population of isolated CD45+cells comprising conventional CD3+T cells (Tcons); and d) a pharmaceutical composition comprising one or more graft versus host disease (GVHD) prophylactic agents, such as a Janus kinase (JAK) inhibitor. Certain aspects of the present disclosure relate to a multi-component cellular therapy product comprising: a) a first population of isolated CD45+cells comprising CD34+hematopoietic stem and progenitor cells (HSPCs); b) a second population of isolated CD45+cells comprising isolated fresh regulatory T cells (Tregs); c) a third population of isolated CD45+cells comprising conventional CD3+T cells (Tcons); and d) a pharmaceutical composition comprising a JAK1 / 2 inhibitor.

[0004] Also provided is method of treating a human subject in need of an allogeneic hematopoietic cell transplant (optionally in the treatment of a hematologic malignancy), wherein the human subject has received, is receiving, or scheduled to receive a JAK inhibitor, optionally wherein the JAK inhibitor comprises a JAK1 / 2 inhibitor, further optionally wherein the JAK inhibitor comprises ruxolitinib, the method comprising administering to a human subject a multi-component cellular therapy product comprising: a) a first population of isolated CD45+ cells comprising CD34+ hematopoietic stem and progenitor cells (HSPCs); b) a second population of isolated CD45+ cells comprising isolated fresh 1IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT regulatory T cells (Tregs); and c) a third population of isolated CD45+ cells comprising conventional CD3+ T cells (Tcons). Also provided is method of treating a human subject in need of an allogeneic hematopoietic cell transplant (optionally in the treatment of a hematologic malignancy), wherein the human subject is scheduled to receive a JAK inhibitor, optionally wherein the JAK inhibitor comprises a JAK1 / 2 inhibitor, further optionally wherein the JAK inhibitor comprises ruxolitinib, the method comprising administering to a human subject a multi-component cellular therapy product comprising: a) a first population of isolated CD45+ cells comprising CD34+ hematopoietic stem and progenitor cells (HSPCs); b) a second population of isolated CD45+ cells comprising isolated fresh regulatory T cells (Tregs); and c) a third population of isolated CD45+ cells comprising conventional CD3+ T cells (Tcons), and wherein the human subject is scheduled to receive the JAK inhibitor following administration of the CD3+ Tcons. Also provided is method of treating a human subject in need of an allogeneic hematopoietic cell transplant (optionally in the treatment of a hematologic malignancy), wherein the human subject is scheduled to receive ruxolitinib, the method comprising administering to a human subject a multi-component cellular therapy product comprising: a) a first population of isolated CD45+ cells comprising CD34+ hematopoietic stem and progenitor cells (HSPCs); b) a second population of isolated CD45+ cells comprising isolated fresh regulatory T cells (Tregs); and c) a third population of isolated CD45+ cells comprising conventional CD3+ T cells (Tcons), and wherein the human subject is scheduled to receive the ruxolitinib following administration of the CD3+ Tcons. Also provided is method of treating a human subject in need of an allogeneic hematopoietic cell transplant (optionally in the treatment of a hematologic malignancy), wherein the human subject is scheduled to receive ruxolitinib and a second GHVD prophylactic agent (optionally, wherein the second GHVD prophylactic agent is tacrolimus), the method comprising administering to a human subject a multi-component cellular therapy product comprising: a) a first population of isolated CD45+ cells comprising CD34+ hematopoietic stem and progenitor cells (HSPCs); b) a second population of isolated CD45+ cells comprising isolated fresh regulatory T cells (Tregs); and c) a third population of isolated CD45+ cells comprising conventional CD3+ T cells (Tcons), and wherein the human subject is scheduled to receive the ruxolitinib and the second GHVD prophylactic agent following administration of the CD3+ Tcons.

[0005] In some embodiments that may be combined with any of the preceding embodiments, the first population of isolated CD45+cells comprises a dose of CD34+HSPCs that ranges from approximately 1.0 x 105or more CD34+HSPCs per kilogram of body weight of a human subject receiving the product to approximately 1.0 x 108or more CD34+HSPCs per kilogram of body weight of the human subject receiving the product. In some embodiments that may be combined with any of the preceding embodiments, the first population of isolated CD45+cells comprises a dose of CD34+HSPCs that ranges from approximately 5.0 x 105or more CD34+HSPCs to approximately 1.5 x 1010or more CD34+HSPCs. In some embodiments that may be combined with any of the preceding embodiments, the first population of isolated CD45+cells comprises a dose of CD34+HSPCs that is at least 1.0 x 106cells per patient kilogram.

[0006] In some embodiments that may be combined with any of the preceding embodiments, the second population of isolated CD45+cells comprises a dose of isolated fresh Tregs that ranges from2IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 1.0 x 105or more isolated fresh Tregs per kilogram of body weight of a human subject receiving the product to approximately 2.0 x 107or more isolated fresh Tregs per kilogram of body weight of the human subject receiving the product. In some embodiments that may be combined with any of the preceding embodiments, the second population of isolated CD45+cells comprises a dose of isolated fresh Tregs that ranges from approximately 5.0 x 105or more isolated fresh Tregs to approximately 3.0 x 109or more isolated fresh Tregs. In some embodiments that may be combined with any of the preceding embodiments, the second population of isolated CD45+cells comprises a dose of isolated fresh Tregs that ranges from .9 x 106to 3.5 x 106cells per patient kilogram. In some embodiments that may be combined with any of the preceding embodiments, the Tregs are CD4+CD25+CD127dlmor CD4+FOXP3+.

[0007] In some embodiments that may be combined with any of the preceding embodiments, the third population of isolated CD45+cells comprises a dose of CD3+Tcons that ranges from approximately 1.0 x 105or more CD3+Tcons per kilogram of body weight of a human subject receiving the product to approximately 4.0 x 107or more CD3+Tcons per kilogram of body weight of the human subject receiving the product. In some embodiments that may be combined with any of the preceding embodiments, the third population of isolated CD45+cells comprises a dose of CD3+Tcons that ranges from 0.9 x 106to 6.9 x 106cells per padent kilogram.

[0008] In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises the JAK inhibitor at a dose that ranges from approximately 2 mg to approximately 20 mg twice per day. In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor dosage comprises approximately 5mg twice per day. In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor dosage comprises approximately 5mg twice per day and tapers to approximately 5mg once per day after approximately 4 weeks and to zero after approximately 8 weeks. In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor is a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor, a JAK1 / 2 inhibitor, a JAK1 / 3 inhibitor, a JAK2 / 3 inhibitor, a JAK1 / 2 / 3 inhibitor, a TYK2 inhibitor, or any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, momelotinib, cerdulatinib, gandotinib, lestaurtinib, , decernotinib, solcitinib, itacitinib, SHR0302, delgocitinib, cucurbitacin I, CHZ868, XL019, AZDI 480, BMS911543, and ilginatinib.

[0009] In some embodiments that may be combined with any of the preceding embodiments, the product further comprises a second pharmaceutical composition comprising a dose of a graft vs host disease (GVHD) prophylactic agent. In some embodiments that may be combined with any of the preceding embodiments, the human subject receiving the multi-component cellular therapy has received, is receiving, or scheduled to receive a GVHD prophylactic agent. In some embodiments that may be combined with any of the preceding embodiments, the human subject receiving the multi-component cellular therapy is scheduled to receive a GVHD prophylactic agent following administration of the CD3+ Tcons of the multi-3IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT component cellular therapy. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is selected from the group consisting of: tacrolimus, cyclosporine A, sirolimus, basiliximab, daclizumab, methotrexate, muromonab-CD3, mycophenolate, antithymocyte globulin, corticosteroids, azathioprine, and mycophenolate mofetil. In some embodiments that may be combined with any of the preceding embodiments, the dose of the GVHD prophylactic agent is sufficient to maintain a trough blood level of approximately 5 ng / mL to approximately 10 ng / mL in a human subject receiving the product. In some embodiments that may be combined with any of the preceding embodiments, the second pharmaceutical composition comprises the GVHD prophylactic agent at a dose that ranges from approximately 0.01 mg per kilogram of body weight of a human subject receiving the product to approximately 0.50 mg per kilogram of body weight of a human subject receiving the product twice per day.

[0010] In some embodiments that may be combined with any of the preceding embodiments, the human subject receiving the multi-component cellular therapy has received, is receiving, or scheduled to receive tacrolimus. In some embodiments that may be combined with any of the preceding embodiments, the human subject receiving the multi-component cellular therapy is scheduled to receive tacrolimus following administration of the CD3+ Tcons of the multi-component cellular therapy. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent comprises tacrolimus at 0.03 mg / kg per day. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent comprises tacrolimus at 0.015 mg / kg per day administered twice a day IV (i.e., for a total dose of 0.03 mg / kg per day). In some embodiments that may be combined with any of the preceding embodiments, the human subject receiving the multi-component cellular therapy has received, is receiving, or scheduled to receive tacrolimus at 0.03 mg / kg per day. In some embodiments that may be combined with any of the preceding embodiments, the human subject receiving the multi-component cellular therapy is scheduled to receive tacrolimus at 0.03 mg / kg per day following administration of the CD3+ Tcons of the multi-component cellular therapy.

[0011] In some embodiments that may be combined with any of the preceding embodiments, the first population of isolated CD45+cells, the second population of isolated CD45+cells, and / or the third population of isolated CD45+cells is formulated at a volume that ranges from approximately 5 mL to approximately 1 L. In some embodiments that may be combined with any of the preceding embodiments, the first population of isolated CD45+cells, the second population of isolated CD45+cells, and / or the third population of isolated CD45+cells is formulated with an excipient at a neutral pH. In some embodiments that may be combined with any of the preceding embodiments, the neutral pH ranges from approximately 6.8 to approximately 7.6. In some embodiments that may be combined with any of the preceding embodiments, the excipient comprises a transport buffer. In some embodiments that may be combined with any of the preceding embodiments, the transport buffer comprises approximately 120 to approximately 160 mEq sodium. In some embodiments that may be combined with any of the preceding embodiments, the transport buffer comprises approximately 270 to approximately 320 mOsmol / L total. In some embodiments that may be combined with any of the preceding embodiments, the transport buffer is selected4IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT from the group consisting of: phosphate -buffered saline (PBS), human serum, PlasmaLyte, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the transport buffer further comprises approximately 0.1% weight by volume to approximately 10% weight by volume of a human carrier protein. In some embodiments that may be combined with any of the preceding embodiments, the human carrier protein is selected from the group consisting of: human serum albumin (HSA), intravenous immune globulin (IVIG), AB serum, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the first population of isolated CD45+cells, the second population of isolated CD45+cells, and / or the third population of isolated CD45+cells is formulated in a single dose transfer bag. In some embodiments that may be combined with any of the preceding embodiments, the single dose transfer bag is a polyvinyl chloride (PVC) transfer bag or an ethylene vinyl acetate (EVA) transfer bag. In some embodiments that may be combined with any of the preceding embodiments, the third population of isolated CD45+cells is formulated with an excipient comprising one more cryoprotectants. In some embodiments that may be combined with any of the preceding embodiments, the one or more cryoprotectants are selected from the group consisting of: sorbitol, dimethyl sulfoxide (DMSO), propylene glycol, glycerol, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG), serum, HSA, hetastarch, CRYOSTOR CS2, CRYOSTOR CS5, and CRYOSTOR CSIO.

[0012] In some embodiments that may be combined with any of the preceding embodiments, the first population of isolated CD45+cells, the second population of isolated CD45+cells, and the third population of isolated CD45+ cells are from an allogeneic donor having at least one HLA mismatch relative to a human subject receiving the product. In some embodiments that may be combined with any of the preceding embodiments, the HLA-mismatched donor is unrelated to a human subject receiving the product. In some embodiments that may be combined with any of the preceding embodiments, the HLA-mismatched donor is related to a human subject receiving the product. In some embodiments that may be combined with any of the preceding embodiments, the at least one HLA mismatch is at an allele selected from the group consisting of: HLA-A, HLA-B, HLA-C, HLA-DRB1, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the cells having at least one HLA mismatch are from a donor that is 6 / 8 HLA-mismatched relative to a human subject receiving the product or is 7 / 8 HLA-mismatched relative to a human subject receiving the product. In some embodiments that may be combined with any of the preceding embodiments, the cells having at least one HLA mismatch are from a donor that is 7 / 8 HLA-mismatched relali vc to a human subject receiving the product. In some embodiments that may be combined with any of the preceding embodiments, the donor that is 7 / 8 HLA-mismatched relative to a human subject receiving the product has a mismatch in HLA-A. In some embodiments that may be combined with any of the preceding embodiments, the donor that is 7 / 8 HLA-mismatched relative to a human subject receiving the product has a mismatch in HLA-B. In some embodiments that may be combined with any of the preceding embodiments, the donor that is 7 / 8 HLA-mismatched relative to a human subject receiving the product has a mismatch in HLA-C. In some embodiments that may be combined with any of the preceding embodiments, the donor that is 7 / 8 HLA-5IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT mismatched relative to a human subject receiving the product has a mismatch in HLA-DRB1. In some embodiments that may be combined with any of the preceding embodiments, the donor that has the at least one HLA mismatch relative to a human subject receiving the product has a mismatched HLA allele as a result of the donor being homozygous for the HLA allele while a human subject receiving the product is heterogeneous for the HLA allele. In some embodiments that may be combined with any of the preceding embodiments, the donor that has at least one HLA mismatch rclalivc to a human subject receiving the product has a mismatched HLA allele as a result of the donor being heterozygous for the HLA allele while a human subject receiving the product is homozygous for the HLA allele. In some embodiments that may be combined with any of the preceding embodiments, the donor that has at least one HLA mismatch relative to a human subject receiving the product has a mismatched HLA allele as a result of both the donor and a human subject receiving the product being heterozygous for the HLA allele. In some embodiments that may be combined with any of the preceding embodiments, upon administration the first population of isolated CD45+cells, the second population of isolated CD45+cells, and the third population of isolated CD45+ cells decrease incidence of non-relapse mortality in a human subject receiving the product and / or increase overall survival in a human subject receiving the product compared to incidence of non-relapse mortality and / or over survival in a corresponding human subject that has been administered a standard myeloablative allogeneic hematopoietic stem cell transplant (alloHSCT) that is 7 / 8 HLA-mismatched relative to the corresponding human subject. In some embodiments that may be combined with any of the preceding embodiments, the product further comprises approximately 1000 mg of mycophenolate mofetil (MMF). In some embodiments that may be combined with any of the preceding embodiments, the body weight of a human subject receiving the product is actual body weight or. In some embodiments that may be combined with any of the preceding embodiments, the body weight of a human subject receiving the product is ideal body weight.

[0013] Other aspects of the present disclosure relate to a method of treating a human subject having or suspected of having a hematologic malignancy, the method comprising administering to a human subject the multi-component cellular therapy product of any one of the preceding embodiments. Other aspects of the present disclosure relate to a method of treating a human subject having or suspected of having a hematologic malignancy, the method comprising administering to a human subject a multi-component pharmaceutical treatment comprising: a) a solution comprising a first population of isolated CD45+cells comprising hematopoietic stem and progenitor cells (HSPCs); b) a solution comprising a second population of isolated CD45+cells comprising regulatory T cells (Tregs); c) a soludon comprising a third population of isolated CD45+cells wherein the third population of isolated CD45+cells comprise CD3+conventional T cells (Tcons); and d) a solution comprising one or more doses of a Janus kinase (JAK) inhibitor.

[0014] In some embodiments that may be combined with any of the preceding embodiments, the hematologic malignancy is selected from the group consisting of: leukemia, acute leukemia, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), mixed phenotype acute leukemia (MP AL), chronic myelogenous leukemia (CML), multiple myeloma, lymphoma, Hodgkin’s lymphoma, non-Hodgkin lymphoma, myelodysplastic syndrome, myeloproliferative syndrome, myelofibrosis, blastic plasmacytoid6IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT dendritic cell neoplasm (BPDCN), and any combinations thereof. In some embodiments that may be combined with any of the preceding embodiments, the administering comprises infusing into a human subject the first population of isolated CD45+cells, the second population of isolated CD45+cells, and the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the third population of isolated CD45+cells is administered from approximately 12 hours to approximately 120 hours after the first population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the third population of isolated CD45+cells is administered from approximately 36 hours to approximately 72 hours after the first population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the third population of isolated CD45+cells is administered from approximately 12 hours to approximately 120 hours after the second population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the third population of isolated CD45+cells is administered from approximately 36 to approximately 72 hours after the second population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the HSPCs are CD34+. In some embodiments that may be combined with any of the preceding embodiments, the first population of isolated CD45+cells comprises from approximately 5 x 105HSPCs per kilogram of actual or ideal body weight of a human subject to approximately 2 x 107HSPCs per kilogram of actual or ideal body weight of the human subject. In some embodiments that may be combined with any of the preceding embodiments, the Tregs are CD4+CD25+CD127dlmor CD4+FOXP3+. In some embodiments that may be combined with any of the preceding embodiments, in the second population of isolated CD45+cells more than approximately 90% of the CD45+cells are Tregs. In some embodiments that may be combined with any of the preceding embodiments, the second population of isolated CD45+cells comprises from approximately 1 x 105Tregs per kilogram of actual or ideal body weight of the human subject to approximately 1 x 107Tregs per kilogram of actual or ideal body weight of the human subject. In some embodiments that may be combined with any of the preceding embodiments, the third population of isolated CD45+cells comprises from approximately 1 x 105Tcons per kilogram of actual or ideal body weight of the human subject to approximately 1 x 107Tcons per kilogram of actual or ideal body weight of the human subject. In some embodiments that may be combined with any of the preceding embodiments, the human subject does not develop higher than stage 2 GVHD within approximately 100 days of the administering of the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the human subject does not develop higher than stage 2 GVHD within approximately 180 days or within approximately 200 days of the administering of the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the human subject does not develop higher than stage 2 GVHD within approximately 1 year of the administering of the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the human subject has previously been or is concurrently being treated for the hematologic malignancy.7IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0015] In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor is a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor, a TYK2 inhibitor, or any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacilinib, Icdralinib, filgotinib, abrocilinib, pacritinib, deucravacitinib, ritlecitinib, momelotinib, cerdulatinib, gandolinib, and lestaurtinib. In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor is initially administered to the human subject at a dose that ranges from approximately 2 mg to approximately 20 mg twice per day. In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor dosage comprises 5 mg twice per day. In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor is initially administered from approximately 2 days to approximately 5 days after the administering of the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the JAK inhibitor is administered for at least approximately 15 days, at least approximately 30 days, at least approximately 45 days, at least approximately 60 days, or at least approximately 90 days after administering the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, adminislralion of the JAK inhibitor is tapered starting at approximately 30 days after initial administration of the JAK inhibitor. In some embodiments that may be combined with any of the preceding embodiments, adminislralion of the JAK inhibitor is tapered to 5mg once per day starting at approximately 30 days after initial administration of the JAK inhibitor. In some embodiments that may be combined with any of the preceding embodiments, administration of the JAK inhibitor is tapered to zero at approximately 60 days after initial adminislralion of the JAK inhibitor. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises administering a graft vs host disease (GVHD) prophylactic agent. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is selected from the group consisting of: tacrolimus, cyclosporine A, sirolimus, basiliximab, daclizumab, methotrexate, muromonab-CD3, my cophenolate, anti-thymocyte globulin, corticosteroids, azathioprine, and mycophenolate mofetil. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is administered at a dose sufficient to maintain a trough blood level of approximately 5 ng / mL to approximately 10 ng / mL in the human subject. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is administered at a dose that ranges from approximately 0.01 mg per kilogram of body weight of the human subject to approximately 0.50 mg per kilogram of body weight of the human subject. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is initially administered from approximately 12 hours to approximately 2 days after the administering of the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is administered for at least approximately 60 days, at least approximately 90 days, at least approximately 120 days, or at most approximately 160 days after administering the third population of isolated CD45+cells. In some embodiments that may be combined8IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT with any of the preceding embodiments, administration of the GVHD prophylactic agent is tapered starring at approximately 60 days, approximately 70 days, approximately 80 days, approximately 90 days, approximately 100 days, approximately 110 days, or approximately 1200 days after initial administration of the GVHD prophylactic agent. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is initially administered to the human subject at a dose that ranges from approximately 0.01 mg to approximately 0.50 mg per kilogram of body weight of the human subject twice per day. In some embodiments that may be combined with any of the preceding embodiments, the dose of the GVHD prophylactic agent is sufficient to maintain a trough blood level of approximately 1 ng / mL to approximately 10 ng / mL in the human subject. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is administered approximately 1 day prior to administration of the JAK inhibitor. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is tacrolimus at 0.03 mg / kg per day. In some embodiments that may be combined with any of the preceding embodiments, the GVHD prophylactic agent is tacrolimus at 0.015 mg / kg per day administered twice a day IV (i.e., for a total of 0.03 mg / kg per day), and administering ruxolitinib at 5mg twice per day.

[0016] In some embodiments that may be combined with any of the preceding embodiments, the method further comprises administering approximately 1000 mg of mycophenolate mofetil (MMF). In some embodiments that may be combined with any of the preceding embodiments, the MMF is initially administered from approximately 12 hours to approximately 24 hours after the administering of the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, administration of the MMF is tapered starting at approximately 30 days, at approximately 35 days, at approximately 40 days, at approximately 41 days, at approximately 42 days, at approximately 43 days, at approximately 44 days, at approximately 45 days, at approximately 46 days, at approximately 47 days, at approximately 48 days, at approximately 49 days, or at approximately 50 days after initial administration of the MMF.

[0017] In some embodiments that may be combined with any of the preceding embodiments, the subject does not receive fluconazole at a dose of greater than 200 mg daily.

[0018] In some embodiments that may be combined with any of the preceding embodiments, the single allogeneic donor that has at least one HLA mismatch is unrelated to the human subject. In some embodiments that may be combined with any of the preceding embodiments, the single allogeneic donor that has at least one HLA mismatch is related to the human subject. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises collecting one or more, or two or more mobilized peripheral blood donations from the donor. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises collecting at most two mobilized peripheral blood donations from the donor. In some embodiments that may be combined with any of the preceding embodiments, the peripheral blood donations are mobilized by granulocyte colonystimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), plerixafor, or any combination thereof. In some embodiments that may be combined with any of the preceding9IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT embodiments, the at least one HLA mismatch is at an allele selected from the group consisting of: HLA-A, HLA-B, HLA-C, HLA-DRB1, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the allogeneic donor that has at least one HLA mismatch is 6 / 8 HLA-mismatched relative to the human subject or is 7 / 8 HLA-mismatched relative to the human subject. In some embodiments that may be combined with any of the preceding embodiments, the allogeneic donor is 7 / 8 HLA-mismatched relali vc to the human subject. In some embodiments that may be combined with any of the preceding embodiments, the allogeneic donor that is 7 / 8 HLA-mismatched relali ve to the human subject has a mismatch in HLA-A. In some embodiments that may be combined with any of the preceding embodiments, the allogeneic donor that is 7 / 8 HLA-mismatched re lali ve to the human subject has a mismatch in HLA-B. In some embodiments that may be combined with any of the preceding embodiments, the allogeneic donor that is 7 / 8 HLA-mismatched relative to the human subject has a mismatch in HLA-C. In some embodiments that may be combined with any of the preceding embodiments, the allogeneic donor that is 7 / 8 HLA-mismatched relali ve to the human subject has a mismatch in HLA-DRB 1. In some embodiments that may be combined with any of the preceding embodiments, the allogeneic donor that has at least one HLA mismatch relative to the human subject has a mismatched HLA allele as a result of the allogeneic donor being homozygous for the HLA allele while the human subject is heterogeneous for the HLA allele. In some embodiments that may be combined with any of the preceding embodiments, the allogeneic donor that has at least one HLA mismatch relative to the human subject has a mismatched HLA allele as a result of the allogeneic donor being heterozygous for the HLA allele while the human subject is homozygous for the HLA allele. In some embodiments that may be combined with any of the preceding embodiments, the allogeneic donor that has at least one HLA mismatch relative to the human subject has a mismatched HLA allele as a result of both the allogeneic donor and the human subject being heterozygous for the HLA allele.

[0019] In some embodiments that may be combined with any of the preceding embodiments, the method further comprises a conditioning regimen, wherein the conditioning regimen is administered before administration of the first population of isolated CD45+cells, the second population of isolated CD45+cells, and / or the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the conditioning regimen is administered from approximately two days to approximately ten days before administration of the first population of isolated CD45+cells, the second population of isolated CD45+cells, and / or the third population of isolated CD45+cells. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises a conditioning regimen, wherein the conditioning regimen is administered before any of (a) to (d). In some embodiments that may be combined with any of the preceding embodiments, the conditioning regimen is administered from approximately two days to approximately ten days before any of (a) to (d). In some embodiments that may be combined with any of the preceding embodiments, the conditioning regimen is a myeloablative conditioning regimen. In some embodiments that may be combined with any of the preceding embodiments, the myeloablative conditioning regimen comprises at least three conditioning reagents, wherein at least one conditioning reagent comprises thiotepa. In some embodiments that may be10IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT combined with any of the preceding embodiments, the myeloablative conditioning regimen comprises one or more doses of busulfan, fludarabine and thiotepa. In some embodiments that may be combined with any of the preceding embodiments, the one or more doses of busulfan, fludarabine and thiotepa comprise from approximately 5 to approximately 12 mg of thiotepa per kg human subject’s actual or ideal body weight, from approximately 7 to approximately 11 mg of busulfan per kg human subject actual or ideal body weight, and from approximately 100 to approximately 200 mg of fludarabine per meter2body surface area respectively. In some embodiments that may be combined with any of the preceding embodiments, the myeloablative conditioning regimen comprises a total body irradiation-based (TBI-based) regimen. In some embodiments that may be combined with any of the preceding embodiments, the TBI-based regimen comprises fractionated total body irradiation (fTBI). In some embodiments that may be combined with any of the preceding embodiments, the fTBI comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more doses. In some embodiments that may be combined with any of the preceding embodiments, the fTBI comprises a total dose that ranges from approximately 500 cGy to approximately 1600 cGy. In some embodiments that may be combined with any of the preceding embodiments, the fTBI-based regimen comprises 4 doses of TBI. In some embodiments that may be combined with any of the preceding embodiments, the TBI-based regimen further comprises one or more conditioning reagents. In some embodiments that may be combined with any of the preceding embodiments, the one or more conditioning reagents are selected from the group consisting of: cyclophosphamide, etoposide, thiotepa, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the TBI-based regimen further comprises one or more doses of cyclophosphamide. In some embodiments that may be combined with any of the preceding embodiments, each of the one or more doses of cyclophosphamide comprise from approximately 10 mg to approximately 100 mg of cyclophosphamide per kilogram of body weight of the human subject.

[0020] Other aspects of the present disclosure relate to a cellular therapy kit comprising the multicomponent cellular therapy product of any one of the preceding embodiments. In some embodiments, the kit further comprises written instructions for using the cellular therapy for treating a hematologic malignancy in a human subject. Other aspects of the present disclosure relate to a unit dose comprising the multi-component cellular therapy product of any one of the preceding embodiments. Other aspects of the present disclosure relate to an article of manufacture comprising the multi-component cellular therapy product of any one of the preceding embodiments.

[0021] Also provided herein is a multi -component cellular therapy product comprising: a) a first population of isolated CD45+ cells comprising CD34+ hematopoietic stem and progenitor cells (HSPCs); b) a second population of isolated CD45+ cells comprising isolated fresh regulatory T cells (Tregs); c) a third population of isolated CD45+ cells comprising conventional CD3+ T cells (Tcons); and d) one or more pharmaceutical compositions separately or combined comprising Mycophenolate mofetil (MMF) and tacrolimus, optionally 1000 mg of MMF, further optionally wherein the MMF is administered twice a day and / or for 8 weeks.11IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0022] It shall be understood that different aspects and / or embodiments of the present disclosure can be appreciated individually, collectively, or in combination with each other. Any description herein concerning a specific composition, multi-component pharmaceutical treatment, multi-component cellular therapy product, cell population, solution, formulation, kit, and / or method apply to and may be used for any other specific composition, multi-component pharmaceutical treatment, multi-component cellular therapy product, cell population, solution, formulation, kit, and / or method. Additionally, any composition disclosed herein is applicable to any herein-disclosed method. In other words, any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.Incorporation by reference

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed descriplion that sets forth illuslralivc embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings of which:

[0025] FIGs. 1A-1B illustrate the schematics of the transplant according to the methods described herein (identified as High-Precision Orca-T or Orca-T) and the differences compared to a standard of care (SOC) cohort (identified as Conventional Transplant or SOC). FIG. 1C illustrates a schematic of graft production and administration.

[0026] FIG. 2A illustrates the weight of patients enrolled in the study disclosed in the Examples.FIGs. 2B-2C illustrate the HSPC and Treg cell dose administered to the patients enrolled in the study disclosed in the Examples. FIG.2D illustrates the purity of Treg cells administered to the patients enrolled in the study disclosed in the Examples.

[0027] FIG. 3A shows the time to platelet engraftment in the study group (identified as Orca-T) and the standard of care (SOC) cohort. FIGs. 3B-3L illustrate engraftment of various cell populations in the patients in the study group disclosed in the Examples. The figures also illustrate the levels of each cell type in the donors before sample collection. Boxplots where shown: boxes show the 75th, 50th, and 25th percentiles; whiskers show the 90th and 10th percentiles. X-axes nomenclature: the leading number (e.g., 01, 02, 025, ...) are mentioned for ordering; following the underscore, Dscrn = healthy donor pre-G-CSF mobilization, Rscrn = recipient within 1 month prior to conditioning, apher = healthy donor blood draw at the time of apheresis, d028 = recipient day 28 post-transplant, d056-d365 = recipient days post-transplant. N’s shown indicate the sample sizes for each timepoint. Symbols indicate values for individual measurements. Cell numbers xlO-3per uL of blood are equivalent to xl,000 cells per uL of blood. FIG.3M-3N show the timeline of lymphocyte and monocyte engraftment in a subset of the study group (Orca- 12IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT T) and the standard of care cohort. FIG. 30 shows representative flow cytometry data for the frequency of CD3+CD4+T cells that were Tregs in two subjects compared to a healthy control. In the healthy control, 3.72% of circulating CD3+CD4+T cells were Tregs (CD25+CD127dlm). In the two graft recipients, 28.1% and 23.7% of CD3+CD4+T cells were Tregs on day +28, 32.3% and 17.8% on day +56, and 19.2% and 20.7% on day +100 post-transplant. FIG.3P shows flow cytometry data for B cell markers from a sample from a recipient of a composition of the present disclosure compared to a healthy control. In all cases, the Y axis is for CD19+staining. The left panels show gating of lymphocytes to identify B cells (CD19+) and T cells (CD3+). 13.4% of lymphocytes in the graft recipient were B cells, compared to 9.84% in the healthy control. The second from left panels show that 98.3-100% of cells gates as CD19+were also CD20+. The panels second from the right show the fraction of B cells that are IgD+, which can be used to identify mature B cells. 92.1% of B cells in the graft recipient were IgD+, and 89.5% in the healthy control. The right-most panels show staining for CD27, which can be used to identify memory B cells, late plasmablasts, and plasma cells, for example. 43.6% of B cells in the graft recipient were CD27+, and 67.1% in the healthy control.

[0028] FIG. 4A shows the onset of grade > 2 aGVHD in the study group (Orca-T) and the standard of care cohort through day +120 post-transplant. At nearly all timepoints, Orca-T data is below the standard of care data. FIG. 4B shows the onset of grade > 3 aGVHD in the study group (Orca T) and the standard of care cohort through day +120 post-transplant. At nearly all timepoints, Orca-T data is below the standard of care data. FIG. 4C shows the onset of moderate to severe cGVHD in the study group (Orca-T) and the standard of care (SOC) cohort through day +365 post-transplant. At nearly all timepoints, Orca-T data is below the standard of care data. FIG. 4D shows the non-relapse related mortality in the study group (Orca-T) and the standard of care (SOC) cohort through day +365 post-transplant. At nearly all liincpoinls, Orca-T data is below the standard of care data. FIG. 4E shows relapse rates in the study group (Orca-T) and the standard of care cohort through day +365 post-transplant. At the final timepoint, Orca-T relapse rate is 16% and the standard of care relapse rate is 19%. FIG. 4F shows GVHD and relapse-free survival rates in the study group (Orca-T) and the standard of care cohort through day +365 post-transplant. At nearly all timepoints, Orca-T data is above the standard of care data. FIG. 4G shows cGVHD-free survival rates in the study group and the standard of care cohort through day +365 post-transplant. At nearly all timepoints, Orca-T data is above the standard of care data. FIG. 4H shows overall survival rates in the study group and the standard of care cohort through day +365 post-transplant. At the final timepoint, Orca-T overall survival rate is 90% and the standard of care overall survival rate is 78%. FIG. 41 shows hospitalization days in a subset of the study group and the standard of care (SOC) cohort through day +365 post-transplant.

[0029] FIG. 5 summarizes the disease status of a small subset of subjects in the study group before transplant and at day +90, +180, and +356 post-transplant. CR signifies complete remission, MRD signifies minimal residual disease.

[0030] FIGs. 6A-6F compare the aGVHD, cGVHD, relapse, relapse-free survival, GVHD and relapse free survival (GRFS) and overall survival rates in a subset of the patients in the study group that received different conditioning regimens.13IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0031] FIGs. 7A-7H compare the aGVHD, cGVHD, non-relapse related mortality, relapse, relapse-free survival, GVHD and relapse free survival (GRFS) and overall survival rates in a subset of the patients in the study group that received different GVHD prophylactic agents.

[0032] FIGs.8A-8C illustrate aGVHD and cGVHD rates in patients with different serum tacrolimus trough levels.

[0033] FIGs. 9A-9B compare the aGVHD and cGVHD levels in patients that had different serum tacrolimus levels. FIGs. 9C-9D compare the aGVHD and cGVHD levels in patients that had different serum tacrolimus levels but were given the same conditioning regimen of busulfan and cyclophosphamide (Bu / Cy). FIGs. 9E-9G compare the aGVHD and cGVHD levels in patients that had different serum tacrolimus levels but were given the same conditioning regimen of Total Body Irradiation (TBI) / Busulfan, Fludarabine, Thiotepa (TBI / BFT). FIG. 9H shows the average trough tacrolimus level through day +30 post-transplant, plotted against the proportion of CD3+cells of donor origin at day +30 (except that chimerism data is from day 90 where indicated by “D90”).

[0034] FIG. 10A illustrates neutrophil and platelet engraftment using Orca-T derived from donors that are 7 / 8 HLA mismatched. Neutrophil and platelet engraftment occurred at median of 12.5 and 15.5 days, respectively. FIG. 10B illustrates percent chimerism of whole blood and T-cell using Orca-T derived from donors that are 7 / 8 HLA mismatched. Whole blood exhibited full chimerism and T-cells exhibited over 90% chimerism 90 days post-transplant.

[0035] FIG. 11A illustrates IL-2 serum plasma levels 14 days post-transplant using Orca-T derived from donors that are 7 / 8 HLA mismatched or 8 / 8 matched. FIG. 11B illustrates IL- 10 serum plasma levels 14 days post-transplant using Orca-T derived from donors that are 7 / 8 HLA mismatched or 8 / 8 matched.

[0036] FIG. 12 illustrates clinical outcomes of six patients that received Orca-T derived from donors that are 7 / 8 HLA mismatched at 90 days, 180 days, and 365 days post-transplant.

[0037] FIG. 13 depicts a timeline schematic of the TBI-based conditioning regimen and administration of the Orca-T cell therapy product.

[0038] FIG. 14 depicts a timeline schematic of the chemotherapy-based conditioning regimen and administration of the Orca-T cell therapy product.

[0039] FIG. 15 depicts a timeline schematic of the Orca-T cell therapy product infusion and dual-agent GVHD prophylaxis.DETAILED DESCRIPTIONIntroduction

[0040] Various embodiments of the present disclosure provide compositions, multi-component pharmaceutical treatments, multi-component cellular therapy products, cell populations, solutions, formulations, kits, and / or methods relating to improved allogeneic hematopoietic stem cell transplantarion (alloHSCT) that includes transplantation of distinct cell populations that are enriched for hematopoietic stem and progenitor cells (HSPCs), regulatory T-cells (Tregs), and conventional T-cells (Tcons).14IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0041] Standard alloHSCT is the transplantation of multipotent hematopoietic stem and progenitor cells (HSPCs), usually derived from donor bone marrow, peripheral blood, or umbilical cord blood, into a recipient. The recipient can be subjected to myeloablative conditioning, which kills hematopoietic cells including tumor cells and host immune cells. The HSPCs transplanted into the recipient then reconstitutes the hematopoietic compartment. HSCT can be useful as a treatment for cancer due to the ability of donor T cells to exert anti-tumor effects, referred to as graft versus tumor (GVT). In patients with hematologic malignancies that are refractory to chemotherapy, HSCT is associated with improved survival.

[0042] Surprisingly, recipients of the improved alloHSCT of the present disclosure who received one or more graft versus host disease (GVHD) prophylactic agents, such as a JAK inhibitor and / or a calcineurin inhibitor (e.g., tacrolimus), had significantly better clinical outcomes than existing standard alloHSCT regimens and standards of care. These recipients experience improved clinical outcomes including, for example, increased overall survival, increased relapse-free survival, increased GVHD- and relapse-free survival (GRFS), more rapid and / or complete engraftment of various hematopoietic components (e.g., neutrophils, platelets, T cells, B cells), improved donor chimerism (e.g., T cell chimerism), decreased relapse, decreased primary graft failure, decreased secondary graft failure, decreased treatment-associated mortality, reduced acute and / or chronic GVHD, and shorter time to discharge from hospital following the one or more GVHD prophylactic agents.

[0043] Although alloHSCT is associated with improved survival in patients with hematologic malignancies that are refractory to chemotherapy, some subjects treated with existing standard alloHSCT regimens exhibit cancer relapse, and a number of complications can limit the efficacy of standard alloHSCT. The effectiveness of standard alloHSCT can be limited by, for example, primary graft failure, secondary graft failure, limited or slow engraftment of various hematopoietic components (e.g., neutrophils, platelets, T cells, or B cells), and limited donor chimerism (e.g., T cell chimerism). Additionally, standard alloHSCT can cause treatment-associated morality or toxicity, for example, However, donor T cells can also attack non-tumor host cells, resulting in graft versus host disease (GVHD). GVHD is a major source of post-HCT complications and can be fatal. Management of GVHD can require immunosuppressive therapy or cytotoxic mediations, which can cause toxicity, increase susceptibility to infection, and / or blunt anli-luinor immunity. The early morbidity and mortality associated with acute graft versus host disease (aGVHD; which occurs within the first 100 days post-transplant) is a major factor limiting the success of HCT, as is the long-term morbidity associated with chronic GVHD (cGVHD). GVHD is a risk for both HLA-matched and HLA-mismatched transplantations. GVHD can occur even if the donor and recipient are HLA-matched, because the immune system can still recognize other differences between in the donor tissues.

[0044] Both GVT and GVHD are largely mediated by conventional T cells (Tcons), which mount immune responses upon recognition of cognate antigen by T cell receptors. Depleting T cells from hematopoietic stem cell transplantation (HCT) grafts can reduce GVHD, but can also result in reduced GVT and increased likelihood of cancer relapse. Besides Tcons, Tregs are an additional subset of T cells that negatively regulate inflammation and that promote immune tolerance. Tregs can prevent or reduce15IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT GVHD through their negative regulation of inflammation, including, for example, inflammation elicited by donor Tcons when they recognize recipient antigens.

[0045] Provided herein are compositions and methods for improved alloHSCT, comprising administering to a subject certain cell populations that comprise populations of cells, including a first population of CD45+cells that comprise, at least, HSPCs, a second population of CD45+cells that comprise, at least Tregs, and a third population of CD45+cells that comprises, at least, Tcons. In some embodiments, the second population of CD45+cells is also referred to a cell population enriched for Tregs. Without wishing to be bound by theory, administering the second population of CD45+cells reduces the incidence and / or severity of GVHD, while administering the third population of CD45+cells, which comprises Tcons, enhances GVT. Thus, embodiments of the present disclosure provide a provides compositions, multi-component pharmaceutical treatments, multi-component cellular therapy products, cell populations, soludons, formulations, kits, and / or methods for administering, at least, both populations of T cells, to enhance GVT while minimizing GVHD. Accordingly, the compositions, multi-component pharmaceutical treatments, multi-component cellular therapy products, cell populations, solutions, formulations, kits, and / or methods disclosed herein can retain the graft-versus-tumor (GVT) effects of alloHSCT administered to a subject having a cancer (e.g., a hematologic cancer), while preventing or reducing graft versus host disease (GVHD) in the subject. In some embodiments, two or more populations of cells are administered at different times, for example, first population of CD45+cells that comprises, at least, HSPCs and the cell population enriched for Tregs can be administered prior to the third population of CD45+cells that comprises, at least, Tcons.Cell Populations

[0046] Embodiments of the present disclosure provide a multi-component pharmaceutical cellular therapy product comprises a) a first population of isolated CD45+cells comprising hematopoietic stem and progenitor cells (HSPCs); b) a second population of isolated CD45+cells comprising fresh regulatory T cells (Tregs); c) a third population of isolated CD45+cells comprising conventional CD3+T cells (Tcons); and (d) a pharmaceutical composition comprising one or more graft versus host disease (GVHD) prophylactic agents, such as a Janus kinase (JAK) inhibitor (e.g., a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor, and / or a TYK2 inhibitor) and / or a calcineurin inhibitor e.g., tacrolimus) .

[0047] In some embodiments, the first population of CD45+cells comprises HSPCs. In other embodiments, the first population of CD45+cells comprises at least one dose of HSPCs. In some embodiments, the first population of CD45+cells comprising HSPCs, or comprising at least one dose of HSPCs, comprises from approximately 1.0 x 105to approximately 5.0 x 108HSPCs per kilogram of body weight of the human subject, from approximately 1.0 x 105to approximately 1.0 x 108HSPCs per kilogram of body weight of the human subject, or from approximately 5.0 x 105to approximately 2.0 x 107HSPCs per kilogram of body weight of the human subject. In some embodiments, the first population of CD45+cells comprising HSPCs, or comprising at least one dose of HSPCs, comprises approximately 1.0 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 1.5 x 105or more HSPCs16IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT per kilogram of body weight of the human subject, approximately 2.0 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 2.5 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 3.0 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 3.5 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 4.0 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 4.5 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 5.0 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 5.5 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 6.0 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 6.5 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 7.0 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 7.5 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 8.0 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 8.5 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 9.0 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 9.5 x 105or more HSPCs per kilogram of body weight of the human subject, approximately 1.0 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 1.25 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 1.5 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 1.75 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 2.0 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 2.25 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 2.5 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 2.75 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 3.0 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 3.25 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 3.5 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 3.75 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 4.0 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 4.25 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 4.5 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 4.75 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 5.0 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 5.25 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 5.5 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 5.75 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 6.0 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 6.25 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 6.5 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 6.75 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 7.0 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 7.25 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 7.5 x 106or more HSPCs per kilogram of body weight17IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT of the human subject, approximately 7.75 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 8.0 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 8.25 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 8.5 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 8.75 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 9.0 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 9.25 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 9.5 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 9.75 x 106or more HSPCs per kilogram of body weight of the human subject, approximately 1.0 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 1.25 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 1.5 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 1.75 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 2.0 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 2.25 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 2.5 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 2.75 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 3.0 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 3.25 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 3.5 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 3.75 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 4.0 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 4.25 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 4.5 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 4.75 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 5.0 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 5.25 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 5.5 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 5.75 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 6.0 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 6.25 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 6.5 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 6.75 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 7.0 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 7.25 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 7.5 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 7.75 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 8.0 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 8.25 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 8.5 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 8.75 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 9.0 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 9.25 x 107or more HSPCs per kilogram of body weight of the human subject, approximately18IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 9.5 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 9.75 x 107or more HSPCs per kilogram of body weight of the human subject, approximately 1.0 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 1.25 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 1.5 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 1.75 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 2.0 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 2.25 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 2.5 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 2.75 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 3.0 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 3.25 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 3.5 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 3.75 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 4.0 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 4.25 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 4.5 x 108or more HSPCs per kilogram of body weight of the human subject, approximately 4.75 x 108or more HSPCs per kilogram of body weight of the human subject, or approximately 5.0 x 108or more HSPCs per kilogram of body weight of the human subject. In some embodiments, the body weight of the human subject is actual body weight of the human subject. In other embodiments, the body weight of the human subject is ideal actual body weight of the human subject.

[0048] In embodiments, the first population of CD45+cells comprises at least about 0.5% granulocytes, at least about 1% granulocytes, at most about 5% granulocytes, at most about 3% granulocytes, at most about 3% monocytes, at most about 2% monocytes, at most about 0.5% lymphocytes, at most about 2% lymphocytes, at least about 15% granulocytes, at least about 20% granulocytes, at most about 35% granulocytes, at most about 30% granulocytes, at most about 25% granulocytes, at least about 15% monocytes, at least about 20% monocytes, at most about 35% monocytes, at most about 30% monocytes, at most about 25% monocytes, at least about 0.5% NK cells, and or at least about 2% NK cells. In various embodiments, the first population of CD45+cells, the population of cells enriched for Tregs, and the third population of CD45+cells are obtained from a single donor. In some embodiments, the first population of CD45+cells, the population of cells enriched for Tregs, and / or the third population of CD45+cells is allogeneic relative to the human subject. In embodiments, the first population of CD45+cells, the population of cells enriched for Tregs, and / or the third population of CD45+cells is obtained from a donor that is HLA-matched relative to the human subject. In various embodiments, the first population of CD45+cells, the population of cells enriched for Tregs, and / or the third population of CD45+cells is obtained from a donor that is HLA-mismatched relative to the human subject. In some embodiments, the first population of CD45+cells, the population of cells enriched for Tregs, and / or the third population of CD45+cells is obtained from a donor that is haploidentical relative to the human subject.

[0049] In some embodiments, the Tregs are CD4+CD25+CD127dlmor CD4+FOXP3+. In some embodiments, the Tregs are CD4+CD25+CD127dlmand are also FOXP3+. In some cases, the population of19IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT cells enriched for Tregs comprises CD45+cells, e.g., more than about 90% of the CD45+cells are Tregs. In other embodiments, the second population of CD45+cells comprises one or more doses of Tregs. In various embodiments, the population of cells enriched for Tregs, or the second population of CD45+cells comprising at least one dose of Tregs, comprises from approximately 1.0 x 105to approximately 1.0 x 108Tregs per kilogram of body weight of the human subject, from approximately 1.0 x 105to approximately 2.0 x 107Tregs per kilogram of body weight of the human subject, from approximately 1.0 x 105to approximately 1.0 x 107Tregs per kilogram of body weight of the human subject, or from approximately 5.0 x 105to approximately 4.0 x 106Tregs per kilogram of body weight of the human subject. In some embodiments, the population of cells enriched for Tregs, or the second population of CD45+cells comprising at least one dose of Tregs, comprises approximately 1.0 x 105or more Tregs per kilogram of body weight of the human subject, approximately 1.5 x 105or more Tregs per kilogram of body weight of the human subject, approximately 2.0 x 105or more Tregs per kilogram of body weight of the human subject, approximately 2.5 x 105or more Tregs per kilogram of body weight of the human subject, approximately 3.0 x 105or more Tregs per kilogram of body weight of the human subject, approximately 3.5 x 105or more Tregs per kilogram of body weight of the human subject, approximately 4.0 x 105or more Tregs per kilogram of body weight of the human subject, approximately 4.5 x 105or more Tregs per kilogram of body weight of the human subject, approximately 5.0 x 105or more Tregs per kilogram of body weight of the human subject, approximately 5.5 x 105or more Tregs per kilogram of body weight of the human subject, approximately 6.0 x 105or more Tregs per kilogram of body weight of the human subject, approximately 6.5 x 105or more Tregs per kilogram of body weight of the human subject, approximately 7.0 x 105or more Tregs per kilogram of body weight of the human subject, approximately 7.5 x 105or more Tregs per kilogram of body weight of the human subject, approximately 8.0 x 105or more Tregs per kilogram of body weight of the human subject, approximately 8.5 x 105or more Tregs per kilogram of body weight of the human subject, approximately 9.0 x 105or more Tregs per kilogram of body weight of the human subject, approximately 9.5 x 105or more Tregs per kilogram of body weight of the human subject, approximately 1.0 x 106or more Tregs per kilogram of body weight of the human subject, approximately 1.25 x 106or more Tregs per kilogram of body weight of the human subject, approximately 1.5 x 106or more Tregs per kilogram of body weight of the human subject, approximately 1.75 x 106or more Tregs per kilogram of body weight of the human subject, approximately 2.0 x 106or more Tregs per kilogram of body weight of the human subject, approximately 2.25 x 106or more Tregs per kilogram of body weight of the human subject, approximately 2.5 x 106or more Tregs per kilogram of body weight of the human subject, approximately 2.75 x 106or more Tregs per kilogram of body weight of the human subject, approximately 3.0 x 106or more Tregs per kilogram of body weight of the human subject, approximately 3.25 x 106or more Tregs per kilogram of body weight of the human subject, approximately 3.5 x 106or more Tregs per kilogram of body weight of the human subject, approximately 3.75 x 106or more Tregs per kilogram of body weight of the human subject, approximately 4.0 x 106or more Tregs per kilogram of body weight of the human subject, approximately 4.25 x 106or more Tregs per kilogram of body weight of the human subject, approximately 4.5 x 106or more Tregs per kilogram of20IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT body weight of the human subject, approximately 4.75 x 106or more Tregs per kilogram of body weight of the human subject, approximately 5.0 x 106or more Tregs per kilogram of body weight of the human subject, approximately 5.25 x 106or more Tregs per kilogram of body weight of the human subject, approximately 5.5 x 106or more Tregs per kilogram of body weight of the human subject, approximately 5.75 x 106or more Tregs per kilogram of body weight of the human subject, approximately 6.0 x 106or more Tregs per kilogram of body weight of the human subject, approximately 6.25 x 106or more Tregs per kilogram of body weight of the human subject, approximately 6.5 x 106or more Tregs per kilogram of body weight of the human subject, approximately 6.75 x 106or more Tregs per kilogram of body weight of the human subject, approximately 7.0 x 106or more Tregs per kilogram of body weight of the human subject, approximately 7.25 x 106or more Tregs per kilogram of body weight of the human subject, approximately 7.5 x 106or more Tregs per kilogram of body weight of the human subject, approximately 7.75 x 106or more Tregs per kilogram of body weight of the human subject, approximately 8.0 x 106or more Tregs per kilogram of body weight of the human subject, approximately 8.25 x 106or more Tregs per kilogram of body weight of the human subject, approximately 8.5 x 106or more Tregs per kilogram of body weight of the human subject, approximately 8.75 x 106or more Tregs per kilogram of body weight of the human subject, approximately 9.0 x 106or more Tregs per kilogram of body weight of the human subject, approximately 9.25 x 106or more Tregs per kilogram of body weight of the human subject, approximately 9.5 x 106or more Tregs per kilogram of body weight of the human subject, approximately 9.75 x 106or more Tregs per kilogram of body weight of the human subject, approximately 1.0 x 107or more Tregs per kilogram of body weight of the human subject, approximately 1.25 x 107or more Tregs per kilogram of body weight of the human subject, approximately 1.5 x 107or more Tregs per kilogram of body weight of the human subject, approximately 1.75 x 107or more Tregs per kilogram of body weight of the human subject, approximately 2.0 x 107or more Tregs per kilogram of body weight of the human subject, approximately 2.25 x 107or more Tregs per kilogram of body weight of the human subject, approximately 2.5 x 107or more Tregs per kilogram of body weight of the human subject, approximately 2.75 x 107or more Tregs per kilogram of body weight of the human subject, approximately 3.0 x 107or more Tregs per kilogram of body weight of the human subject, approximately 3.25 x 107or more Tregs per kilogram of body weight of the human subject, approximately 3.5 x 107or more Tregs per kilogram of body weight of the human subject, approximately 3.75 x 107or more Tregs per kilogram of body weight of the human subject, approximately 4.0 x 107or more Tregs per kilogram of body weight of the human subject, approximately 4.25 x 107or more Tregs per kilogram of body weight of the human subject, approximately 4.5 x 107or more Tregs per kilogram of body weight of the human subject, approximately 4.75 x 107or more Tregs per kilogram of body weight of the human subject, approximately 5.0 x 107or more Tregs per kilogram of body weight of the human subject, approximately 5.25 x 107or more Tregs per kilogram of body weight of the human subject, approximately 5.5 x 107or more Tregs per kilogram of body weight of the human subject, approximately 5.75 x 107or more Tregs per kilogram of body weight of the human subject, approximately 6.0 x 107or more Tregs per kilogram of body weight of the human subject, approximately 6.25 x 107or more Tregs per kilogram of body weight of the human subject,21IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 6.5 x 107or more Tregs per kilogram of body weight of the human subject, approximately 6.75 x 107or more Tregs per kilogram of body weight of the human subject, approximately 7.0 x 107or more Tregs per kilogram of body weight of the human subject, approximately 7.25 x 107or more Tregs per kilogram of body weight of the human subject, approximately 7.5 x 107or more Tregs per kilogram of body weight of the human subject, approximately 7.75 x 107or more Tregs per kilogram of body weight of the human subject, approximately 8.0 x 107or more Tregs per kilogram of body weight of the human subject, approximately 8.25 x 107or more Tregs per kilogram of body weight of the human subject, approximately 8.5 x 107or more Tregs per kilogram of body weight of the human subject, approximately 8.75 x 107or more Tregs per kilogram of body weight of the human subject, approximately 9.0 x 107or more Tregs per kilogram of body weight of the human subject, approximately 9.25 x 107or more Tregs per kilogram of body weight of the human subject, approximately 9.5 x 107or more Tregs per kilogram of body weight of the human subject, approximately 9.75 x 107or more Tregs per kilogram of body weight of the human subject, approximately 1.0 x 108or more Tregs per kilogram of body weight of the human subject. In some embodiments, the body weight of the human subject is actual body weight of the human subject. In other embodiments, the body weight of the human subject is ideal actual body weight of the human subject.

[0050] In some embodiments, the third population of CD45+cells comprises Tcons. In other embodiments, the third population of CD45+comprises at least one dose of Tcons. In some embodiments, the third population of CD45+cells comprising Tcons, or comprising at least one dose of Tcons, comprises from approximately 1.0 x 105to approximately 1.0 x 108Tcons per kilogram of body weight of the human subject, from approximately 1.0 x 105to approximately 4.0 x 107Tcons per kilogram of body weight of the human subject, from approximately 1.0 x 105to approximately 1.0 x 107Tcons per kilogram of body weight of the human subject, or from approximately 5.0 x 105to approximately 5 x 106Tcons per kilogram of body weight of the human subject. In some embodiments, the third population of CD45+cells comprising Tcons, or comprising at least one dose of Tcons, comprises approximately 1.0 x 105or more Tcons per kilogram of body weight of the human subject, approximately 1.5 x 105or more Tcons per kilogram of body weight of the human subject, approximately 2.0 x 105or more Tcons per kilogram of body weight of the human subject, approximately 2.5 x 105or more Tcons per kilogram of body weight of the human subject, approximately 3.0 x 105or more Tcons per kilogram of body weight of the human subject, approximately 3.5 x 105or more Tcons per kilogram of body weight of the human subject, approximately 4.0 x 105or more Tcons per kilogram of body weight of the human subject, approximately 4.5 x 105or more Tcons per kilogram of body weight of the human subject, approximately 5.0 x 105or more Tcons per kilogram of body weight of the human subject, approximately 5.5 x 105or more Tcons per kilogram of body weight of the human subject, approximately 6.0 x 105or more Tcons per kilogram of body weight of the human subject, approximately 6.5 x 105or more Tcons per kilogram of body weight of the human subject, approximately 7.0 x 105or more Tcons per kilogram of body weight of the human subject, approximately 7.5 x 105or more Tcons per kilogram of body weight of the human subject, approximately 8.0 x 105or more Tcons per kilogram of body weight of the human subject, approximately 8.5 x 105or22IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT more Tcons per kilogram of body weight of the human subject, approximately 9.0 x 105or more Tcons per kilogram of body weight of the human subject, approximately 9.5 x 105or more Tcons per kilogram of body weight of the human subject, approximately 1.0 x 106or more Tcons per kilogram of body weight of the human subject, approximately 1.25 x 106or more Tcons per kilogram of body weight of the human subject, approximately 1.5 x 106or more Tcons per kilogram of body weight of the human subject, approximately 1.75 x 106or more Tcons per kilogram of body weight of the human subject, approximately 2.0 x 106or more Tcons per kilogram of body weight of the human subject, approximately 2.25 x 106or more Tcons per kilogram of body weight of the human subject, approximately 2.5 x 106or more Tcons per kilogram of body weight of the human subject, approximately 2.75 x 106or more Tcons per kilogram of body weight of the human subject, approximately 3.0 x 106or more Tcons per kilogram of body weight of the human subject, approximately 3.25 x 106or more Tcons per kilogram of body weight of the human subject, approximately 3.5 x 106or more Tcons per kilogram of body weight of the human subject, approximately 3.75 x 106or more Tcons per kilogram of body weight of the human subject, approximately 4.0 x 106or more Tcons per kilogram of body weight of the human subject, approximately 4.25 x 106or more Tcons per kilogram of body weight of the human subject, approximately 4.5 x 106or more Tcons per kilogram of body weight of the human subject, approximately 4.75 x 106or more Tcons per kilogram of body weight of the human subject, approximately 5.0 x 106or more Tcons per kilogram of body weight of the human subject, approximately 5.25 x 106or more Tcons per kilogram of body weight of the human subject, approximately 5.5 x 106or more Tcons per kilogram of body weight of the human subject, approximately 5.75 x 106or more Tcons per kilogram of body weight of the human subject, approximately 6.0 x 106or more Tcons per kilogram of body weight of the human subject, approximately 6.25 x 106or more Tcons per kilogram of body weight of the human subject, approximately 6.5 x 106or more Tcons per kilogram of body weight of the human subject, approximately 6.75 x 106or more Tcons per kilogram of body weight of the human subject, approximately 7.0 x 106or more Tcons per kilogram of body weight of the human subject, approximately 7.25 x 106or more Tcons per kilogram of body weight of the human subject, approximately 7.5 x 106or more Tcons per kilogram of body weight of the human subject, approximately 7.75 x 106or more Tcons per kilogram of body weight of the human subject, approximately 8.0 x 106or more Tcons per kilogram of body weight of the human subject, approximately 8.25 x 106or more Tcons per kilogram of body weight of the human subject, approximately 8.5 x 106or more Tcons per kilogram of body weight of the human subject, approximately 8.75 x 106or more Tcons per kilogram of body weight of the human subject, approximately 9.0 x 106or more Tcons per kilogram of body weight of the human subject, approximately 9.25 x 106or more Tcons per kilogram of body weight of the human subject, approximately 9.5 x 106or more Tcons per kilogram of body weight of the human subject, approximately 9.75 x 106or more Tcons per kilogram of body weight of the human subject, approximately 1.0 x 107or more Tcons per kilogram of body weight of the human subject, approximately 1.25 x 107or more Tcons per kilogram of body weight of the human subject, approximately 1.5 x 107or more Tcons per kilogram of body weight of the human subject, approximately 1.75 x 107or more Tcons per kilogram of body weight of the human subject, approximately 2.0 x 107or more Tcons per kilogram of body weight of23IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT the human subject, approximately 2.25 x 107or more Tcons per kilogram of body weight of the human subject, approximately 2.5 x 107or more Tcons per kilogram of body weight of the human subject, approximately 2.75 x 107or more Tcons per kilogram of body weight of the human subject, approximately 3.0 x 107or more Tcons per kilogram of body weight of the human subject, approximately 3.25 x 107or more Tcons per kilogram of body weight of the human subject, approximately 3.5 x 107or more Tcons per kilogram of body weight of the human subject, approximately 3.75 x 107or more Tcons per kilogram of body weight of the human subject, approximately 4.0 x 107or more Tcons per kilogram of body weight of the human subject, approximately 4.25 x 107or more Tcons per kilogram of body weight of the human subject, approximately 4.5 x 107or more Tcons per kilogram of body weight of the human subject, approximately 4.75 x 107or more Tcons per kilogram of body weight of the human subject, approximately 5.0 x 107or more Tcons per kilogram of body weight of the human subject, approximately 5.25 x 107or more Tcons per kilogram of body weight of the human subject, approximately 5.5 x 107or more Tcons per kilogram of body weight of the human subject, approximately 5.75 x 107or more Tcons per kilogram of body weight of the human subject, approximately 6.0 x 107or more Tcons per kilogram of body weight of the human subject, approximately 6.25 x 107or more Tcons per kilogram of body weight of the human subject, approximately 6.5 x 107or more Tcons per kilogram of body weight of the human subject, approximately 6.75 x 107or more Tcons per kilogram of body weight of the human subject, approximately 7.0 x 107or more Tcons per kilogram of body weight of the human subject, approximately 7.25 x 107or more Tcons per kilogram of body weight of the human subject, approximately 7.5 x 107or more Tcons per kilogram of body weight of the human subject, approximately 7.75 x 107or more Tcons per kilogram of body weight of the human subject, approximately 8.0 x 107or more Tcons per kilogram of body weight of the human subject, approximately 8.25 x 107or more Tcons per kilogram of body weight of the human subject, approximately 8.5 x 107or more Tcons per kilogram of body weight of the human subject, approximately 8.75 x 107or more Tcons per kilogram of body weight of the human subject, approximately 9.0 x 107or more Tcons per kilogram of body weight of the human subject, approximately 9.25 x 107or more Tcons per kilogram of body weight of the human subject, approximately 9.5 x 107or more Tcons per kilogram of body weight of the human subject, approximately 9.75 x 107or more Tcons per kilogram of body weight of the human subject, approximately 1.0 x 108or more Tcons per kilogram of body weight of the human subject. In some embodiments, the body weight of the human subject is actual body weight of the human subject. In other embodiments, the body weight of the human subject is ideal actual body weight of the human subject.

[0051] In embodiments, the third population of CD45+cells comprises at least about 0.1% CD34+cells or from approximately 0.2% to approximately 20% CD34+cells and / or at least about 0.1% Tregs. In various embodiments, the third population of CD45+cells comprises a population of memory T cells (Tmems), e.g., Tmems that are CD3+CD45RA- CD45RO+. In some embodiments, the population of Tmems comprises more than about 3 x 105Tmems per kilogram of ideal body actual or ideal body weight of the human subject. In embodiments, the population of Tmems comprises from approximately 3 x 105to approximately 1 x 109Tmems per kilogram of ideal body actual or ideal body weight of the human subject.24IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT In various embodiments, the third population of CD45+cells comprises a population of invariant natural killer T cells (iNKTs), e.g., iNKTs that are CD3+Va24Jal8+. In some embodiments, the population of iNKTs comprises more than about 5 x 102iNKTs per kilogram of ideal body actual or ideal body weight of the human subject. In embodiments, the population of iNKTs comprises from approximately 5 x 102to approximately 1 x 107iNKTs per kilogram of ideal body actual or ideal body weight of the human subject. In some embodiments, the third population of CD45+cells is co-cultured with donor cancer anligcns / pcplides and / or antigen-presenting cells.

[0052] In some embodiments, a cell population of the present disclosure, for example the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells, may comprise one or more cells comprising one or more chimeric receptors. In some embodiments, the one or more cells may be T cells, NK cells, CTLs, Tregs, and / or NKT cells. In some embodiments, the one or more chimeric receptors comprise one or more extracellular antigen-binding domains that bind to one or more cancer-associated antigens. Any suitable cancer-associated antigen known in the art may be targeted. In some embodiments, the chimeric receptor is a chimeric antigen receptor or a T cell receptor.

[0053] Provided herein are compositions, multi-component pharmaceutical treatments, multicomponent cellular therapy products, cell populations, soludons, formulations, kits, and / or methods for improved hematopoietic stem cell transplantation (HCT), for example, allogeneic hematopoietic stem cell transplantation (alloHSCT). Compositions, multi-component pharmaceutical treatments, multi-component cellular therapy products, cell populations, solutions, formulations, kits, and / or methods disclosed herein can comprise one or more cell populations that can be administered in combination with a GVHD prophylactic agent to achieve positive clinical outcomes. A cell population can comprise one or more types of cells, for example, hematopoietic stem and progenitor cells (HSPCs), conventional T cells (Tcons), regulatory T cells (Tregs), invariant natural killer T cells (iNKTs), memory T cells (Tmems), and combinations thereof.

[0054] The disclosure provides parameters for cell populations and methods of administering cell populations that can contribute to successful clinical outcomes in HCT recipient subjects. Without wishing to be bound by theory, parameters that can contribute to successful clinical outcomes in HCT recipient subjects include, for example, co-administration of on or more GVHD prophylactic agents as described herein (e.g., a JAK inhibitor and / or tacrolimus), populations administered, order and timing for the administration of different populations, purity standards for populations, methods for obtaining populations, methods of handling or storing populations, dosages of populations administered, methods for obtaining populations, and combinations thereof.

[0055] In various embodiments, administering comprises infusing into the human subject the first population of CD45+cells, the population of cells enriched for Tregs (e.g., the second population of CD45+cells), and the third population of CD45+cells.

[0056] In some embodiments, the population of cells enriched for Tregs or the second population of CD45+cells (as disclosed herein) is administered from approximately 5 minutes to approximately 5 hours after administration of the first population of CD45+cells (as disclosed herein). In some embodiments, the25IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT population of cells enriched for Tregs or the second population of CD45+cells is administered approximately 5 minutes, approximately 6 minutes, approximately 7 minutes, approximately 8 minutes, approximately 9 minutes, approximately 10 minutes, approximately 11 minutes, approximately 12 minutes, approximately 13 minutes, approximately 14 minutes, approximately 15 minutes, approximately 16 minutes, approximately 17 minutes, approximately 18 minutes, approximately 19 minutes, approximately 20 minutes, approximately 21 minutes, approximately 22 minutes, approximately 23 minutes, approximately 24 minutes, approximately 25 minutes, approximately 26 minutes, approximately 27 minutes, approximately 28 minutes, approximately 29 minutes, approximately 30 minutes, approximately 31 minutes, approximately 32 minutes, approximately 33 minutes, approximately 34 minutes, approximately 35 minutes, approximately 36 minutes, approximately 37 minutes, approximately 38 minutes, approximately 39 minutes, approximately 40 minutes, approximately 41 minutes, approximately 42 minutes, approximately 43 minutes, approximately 44 minutes, approximately 45 minutes, approximately 46 minutes, approximately 47 minutes, approximately 48 minutes, approximately 49 minutes, approximately 50 minutes, approximately 51 minutes, approximately 52 minutes, approximately 53 minutes, approximately 54 minutes, approximately 55 minutes, approximately 56 minutes, approximately 57 minutes, approximately 58 minutes, approximately 59 minutes, approximately 1 hour, approximately 1.10 hours, approximately 1.20 hours, approximately 1.30 hours, approximately 1.40 hours, approximately 1.50 hours, approximately 1.60 hours, approximately 1.70 hours, approximately 1.80 hours, approximately 1.90 hours, approximately 2 hours, approximately 2.10 hours, approximately 2.20 hours, approximately 2.30 hours, approximately 2.40 hours, approximately 2.50 hours, approximately 2.60 hours, approximately 2.70 hours, approximately 2.80 hours, approximately 2.90 hours, approximately 3 hours, approximately 3.10 hours, approximately 3.20 hours, approximately 3.30 hours, approximately 3.40 hours, approximately 3.50 hours, approximately 3.60 hours, approximately 3.70 hours, approximately 3.80 hours, approximately 3.90 hours, approximately 4 hours, approximately 4.10 hours, approximately 4.20 hours, approximately 4.30 hours, approximately 4.40 hours, approximately 4.50 hours, approximately 4.60 hours, approximately 4.70 hours, approximately 4.80 hours, approximately 4.90 hours, or approximately 5 hours after administration of the first population of CD45+cells.

[0057] In some embodiments, the third population of CD45+cells (as disclosed herein) is administered at least about 12 hours after the first population of CD45+cells (as disclosed herein), the third population of CD45+cells is administered from approximately 24 to approximately 96 hours after the first population of CD45+cells, the third population of CD45+cells is administered from approximately 36 to approximately 60 hours after the first population of CD45+cells, the third population of CD45+cells is administered at least about 12 hours after the population of cells enriched for Tregs (as disclosed herein), the third population of CD45+cells is administered from approximately 24 to approximately 96 hours after the population of cells enriched for Tregs, and / or the third population of CD45+cells is administered from approximately 36 to approximately 60 hours after the population of cells enriched for Tregs or the second population of CD45+cells.26IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0058] In some embodiments, the third population of CD45+cells (as disclosed herein) is administered from approximately 12 hours to approximately 120 hours after administration of the first population of isolated CD45+cells (as disclosed herein). In some embodiments, the third population of CD45+cells is administered approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately, 24 hours, approximately 25 hours, approximately 26 hours, approximately 27 hours, approximately 28 hours, approximately 29 hours, approximately 30 hours, approximately 31 hours, approximately 32 hours, approximately 33 hours, approximately, 34 hours, approximately 35 hours, approximately 36 hours, approximately 37 hours, approximately 38 hours, approximately 39 hours, approximately 40 hours, approximately 41 hours, approximately 42 hours, approximately 43 hours, approximately, 44 hours, approximately 45 hours, approximately 46 hours, approximately 47 hours, approximately 48 hours, approximately 49 hours, approximately 50 hours, approximately 51 hours, approximately 52 hours, approximately 53 hours, approximately, 54 hours, approximately 55 hours, approximately 56 hours, approximately 57 hours, approximately 58 hours, approximately 59 hours, approximately 60 hours, approximately 61 hours, approximately 62 hours, approximately 63 hours, approximately, 64 hours, approximately 65 hours, approximately 66 hours, approximately 67 hours, approximately 68 hours, approximately 69 hours, approximately 70 hours, approximately 71 hours, approximately 72 hours, approximately 73 hours, approximately, 74 hours, approximately 75 hours, approximately 76 hours, approximately 77 hours, approximately 78 hours, approximately 79 hours, approximately 80 hours, approximately 81 hours, approximately 82 hours, approximately 83 hours, approximately, 84 hours, approximately 85 hours, approximately 86 hours, approximately 87 hours, approximately 88 hours, approximately 89 hours, approximately 90 hours, approximately 91 hours, approximately 92 hours, approximately 93 hours, approximately, 94 hours, approximately 95 hours, approximately 96 hours, approximately 97 hours, approximately 98 hours, approximately 99 hours, approximately 100 hours, approximately 101 hours, approximately 102 hours, approximately 103 hours, approximately, 104 hours, approximately 105 hours, approximately 106 hours, approximately 107 hours, approximately 108 hours, approximately 109 hours, approximately 110 hours, approximately 111 hours, approximately 112 hours, approximately 113 hours, approximately, 114 hours, approximately 115 hours, approximately 116 hours, approximately 117 hours, approximately 118 hours, approximately 119 hours, or approximately 120 hours after administration of the first population of CD45+cells.

[0059] In some embodiments, the third population of CD45+cells (as disclosed herein) is administered from approximately 12 hours to approximately 120 hours after administration of the population of cells enriched for Tregs or the second population of CD45+cells (as disclosed herein). In some embodiments, the third population of CD45+cells is administered approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately, 24 hours, approximately 25 hours,27IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 26 hours, approximately 27 hours, approximately 28 hours, approximately 29 hours, approximately 30 hours, approximately 31 hours, approximately 32 hours, approximately 33 hours, approximately, 34 hours, approximately 35 hours, approximately 36 hours, approximately 37 hours, approximately 38 hours, approximately 39 hours, approximately 40 hours, approximately 41 hours, approximately 42 hours, approximately 43 hours, approximately, 44 hours, approximately 45 hours, approximately 46 hours, approximately 47 hours, approximately 48 hours, approximately 49 hours, approximately 50 hours, approximately 51 hours, approximately 52 hours, approximately 53 hours, approximately, 54 hours, approximately 55 hours, approximately 56 hours, approximately 57 hours, approximately 58 hours, approximately 59 hours, approximately 60 hours, approximately 61 hours, approximately 62 hours, approximately 63 hours, approximately, 64 hours, approximately 65 hours, approximately 66 hours, approximately 67 hours, approximately 68 hours, approximately 69 hours, approximately 70 hours, approximately 71 hours, approximately 72 hours, approximately 73 hours, approximately, 74 hours, approximately 75 hours, approximately 76 hours, approximately 77 hours, approximately 78 hours, approximately 79 hours, approximately 80 hours, approximately 81 hours, approximately 82 hours, approximately 83 hours, approximately, 84 hours, approximately 85 hours, approximately 86 hours, approximately 87 hours, approximately 88 hours, approximately 89 hours, approximately 90 hours, approximately 91 hours, approximately 92 hours, approximately 93 hours, approximately, 94 hours, approximately 95 hours, approximately 96 hours, approximately 97 hours, approximately 98 hours, approximately 99 hours, approximately 100 hours, approximately 101 hours, approximately 102 hours, approximately 103 hours, approximately, 104 hours, approximately 105 hours, approximately 106 hours, approximately 107 hours, approximately 108 hours, approximately 109 hours, approximately 110 hours, approximately 111 hours, approximately 112 hours, approximately 113 hours, approximately, 114 hours, approximately 115 hours, approximately 116 hours, approximately 117 hours, approximately 118 hours, approximately 119 hours, or approximately 120 hours after administration of the population of cells enriched for Tregs or the second population of CD45+cells.

[0060] HSPCs can have extensive self-renewal capacity, and an ability to dil'lcrcnlialc into specialized cell types, for example, an ability to reconstitute all hematopoietic cell lineages. HSPCs can undergo asynchronous replication, where two daughter cells are produced with different phenotypes. HSPCs cells can exist in a mitotically quiescent form. HSPCs can be derived from bone marrow, peripheral blood, and / or umbilical cord blood.

[0061] Subsets of immune cells, such as conventional T cells (Tcons), regulatory T cells (Tregs), invariant natural killer T cells (iNKTs), and memory T cells (Tmems) can contribute to aspects of GVHD following HCT, and can also contribute to, for example, GVT immune responses, immune reconstitution, infection susceptibility, and padent survival.

[0062] GVHD can be mediated in large part by donor T cells, which can elicit inflammatory responses upon recognition of recipient antigens. T cell depletion (TCD) of cell populations for transplantation to a subject can be undertaken to decrease the likelihood of acute and / or chronic GVHD. T cells can be depleted using methods including, but not limited to, physical adsorption of T cells to protein28IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT ligands such as lectins, immunodepletion with T cell specific antibodies, and immunoaffinity techniques (for example, use of T cell or lymphocyte-specific antibodies in immunoadsorption columns, magnetic activated cell sorting (MACS), or fluorescent activated cell sorting (FACS)). Applying TCD techniques to donor grafts can result in, for example, 10-fold to 105-fold depletion of T cells, and reduced incidence of GVHD. However, TCD can also result in increased incidence of cancer relapse, as the lack of T cells can reduce a graft-versus-tumor (GVT) immune response. Additionally, TCD can result in impaired immune recovery, and increased susceptibility to infections.

[0063] Both GVT and GVHD can be largely mediated by conventional T cells (Tcons), which mount immune responses upon recognition of cognate antigen by T cell receptors (tumor antigens for GVT, nontumor recipient antigens for GVHD). Tcons can, for example, contribute to GVT, GVHD, or a combination thereof. In some embodiments, administration of Tcons after administration of Tregs can enhance GVT immunity, and / or reduce susceptibility to infection.

[0064] Tcons can broadly refer to all CD3+T cells, cells expressing CD3 and CD4 or cells expressing CD3 and CD8, cells expressing medium to high levels of CD127, cells expressing CD3 and medium to high levels of CD127, cells expressing CD3, cells expressing medium to high levels of CD127, and cells expressing CD4 or CD8. In some embodiments, Tcons do not express Va24Jal8 TCR. Tcons and Regulatory T cells (“Tregs”) can be non-mutually-exclusive cell populations. In some embodiments, Tcons and Tregs are mutually exclusive cell populations.

[0065] Regulatory T cells (“Tregs”) are a specialized subpopulation of T cells that negatively regulate (e.g., suppress) activation of the immune system and thereby promote immune tolerance. Without wishing to be bound by theory, cell populations of the disclosure enriched for Tregs contribute to positive clinical outcomes by, for example, reducing the incidence and / or severity of GVHD in a transplant recipient subject, and / or improving immune reconstitution in a transplant recipient. Administering cell population enriched for Tregs with a population of CD45+cells that comprises, at least, HSPCs can, for example, facilitate retention of graft versus tumor (GVT) and reduced incidence and / or severity of GVHD. Without wishing to be bound by theory, administering population of cells enriched for Tregs can prevent GVHD, and administering third population of CD45+cells that comprises, at least, Tcons can promote GVT effects, for example, relative to alternate hematopoietic stem cell transplantation (HCT) methods, i.e., methods that are distinct from the compositions, multi-component pharmaceutical treatments, multi-component cellular therapy products, cell populations, solutions, formulations, kits, and / or methods disclosed herein. In some embodiments, administering a population of cells enriched for Tregs reduces the risk of developing GVHD, and administering third population of CD45+cells that comprises, at least, Tcons promotes GVT effects relative to alternate HCT methods, i.e., methods that are distinct from the compositions, multi-component pharmaceutical treatments, multi-component cellular therapy products, cell populations, solutions, formulations, kits, and / or methods disclosed herein.

[0066] As used herein, an alternate composition lacks one or more cell populations and / or prophylactic agent that are disclosed herein and / or recited in the claims. As examples, an alternate composition lacks one or more of a cell population comprising HSPCs, a cell population comprising Tregs,29IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT a cell population comprising Tcons, and a prophylactic agent. In some embodiments, an alternate composition or treatment regimen comprises an additional cell population or agent compared to a composition or treatment regimen of the disclosure, e.g., a an additional or different GVHD prophylactic agent.

[0067] There are a number of subsets of Tregs, for example, TCRa+CD4+regulatory T cells, which include natural regulatory T cells (nTregs) and induced regulatory T cells (iTregs). nTregs can be T cells produced in the thymus and delivered to the periphery as a long-lived lineage of self-antigen-specific lymphocytes. iTregs can be recruited from circulating lymphocytes and acquire regulatory properties under particular conditions of stimulation in the periphery. nTregs and iTregs are CD4+CD25+; both can inhibit proliferation of CD4+CD25- T cells in a dose-dependent manner. In some embodiments, Tregs are anergic and do not proliferate upon TCR stimulation. In addition to being positive for CD4 and CD25, Tregs can be positive for the transcription factor FOXP3, an intracellular marker. Tregs can be identified or selected based on various marker expression profiles. Non-limiting examples of marker expression profiles that can be used to select Tregs include (1) CD4+CD25+CD127dim, (2) CD4+FOXP3+, (3) CD3+CD4+CD25+, (4) CD3+CD4+CD25+CD127dim, (5) CD3+CD4+CD25+CD127dimFOXP3+, (6) CD3+FOXP3+, (7) CD3+CD4+FOXP3+, (8) CD3+CD4+CD25+FOXP3+, (9) CD3+CD25+FOXP3+, (10) CD3+CD25+CD127dim, (11) CD4+CD25+, (12) CD4+CD25+CD127dimFOXP3+, (13) FOXP3+, (14) CD4+FOXP3+, (15) CD4+CD25+FOXP3+, (16) CD25+FOXP3+, and (17) CD25+CD127dim.

[0068] Selection based on certain expression profiles can be achieved based on extracellular markers and without requiring cell permeabilization, for example, selection based on CD4+CD25+CD127dlm.

[0069] A cell population that comprises Tregs can, for example, reduce the incidence of graft rejection, reduce the incidence and / or severity of GVHD, promote hematopoietic reconstitution, promote immune reconstitution, promote mixed chimerism, or a combination thereof.

[0070] A cell population of the disclosure can comprise invariant natural killer T cells (iNKTs). iNKTs are subclass of CD Id-restricted Natural Killer T (NKT) cells that express a highly conserved af>-T cell receptor that comprises of Va24Jal8 TCRa chain in humans (referred to herein as “Va24Jal8+”). iNKT cells can be identified by binding with CDld-multimers like that are loaded with a-galactosylceramide (GalCer), PBS-57, PBS-44 or other natural or synthetic glycolipids. Another method of identification is an antibody or combination of antibodies that specifically recognize the Va24Jal8 region. Examples include a Va24 antibody, a Jal 8 antibody, or the monoclonal antibody clone 6B 11 which binds specifically to a unique region of the Va24Jal8 TCR and can be used to identify iNKT cells. iNKTs can be CD3+Va24Jal8+.

[0071] In some embodiments, iNKTs can promote engraftment, promote GVT, reduce incidence and / or severity of GVHD, decrease susceptibility to cancer relapse, decrease susceptibility to infection, or a combination thereof. In some embodiments, iNKTs promote the activity of Tregs. In some embodiments, iNKTs promote the activity of HSPCs.

[0072] A cell population of the disclosure can comprise memory T cells (Tmems). Tmems can refer to antigen-experienced T cells that express, for example, the phenotypic markers CD45RO, TCRa, TCR ,30IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT CD3, CD4, CD95, and IL-2R or the phenotypic markers CD45RO, TCRa, TCR , CD3, CD8, CD95, and IL-2R . Tmems provide immunity and are capable of persisting for a long period of time in an inactive state. Tmems are able to rapidly acquire effector functions upon re-challenge with antigen. A population of Tmems can include any combination of the subclasses T central memory cells and T effector memory cells. In some embodiments, Tmems are CD3+CD45RA CD45RO+. In various methods, Tmems administered to a subject receiving HCT can, for example, promote GVT, reduce GVHD, decrease susceptibility to cancer relapse, decrease susceptibility to infection, or a combination thereof.Acquisition, processing, and preparation of cells

[0073] Certain aspects of the present disclosure relate to methods for preparing the compositions, multi-component pharmaceutical treatments, multi-component cellular therapy products, cell populations, solutions, formulations, and / or kits of the present disclosure.

[0074] In some embodiments, at least one mobilized peripheral blood donation is collected from a donor or at most two mobilized peripheral blood donations are collected from the donor. In some embodiments, the mobilized peripheral blood donation is an HSPC-mobilized peripheral blood apheresis donation. In some embodiments, prior to peripheral blood donation, the donor may be vaccinated with a tumor antigen and / or a pathogen to enhance graft versus infection.

[0075] In embodiments, at least one of the mobilized peripheral blood donations is processed and sorted to enrich CD34+cells and Tregs. In some embodiments, the peripheral blood donalion is further processed and sorted or alternatively processed and sorted to enrich CD3+cells (e.g., Tcons). In embodiments, at least one of the mobilized peripheral blood donations is processed and sorted to enrich CD34+cells, Tregs, and / or Tcons. The processing and sorting for the CD34+cells, Tregs, and Tcons maybe done in any odder. For example, at least one of the mobilized peripheral blood donations may be processed and sorted to first enrich CD34+cells, then Tcons, and then Tregs. Alternatively, at least one of the mobilized peripheral blood donations may be processed and sorted to first enrich CD34+cells, then Tregs, and then Tcons. Alternatively, at least one of the mobilized peripheral blood donations may be processed and sorted to first enrich Tregs, then CD34+cells, and then Tcons. Alternatively, at least one of the mobilized peripheral blood donations may be processed and sorted to first enrich Tregs, then Tcons, and then CD34+cells. Allernali vely, at least one of the mobilized peripheral blood donalions may be processed and sorted to first enrich Tcons, then CD34+cells, and then Tregs. Alternatively, at least one of the mobilized peripheral blood donations may be processed and sorted to first enrich Tcons, then Tregs, and then CD34+cells. In some embodiments, at least one of the mobilized peripheral blood donations may be processed and sorted to first enrich Tregs and then CD34+cells, without enrichment for Tcons. In other embodiments, at least one of the mobilized peripheral blood donations may be processed and sorted to first enrich CD34+cells and then Tregs without enrichment for Tcons.

[0076] In some embodiments, the processing and sorting time of the one or more of the mobilized peripheral blood donations is less than about 40 hours, the processing and sorting time of the one or more of the mobilized peripheral blood donations is less than about 35 hours, the processing and sorting time of the one or more of the mobilized peripheral blood donations is less than about 30 hours, the processing and 31IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT sorting time of the one or more of the mobilized peripheral blood donations is less than about 25 hours, the processing and sorting rime of the one or more of the mobilized peripheral blood donations is less than about 20 hours, the processing and sorting time of the one or more of the mobilized peripheral blood donations is less than about 15 hours, and / or the processing and sorting time of the one or more of the mobilized peripheral blood donations is at most about 35 hours, the processing and sorting time of the one or more of the mobilized peripheral blood donations is at most about 30 hours, the processing and sorting time of the one or more of the mobilized peripheral blood donations is at most about 25 hours, the processing and sorting time of the one or more of the mobilized peripheral blood donations is at most about 20 hours, or the processing and sorting time of the one or more of the mobilized peripheral blood donations is at most about 15 hours.

[0077] In various embodiments, the one or more of the mobilized peripheral blood donations is processed and sorted using one or more immune-separation particles (ISPs), e.g., ISPs comprise affinity reagents such as immuno-magnetic separation particles which may be antibodies each conjugated to an iron-containing particle, and / or immuno-fluorescent separation particles which may be antibodies each conjugated to a fluorophore or other fluorescent particle.

[0078] In some embodiments, the affinity reagents comprise a plurality of CD34-reagents (e.g., an anti-CD34 antibody) that binds to one or more CD34 receptors on a HSPC. In some cases, at least a portion of the plurality of ISPs are attached to CD34+receptors on the HPSC’s of the HSPC cell population; optionally, an average number of ISPs per HSPC in the HSPC cell population is less than about 20,000, an average number of ISPs per HSPC in the HSPC cell population is equal to or less than about 10,000, and / or an average number of ISPs per HSPC in the HSPC cell population is from approximately 1000 to approximately 20,000.

[0079] In some embodiments, an average number of ISPs per HSPC in the HSPC cell population may be about 1,500 to approximately 20,000. In some embodiments, an average number of ISPs per HSPC in the HSPC cell population may be at least about 1,500. In some embodiments, an average number of ISPs per HSPC in the HSPC cell population may be at most about 20,000. In some embodiments, an average number of ISPs per HSPC in the HSPC cell population may be about 1,500 to approximately 2,000, about 1,500 to approximately 5,000, about 1,500 to approximately 6,000, about 1,500 to approximately 10,000, about 1,500 to approximately 12,000, about 1,500 to approximately 15,000, about 1,500 to approximately 20,000, about 2,000 to approximately 5,000, about 2,000 to approximately 6,000, about 2,000 to approximately 10,000, about 2,000 to approximately 12,000, about 2,000 to approximately 15,000, about 2,000 to approximately 20,000, about 5,000 to approximately 6,000, about 5,000 to approximately 10,000, about 5,000 to approximately 12,000, about 5,000 to approximately 15,000, about 5,000 to approximately 20,000, about 6,000 to approximately 10,000, about 6,000 to approximately 12,000, about 6,000 to approximately 15,000, about 6,000 to approximately 20,000, about 10,000 to approximately 12,000, about 10,000 to approximately 15,000, about 10,000 to approximately 20,000, about 12,000 to approximately 15,000, about 12,000 to approximately 20,000, or about 15,000 to approximately 20,000. In some embodiments, an average number of ISPs per HSPC in the HSPC cell population may be about 1,500,32IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT about 2,000, about 5,000, about 6,000, about 10,000, about 12,000, about 15,000, or about 20,000. In some embodiments, an average number of ISPs per HSPC in the HSPC cell population may be at least 1,500, 2,000, 5,000, 6,000, 10,000, 12,000, 15,000, or 20,000. In some embodiments, an average number of ISPs per HSPC in the HSPC cell population may be at most 1,500, 2,000, 5,000, 6,000, 10,000, 12,000, 15,000, or 20,000.

[0080] In some embodiments, the affinity reagents comprise a plurality of CD25-reagents (e.g., an anti-CD25 antibody) that binds to one or more CD25 receptors on a cell. In some cases, at least a portion of the plurality of ISPs are attached to CD25+receptors on the cells of the Treg cell population; optionally, an average number of ISPs per T-reg cell in the Treg population is equal or less than about 4000 or an average number of ISPs per T-reg cell in the Treg population is from approximately 1500 to approximately 2500. In some cases, at least a portion of the plurality of ISPs are attached to CD3+receptors on the cells of the heterogenous cell population; optionally, an average number of ISPs per cell in population of T heterogenous is less than about 4,000. In some embodiments, an average number of ISPs per Tcon cell in the Tcon cell population may be about 100 to approximately 1,000. In some embodiments, an average number of ISPs per Tcon cell in the Tcon cell population may be at least about 100. In some embodiments, an average number of ISPs per Tcon cell in the Tcon cell population may be at most about 1,000. In some embodiments, an average number of ISPs per Tcon cell in the Tcon cell population may be about 100 to approximately 200, about 100 to approximately 500, about 100 to approximately 1,000, about 200 to approximately 500, about 200 to approximately 1,000, or about 500 to approximately 1,000. In some embodiments, an average number of ISPs per Tcon cell in the Tcon cell population may be about 100, about 200, about 500, or about 1,000. In some embodiments, an average number of ISPs per Tcon cell in the Tcon cell population may be at least 100, 200, 500, or 1,000. In some embodiments, an average number of ISPs per Tcon cell in the Tcon cell population may be at most 100, 200, 500, or 1,000.

[0081] In some embodiments, an average number of ISPs per Treg cells in the Treg cell population may be about 500 to approximately 4,000. In some embodiments, an average number of ISPs per Treg cells in the Treg cell population may be at least about 500. In some embodiments, an average number of ISPs per Treg cells in the Treg cell population may be at most about 4,000. In some embodiments, an average number of ISPs per Treg cells in the Treg cell population may be about 500 to approximately 1,000, about 500 to approximately 1,500, about 500 to approximately 2,000, about 500 to approximately 2,500, about 500 to approximately 3,000, about 500 to approximately 4,000, about 1,000 to approximately 1,500, about 1,000 to approximately 2,000, about 1,000 to approximately 2,500, about 1,000 to approximately 3,000, about 1,000 to approximately 4,000, about 1,500 to approximately 2,000, about 1,500 to approximately 2,500, about 1,500 to approximately 3,000, about 1,500 to approximately 4,000, about 2,000 to approximately 2,500, about 2,000 to approximately 3,000, about 2,000 to approximately 4,000, about 2,500 to approximately 3,000, about 2,500 to approximately 4,000, or about 3,000 to approximately 4,000. In some embodiments, an average number of ISPs per Treg cells in the Treg cell population may be about 500, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, or about 4,000. In some embodiments, an average number of ISPs per Treg cells in the Treg cell population may be at least 500,33IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 1,000, 1,500, 2,000, 2,500, 3,000, or 4,000. In some embodiments, an average number of ISPs per Treg cells in the Treg cell population may be at most 500, 1,000, 1,500, 2,000, 2,500, 3,000, or 4,000.

[0082] In some embodiments, CD25-enriched cell populations are further processed by sorting with one or more ISPs comprising immuno-fluorescent separation particles (e.g., antibodies conjugated to fluorophores) specific for CD127 and CD4. In some embodiments, a CD25-enriched Treg population of the present disclosure is sorted using the CD 127- specific ISPs and CD4-specific ISPs to obtain a CD25+CD4+CD127dlmTreg population. In some embodiments, the Treg population is also FOXP3+. In some embodiments, a CD127dlmcell population may include a CD127" population and / or a CD127lowpopulation.

[0083] In some embodiments, the affinity reagents comprise a plurality of CD3-reagents (e.g., an anti-CD3 antibody) that binds to one or more CD3 receptors on a cell (e.g., a Tcon). In some cases, at least a portion of the plurality of ISPs are attached to CD3+receptors on the cells of the Tcon cell population; optionally, an average number of ISPs per T cell in the Tcon population is equal or less than about 4000 or an average number of ISPs per T cell in the Tcon population is from approximately 1500 to approximately 2500. In some cases, at least a portion of the plurality of ISPs are attached to CD3+receptors on the cells of the heterogenous cell population; optionally, an average number of ISPs per cell in population of T heterogenous is less than about 4,000.

[0084] In various embodiments, cells of the mobilized peripheral blood donation are sorted such that the first population of CD45+cells comprises at most about 10% granulocytes. In some cases, cells of the mobilized peripheral blood donation are sorted such that the first population of CD45+cells comprises at most about 7% granulocytes.

[0085] In some embodiments, cells of the mobilized donor peripheral blood donation are sorted such that the first population of CD45+cells comprises at most about 4% monocytes. In some cases, cells of the mobilized donor peripheral blood donation are sorted such that the first population of CD45+cells comprises at least about 0.1 % monocytes.

[0086] In embodiments, cells of the mobilized donor peripheral blood donation are sorted such that the population enriched for Tregs comprises at most about 10% CD25- cells.

[0087] In some embodiments, a cell population of the disclosure is obtained from whole blood. A cell population of the disclosure can be obtained from a peripheral blood apheresis product, for example, a mobilized peripheral blood apheresis product, e.g., mobilized by administration of GCSF, GM-CSF, MOZOBIL® (plerixafor), and combinations thereof, to a donor. A cell population of the disclosure can be obtained from at least one apheresis product, two apheresis products, three apheresis products, four apheresis products, five apheresis products, six apheresis products, or more. In some embodiments, a cell population of the disclosure is obtained from one apheresis product. In some embodiments, a cell population of the disclosure is obtained from two apheresis products. In some embodiments, a cell population of the disclosure is obtained from an apheresis product from one donor and an apheresis product from an at least second donor.34IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0088] In some embodiments, a cell population of the disclosure is obtained from bone marrow sample. In some embodiments, a cell population of the disclosure can be obtained from a bone marrow ample, for example, a mobilized bone marrow sample, e.g., mobilized by administration of GCSF, GM-CSF, MOZOBIL® (plerixafor), and combinations thereof, to a donor.

[0089] In some embodiments, a cell population of the disclosure is obtained from umbilical cord blood.

[0090] A cell population of the disclosure can be refined by selection from a population of cells, for example, peripheral blood or a peripheral blood apheresis product. Selection methods for cell populations can comprise methods involving positive or negative selection of a cell population of interest. Selection methods for cell populations can comprise affinity reagents, including but not limited to an antibody, a full-length antibody, a fragment of an antibody, a naturally occurring antibody, a synthetic antibody, an engineered antibody, a full-length affibody, a fragment of an affibody, a full-length affilin, a fragment of an affilin, a full-length anticalin, a fragment of an anticalin, a full-length avimer, a fragment of an avimer, a full-length DARPin, a fragment of a DARPin, a full-length fynomer, a fragment of a fynomer, a full-length kunitz domain peptide, a fragment of a kunitz domain peptide, a full-length monobody, a fragment of a monobody, a peptide, or a polyaminoacid. In some embodiments, the affinity reagent is directly conjugated to a detection reagent and / or purification reagent. In some cases, the detection reagent and purification reagent are the same. In some cases, the detection reagent and purification reagent are different. For example, the detection reagent and / or purification reagent is fluorescent, magnetic, or the like. In some cases, the detection reagent and / or purification reagent is a magnetic particle for column purification. For example, magnetic column purification may be performed using the Miltenyi system (CliniMACs) of columns, antibodies, buffers, preparation materials and reagents.

[0091] In various embodiments, at least one of the cell populations have a plurality of immunoseparation particles (ISPs) attached to receptors on the cells of the cell population. In some cases, the plurality of ISPs are immuno-magnetic separation particles. In some embodiments, the plurality of ISPs comprise an antibody conjugated to an iron containing particle. In some cases, at least a portion of the plurality of ISPs are attached to CD34+receptors on the HPSC’s of the HSPC cell population; optionally, an average number of ISPs per HSPC in the HSPC cell population is less than about 6,000, an average number of ISPs per HSPC in the HSPC cell population is equal to or less than about 3,000, and / or an average number of ISPs per HSPC in the HSPC cell population is from approximately 1700 to approximately 3,000. In some cases, at least a portion of the plurality of ISPs are attached to CD25+receptors on the cells of the Treg cell population; optionally, an average number of ISPs per T-reg cell in the Treg population is equal or less than about 1700 or an average number of ISPs per T-reg cell in the Treg population is from approximately 1400 to approximately 1700. In some cases, at least a portion of the plurality of ISPs are attached to CD3+receptors on the cells of the heterogenous cell population; optionally, an average number of ISPs per cell in population of T heterogenous is less than about 1,000.

[0092] Affinity reagents can comprise immunoaffinity reagents, utilizing the binding specificity of antibodies or fragments or derivatives thereof to positively or negatively select for a cell population of35IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT interest. Selection methods for cell populations can comprise an affinity agent and a column, such as magnetic activated cell sorting (MACS) with specific antibodies and microbeads. Selection methods for cell populations can comprise fluorescent activated cell sorting (FACS), with cell populations sorted based on staining profiles with one or more fluorescently-conjugated antibodies. Selection methods for cell populations can comprise physical adsorption, for example, physical adsorption of T cells to protein ligands such as lectins.

[0093] HSPCs can be obtained by harvesting from bone marrow or from peripheral blood. Bone marrow can be aspirated from the posterior iliac crest or the anterior iliac crest while the donor is under either local or general anesthesia. HSPCs can be obtained by harvesting from peripheral blood, for example, by peripheral blood apheresis. The number of stem cells harvested can be increased by treating the donor with a mobilization agent, i.e., an agent that mobilizes stem cells from the bone marrow into peripheral blood. Non-limiting examples of mobilization agents include granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), a SDF-1 antagonist, a CXCR4 antagonist (e.g., POL6326, BKT-140, TG-0054, NOX-A12), MOZOBIL® (plerixafor), a CXCR2 ligand (e.g., GROP), a sphingosine- 1 -phosphatase (SIP) agonist, (e.g., SEW2871), a VCAM / VLA-4 inhibitor (e.g., BIO5192), a proteosome inhibitor e.g., Bortezomib), parathyroid hormone, a hypoxia inducible factor (HIF) stabilizer (e.g., FG-4497), and combinations thereof. Techniques to mobilize stem cells into peripheral blood can comprise administering to a donor, for example, 10 to 40 p / kg / day of a mobilization agent. A mobilization agent can be administered to the donor in, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. An apheresis product can be isolated from a donor about, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, or 30 hour(s) after a dose of mobilization agent.

[0094] A population of CD45+cells of the disclosure can comprise a HSPCs. The HSPCs can be selected based on expression of CD34. For example, the HSPCs of the disclosure can be selected using anti-CD34 antibodies as part of a magnetic activated cell sorting (MACS) or fluorescent activated cell sorting (FACS) system.

[0095] The number of HPSCs in a population of CD45+cells can be determined, for example, by quantifying CD34+cells via flow cytometry. In some embodiments, dose calculations are adjusted based on measures of cell viability measurements, e.g., viability determined via flow cytometry with propidium iodide or 7-AAD, or via trypan blue exclusion.

[0096] A cell population of the disclosure can be enriched for Tregs (e.g., the second population of CD45+cells). Tregs can be selected based on expression of markers including CD3, CD4, CD25, CD127, FOXP3, and combinations thereof.

[0097] Tregs can be selected using magnetic activated cell sorting (MACS). Tregs can be selected using fluorescent activated cell sorting (FACS). Tregs can be selected using multiple procedures, for example, multiple MACS selections, multiple FACS selections, or a combination of MACS and FACS selections. For example, a first selection may be performed for expression of CD25, isolating CD25+cells from a hematopoietic cell sample, for example with MACS. A second selection may be performed by36IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT contacting the CD25+cells with antibodies specific for CD4 and for CD127, where FACS is used to isolate cells that are CD4+CD127dim.

[0098] Tregs can be isolated from whole blood. Tregs can be isolated from a peripheral blood apheresis product. Tregs can be isolated from a population of cells previously enriched and / or depleted for one or more other cell types, e.g., isolated from a population of cells depleted of CD34+cells. In some embodiments, Tregs are isolated from the flow-through fraction of a CD34+MACS selection.

[0099] The number of Tregs in a population of cells can be determined, for example, by flow cytometry, where Tregs can be identified as, for example, CD4+CD25+CD127dlmor CD4+FOXP3+. Dose calculations can be adjusted based on measures of cell viability measurements, e.g., viability determined via flow cytometry with propidium iodide or 7-AAD, or via trypan blue exclusion.

[0100] In some embodiments, the third population of CD45+cells can comprise a population of Tcons. The third population of CD45+cells that comprises, at least, Tcons can be sourced from peripheral blood. The third population of CD45+cells can be sourced from a peripheral blood apheresis product.

[0101] In some embodiments, no selection steps are carried out, and a population of CD45+cells that comprises, at least, Tcons is sourced directly from an aliquot of peripheral blood or apheresis product. In some embodiments, a population of cells can be enriched for Tcons, for example, by sorting based on the expression of various markers using MACS, FACS, or a combination thereof. In some embodiments, a third population of CD45+cells can be enriched by sorting for CD3+cells. In some embodiments, a third population of CD45+cells can be enriched by sorting for CD4+and CD8+cells. In some embodiments, a third population of CD45+cells can be enriched by negative selection, where non-Tcon cells are removed, for example, by MACS depletion of cells expressing CD34, CD19, CD25, or a combination thereof.

[0102] The number of Tcons present in a third population of CD45+cells can be quantified, for example, by quantifying CD3+cells via flow cytometry. The number of CD3+cells in an aliquot can be determined and a volume comprising an appropriate dose of CD3 cells administered to the recipient. Dose calculations can be adjusted based on measures of cell viability, e.g., viability determined via flow cytometry with propidium iodide or 7-AAD, or via trypan blue exclusion.

[0103] An apheresis product of the disclosure can be split into two portions, one portion used to provide the third population of CD45+cells that comprises, at least, Tcons and the other portion to isolate and purify the population of CD45+cells that comprises, at least, HSPCs, and the cell population enriched for Tregs. In alternate embodiments, CD34+cells are isolated and purified from the apheresis product, creating a CD34-negative cell fraction from which the cell Treg are then isolated to help provide the cell population enriched for Tregs.

[0104] A cell population of the disclosure can comprise a population of iNKTs. A population of iNKTs can be sourced from peripheral blood. A population of iNKTs can be sourced from a peripheral blood apheresis product.

[0105] A population of cells can be enriched for iNKTs, for example, by sorting based on the expression of various markers using MACS, FACS, or a combination thereof. A population of iNKTs can be enriched, for example, by sorting for CD3+Va24Jal8+cells.37IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0106] The number of iNKTs present in a population can be quantified, for example, by quantifying CD3+Va24Jal8+cells via flow cytometry. The number of CD3+Va24Jal8+cells in an aliquot can be determined and a volume comprising an appropriate dose of iNKTs administered to the recipient. In some embodiments, dose calculations are adjusted based on measures of cell viability measurements, e.g., viability determined via flow cytometry with propidium iodide or 7-AAD, or via trypan blue exclusion.

[0107] A cell population of the disclosure can comprise a population of Tmems. A population of Tmems can be sourced from peripheral blood. A population of Tmems can be sourced from a peripheral blood apheresis product.

[0108] A population of cells can be enriched for Tmems, for example, by sorting based on the expression of various markers using MACS, FACS, or a combination thereof. A population of Tmems can be enriched, for example, by sorting for CD3+CD45RA-CD45RO+cells.

[0109] The number of Tmems present in a population can be quantified, for example, by quantifying CD3+CD45RA-CD45RO+cells via flow cytometry. The number of CD3+CD45RA-CD45RO+cells in an aliquot can be determined and a volume comprising an appropriate dose of Tmems administered to the recipient. Dose calculations can be adjusted based on measures of cell viability measurements, e.g., viability determined via flow cytometry with propidium iodide or 7-AAD, or via trypan blue exclusion.

[0110] A cell population of the disclosure or a cell population of the disclosure can be administered freshly after isolation, or after cryopreservation and subsequent thawing.

[0111] Cells freshly isolated from a donor (“fresh cells”) can be administered to a recipient subject. Fresh cells can be stored in a buffer, for example, CliniMACs PBS-EDTA Buffer with 0.5% human serum albumin, or Plasma-Lyte-A, pH 7.4 supplemented with 2% human serum albumin. Fresh cells can be stored at a reduced temperature (e.g., 2-8 °C), and without being cryopreserved / frozen.

[0112] After acquiring a fresh population of cells from a donor, the fresh cells can be stored for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 37 hours, at least about 38 hours, at least about 39 hours, at least about 40 hours, at least about 44 hours, at least about 48 hours, at least about 50 hours, at least about 55 hours, at least about 60 hours, at least about 61 hours, at least about 62 hours, at least about 65 hours, at least about 70 hours, at least about 72 hours, at least about 80 hours, at least about 90 hours, at least about 96 hours, at least about 120 hours, at least about 150 hours, at least about 200 hours, at least about 300 hours, or more prior to administration to a subject.38IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0113] After acquiring a fresh population of cells from a donor, the fresh cells can be stored for at most about 1 hour, at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 6 hours, at most about 7 hours, at most about 8 hours, at most about 9 hours, at most about 10 hours, at most about 12 hours, at most about 14 hours, at most about 16 hours, at most about 18 hours, at most about 20, at most 22 hours, at most about 24 hours, at most about 30 hours, at most about 36 hours, at most about 40 hours, at most about 48 hours, at most about 60 hours, at most about 70 hours, at most about 72 hours, at most about 80 hours, at most about 90 hours, at most about 96 hours, at most about 120 hours, at most about 150 hours, at most about 200 hours, or at most about 300 hours prior to administration to a subject.

[0114] In some embodiments, after processing, one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells, may be formulated with one or more excipients and / or cryoprotectants. In some embodiments, the one or more excipients comprise buffers, such as transport or infusion buffers. In some embodiments, the cell populations are formulated at a neutral pH. In some embodiments, the HSPCs of the present disclosure are formulated with one or more excipients at a neutral pH. In some embodiments, the Tregs of the present disclosure are formulated with one or more excipients at a neutral pH. In some embodiments, the Tcons of the present disclosure are formulated with one or more excipients at a neutral pH. In some embodiments, the HSPCs and Tregs of the present disclosure are formulated together with one or more excipients at a neutral pH. In some embodiments, the HSPCs and Tcons of the present disclosure are formulated together with one or more excipients at a neutral pH. In some embodiments, the Tregs and Tcons of the present disclosure are formulated together with one or more excipients at a neutral pH. In some embodiments, the HSPCs, Tregs, and Tcons of the present disclosure are formulated together with one or more excipients at a neutral pH. In some embodiments, the one or more excipients comprise one or more transport buffers.

[0115] In some embodiments, a neutral pH ranges from approximately 6.8 to approximately 7.6. In some embodiments, a neutral pH is approximately 6.8, approximately 6.85, approximately 6.9, approximately 6.95, approximately 7, approximately 7.05, approximately 7.1, approximately 7.15, approximately 7.2, approximately 7.25, approximately 7.3, approximately 7.35, approximately 7.4, approximately 7.45, approximately 7.5, approximately 7.55, or approximately 7.6.

[0116] In some embodiments, one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells, may be formulated in one or more excipients. In some embodiments, the one or more excipients may comprise one or more buffers. The buffers may be transport buffers or infusion buffers. Transport buffers suitable for use with the cell populations of the present disclosure include buffers that have a milliequivalent (mEq) of sodium that ranges from approximately 120 mEq sodium to approximately 160 mEq sodium. In some embodiments, a transport buffer of the present disclosure comprises approximately 120 mEq sodium, approximately 125 mEq sodium, approximately 130 mEq sodium, approximately 135 mEq sodium, approximately 140 mEq sodium, approximately 145 mEq sodium, approximately 120 mEq39IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT sodium, approximately 150 mEq sodium, approximately 155 mEq sodium, or approximately 160 mEq sodium.

[0117] Transport buffers suitable for use with the cell populations of the present disclosure include buffers that have an osmotic concentration that ranges from approximately 270 mOsmol / L to approximately 320 mOsmol / L. In some embodiments, a transport buffer of the present disclosure have an osmotic concentration that is approximately 270 mOsmol / L, approximately 275 mOsmol / L, approximately 280 mOsmol / L, approximately 285 mOsmol / L, approximately 290 mOsmol / L, approximately 295 mOsmol / L, approximately 300 mOsmol / L, approximately 305 mOsmol / L, approximately 310 mOsmol / L, approximately 315 mOsmol / L, or approximately 320 mOsmol / L.

[0118] In some embodiments, transport buffers suitable for use with one or more cell populations of the present disclosure e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells, include, without I imitation , phosphate-buffered saline (PBS), human serum, PlasmaLyte, and any combination thereof. In some embodiments, one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells are formulated with a transport buffer. In some embodiments, one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells are formulated with a sufficient amount of PBS. In some embodiments, one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells are formulated with a sufficient amount of human serum. In some embodiments, one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells are formulated with a sufficient amount of PlasmaLyte (e.g., PlasmaLyte A).

[0119] In some embodiments, a transport buffer of the present disclosure may further include a human carrier protein, including without limitation, human serum albumin (HSA), intravenous immune globulin (IVIG), AB serum, or any combination thereof. In some embodiments, the human carrier protein has a concentration that ranges from approximately 0.1% weight by volume to approximately 10% weight by volume of the human carrier protein. In some embodiments, the human carrier protein has a concentration that is approximately 0.1% weight by volume, approximately 0.2% weight by volume, approximately 0.3% weight by volume, approximately 0.4% weight by volume, approximately 0.5% weight by volume, approximately 0.6% weight by volume, approximately 0.7% weight by volume, approximately 0.8% weight by volume, approximately 0.9 weight by volume, approximately 1.0% weight by volume, approximately 1.1% weight by volume, approximately 1.2% weight by volume, approximately 1.3% weight by volume, approximately 1.4% weight by volume, approximately 1.5% weight by volume, approximately 1.6% weight by volume, approximately 1.7% weight by volume, approximately 1.8% weight by volume, approximately 1.9% weight by volume, approximately 2.0% weight by volume, approximately 2.1% weight by volume, approximately 2.2% weight by volume, approximately 2.3% weight by volume, approximately 2.4% weight by volume, approximately 2.5% weight by volume,40IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 2.6% weight by volume, approximately 2.7% weight by volume, approximately 2.8% weight by volume, approximately 2.9% weight by volume, approximately 3.0% weight by volume, approximately 3.1% weight by volume, approximately 3.2% weight by volume, approximately 3.3% weight by volume, approximately 3.4% weight by volume, approximately 3.5% weight by volume, approximately 3.6% weight by volume, approximately 3.7% weight by volume, approximately 3.8% weight by volume, approximately 3.9% weight by volume, approximately 4.0% weight by volume, approximately 4.1% weight by volume, approximately 4.2% weight by volume, approximately 4.3% weight by volume, approximately 4.4% weight by volume, approximately 4.5% weight by volume, approximately 4.6% weight by volume, approximately 4.7% weight by volume, approximately 4.8% weight by volume, approximately 4.9% weight by volume, approximately 5.0% weight by volume, approximately 5.1% weight by volume, approximately 5.2% weight by volume, approximately 5.3% weight by volume, approximately 5.4% weight by volume, approximately 5.5% weight by volume, approximately 5.6% weight by volume, approximately 5.7% weight by volume, approximately 5.8% weight by volume, approximately 5.9% weight by volume, approximately 6.0% weight by volume, approximately 6.1% weight by volume, approximately 6.2% weight by volume, approximately 6.3% weight by volume, approximately 6.4% weight by volume, approximately 6.5% weight by volume, approximately 6.6% weight by volume, approximately 6.7% weight by volume, approximately 6.8% weight by volume, approximately 6.9% weight by volume, approximately 7.0% weight by volume, approximately 7.1% weight by volume, approximately 7.2% weight by volume, approximately 7.3% weight by volume, approximately 7.4% weight by volume, approximately 7.5% weight by volume, approximately 7.6% weight by volume, approximately 7.7% weight by volume, approximately 7.8% weight by volume, approximately 7.9% weight by volume, approximately 8.0% weight by volume, approximately 8.1% weight by volume, approximately 8.2% weight by volume, approximately 8.3% weight by volume, approximately 8.4% weight by volume, approximately 8.5% weight by volume, approximately 8.6% weight by volume, approximately 8.7% weight by volume, approximately 8.8% weight by volume, approximately 8.9% weight by volume, approximately 9.0% weight by volume, approximately 9.1% weight by volume, approximately 9.2% weight by volume, approximately 9.3% weight by volume, approximately 9.4% weight by volume, approximately 9.5% weight by volume, approximately 9.6% weight by volume, approximately 9.7% weight by volume, approximately 9.8% weight by volume, approximately 9.9% weight by volume, or approximately 10% weight by volume of the human carrier protein.

[0120] In some embodiments, one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells are formulated with an excipient, such as a transport buffer of the present disclosure, in an amount sufficient to yield a volume that ranges from approximately 5 mL to 1 L. In some embodiments, the first population of CD45+cells is formulated with an excipient, such as a transport buffer of the present disclosure, in an amount sufficient to yield a volume that ranges from approximately 5 mL to 1 L. In some embodiments, the second population of CD45+cells is formulated with an excipient, such as a transport41IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT buffer of the present disclosure, in an amount sufficient to yield a volume that ranges from approximately 5 mL to 1 L. In some embodiments, the third population of CD45+cells is formulated with an excipient, such as a transport buffer of the present disclosure, in an amount sufficient to yield a volume that ranges from approximately 5 mL to 1 L. In some embodiments, the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells is formulated (e.g., with one or more excipients, such as a transport buffer) at volume of approximately 5 mL, approximately lOmL, approximately 15 mL, approximately 20 mL, approximately 25 mL, approximately 30 mL, approximately 35 mL, approximately 40 mL, approximately 45 mL, approximately 50 mL, approximately 55 mL, approximately 60 mL, approximately 65 mL, approximately 70 mL, approximately 75 mL, approximately 80 mL, approximately 85 mL, approximately 90 mL, approximately 95 mL, approximately 100 mL, approximately 105 mL, approximately 110 mL, approximately 115 mL, approximately 120 mL, approximately 125 mL, approximately 130 mL, approximately 135 mL, approximately 140 mL, approximately 145 mL, approximately 150 mL, approximately 155 mL, approximately 160 mL, approximately 165 mL, approximately 170 mL, approximately 175 mL, approximately 180 mL, approximately 185 mL, approximately 190 mL, approximately 195 mL, approximately 200 mL, approximately 205 mL, approximately 210 mL, approximately 215 mL, approximately 220 mL, approximately 225 mL, approximately 230 mL, approximately 235 mL, approximately 240 mL, approximately 245 mL, approximately 250 mL, approximately 255 mL, approximately 260 mL, approximately 265 mL, approximately 270 mL, approximately 275 mL, approximately 280 mL, approximately 285 mL, approximately 290 mL, approximately 295 mL, approximately 300 mL, approximately 305 mL, approximately 310 mL, approximately 315 mL, approximately 320 mL, approximately 325 mL, approximately 330 mL, approximately 335 mL, approximately 340 mL, approximately 345 mL, approximately 350 mL, approximately 355 mL, approximately 360 mL, approximately 365 mL, approximately 370 mL, approximately 375 mL, approximately 380 mL, approximately 385 mL, approximately 390 mL, approximately 395 mL, approximately 400 mL, approximately 405 mL, approximately 410 mL, approximately 415 mL, approximately 420 mL, approximately 425 mL, approximately 430 mL, approximately 435 mL, approximately 440 mL, approximately 445 mL, approximately 450 mL, approximately 455 mL, approximately 460 mL, approximately 465 mL, approximately 470 mL, approximately 475 mL, approximately 480 mL, approximately 485 mL, approximately 490 mL, approximately 495 mL, approximately 500 mL, approximately 505 mL, approximately 510 mL, approximately 515 mL, approximately 520 mL, approximately 525 mL, approximately 530 mL, approximately 535 mL, approximately 540 mL, approximately 545 mL, approximately 550 mL, approximately 555 mL, approximately 560 mL, approximately 565 mL, approximately 570 mL, approximately 575 mL, approximately 580 mL, approximately 585 mL, approximately 590 mL, approximately 595 mL, approximately 600 mL, approximately 605 mL, approximately 610 mL, approximately 615 mL, approximately 620 mL, approximately 625 mL, approximately 630 mL, approximately 635 mL, approximately 640 mL, approximately 645 mL, approximately 650 mL, approximately 655 mL, approximately 660 mL,42IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 665 mL, approximately 670 mL, approximately 675 mL, approximately 680 mL, approximately 685 mL, approximately 690 mL, approximately 695 mL, approximately 700 mL, approximately 705 mL, approximately 710 mL, approximately 715 mL, approximately 720 mL, approximately 725 mL, approximately 730 mL, approximately 735 mL, approximately 740 mL, approximately 745 mL, approximately 750 mL, approximately 755 mL, approximately 760 mL, approximately 765 mL, approximately 770 mL, approximately 775 mL, approximately 780 mL, approximately 785 mL, approximately 790 mL, approximately 795 mL, approximately 800 mL, approximately 805 mL, approximately 810 mL, approximately 815 mL, approximately 820 mL, approximately 825 mL, approximately 830 mL, approximately 835 mL, approximately 840 mL, approximately 845 mL, approximately 850 mL, approximately 855 mL, approximately 860 mL, approximately 865 mL, approximately 870 mL, approximately 875 mL, approximately 880 mL, approximately 885 mL, approximately 890 mL, approximately 895 mL, approximately 900 mL, approximately 905 mL, approximately 910 mL, approximately 915 mL, approximately 920 mL, approximately 925 mL, approximately 930 mL, approximately 935 mL, approximately 940 mL, approximately 945 mL, approximately 950 mL, approximately 955 mL, approximately 960 mL, approximately 965 mL, approximately 970 mL, approximately 975 mL, approximately 980 mL, approximately 985 mL, approximately 990 mL, approximately 995 mL, or approximately 1 L. In some embodiments, a formulation of the present disclosure further comprises a human carrier protein of the present disclosure. In some embodiments, a formulation of the present disclosure further comprises one or more cryoprotectants.

[0121] Cells populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells, can be cryopreserved. In some embodiments, cryopreservation can be beneficial to the methods disclosed herein. For example, cryopreservation of the third population of CD45+cells that comprises, at least, Tcons prior to subsequent thawing and administering to a subject may reduce GVHD.

[0122] An additional aspect provides a method of iransplanling a conventional T cell (Tcon) population into a human subject without eliciting a stage 2 or higher graft versus host disease (GVHD) response up to approximately 100 days after transplanting. The method comprising: (i). administering a soludon comprising a population of conventional T cells (Tcons); and (ii). administering a solution comprising a population of regulatory T cells (Tregs). In this method, the population of Tcons is cryopreserved for at least about 4 hours; and the solution comprising the population of Tcons and the solution comprising the population of Tregs comprise less than about 5 EU of endotoxins per ml of the solution.

[0123] Cryopreservation can comprise addition of a preservative agent (e.g., DMSO) or one or more cryoprotectants, and gradual cooling of cells in a controlled-rate freezer to prevent osmotic cellular injury resulting from ice crystal formation. Cryopreservation can comprise commercial cryopreservation reagents and materials (e.g., cryoprotectants), for example, cryobags sorbitol, dimethyl sulfoxide (DMSO),43IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT propylene glycol, glycerol, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG), serum, HSA, hetastarch, CRYOSTOR CS2, CRYOSTOR CS5, and CRYOSTOR CS10, or any combination thereof.

[0124] In certain embodiments, a cryoprotectant of the present disclosure comprises DMSO. For example, in some embodiments the third population of CD45+cells may be formulated for cryopreservation with a cryoprotectant that comprises DMSO in an amount that ranges from approximately 1% volume by volume to approximately 15% volume by volume. In some embodiments, the cryoprotectant comprises DMSO in an amount that is approximately 1 % volume by volume, approximately 1.2% volume by volume, approximately 1.4% volume by volume, approximately 1.6% volume by volume, approximately 1.8% volume by volume, approximately 2% volume by volume, approximately 2.2% volume by volume, approximately 2.4% volume by volume, approximately 2.6% volume by volume, approximately 2.8% volume by volume, approximately 3% volume by volume, approximately 3.2% volume by volume, approximately 3.4% volume by volume, approximately 3.6% volume by volume, approximately 3.8% volume by volume, approximately 4% volume by volume, approximately 4.2% volume by volume, approximately 4.4% volume by volume, approximately 4.6% volume by volume, approximately 4.8% volume by volume, approximately 5% volume by volume, approximately 5.2% volume by volume, approximately 5.4% volume by volume, approximately 5.6% volume by volume, approximately 5.8% volume by volume, approximately 6% volume by volume, approximately 6.2% volume by volume, approximately 6.4% volume by volume, approximately 6.6% volume by volume, approximately 6.8% volume by volume, approximately 7% volume by volume, approximately 7.2% volume by volume, approximately 7.4% volume by volume, approximately 7.6% volume by volume, approximately 7.8% volume by volume, approximately 8% volume by volume, approximately 8.2% volume by volume, approximately 8.4% volume by volume, approximately 8.6% volume by volume, approximately 8.8% volume by volume, approximately 9% volume by volume, approximately 9.2% volume by volume, approximately 9.4% volume by volume, approximately 9.6% volume by volume, approximately 9.8% volume by volume, approximately 10% volume by volume, approximately 10.2% volume by volume, approximately 10.4% volume by volume, approximately 10.6% volume by volume, approximately 10.8% volume by volume, approximately 11% volume by volume, approximately 11.2% volume by volume, approximately 11.4% volume by volume, approximately 11.6% volume by volume, approximately 11.8% volume by volume, approximately 12% volume by volume, approximately 12.2% volume by volume, approximately 12.4% volume by volume, approximately 12.6% volume by volume, approximately 12.8% volume by volume, approximately 13% volume by volume, approximately 13.2% volume by volume, approximately 13.4% volume by volume, approximately 13.6% volume by volume, approximately 13.8% volume by volume, approximately 14% volume by volume, approximately 14.2% volume by volume, approximately 14.4% volume by volume, approximately 14.6% volume by volume, approximately 14.8% volume by volume, or approximately 15% volume by volume.

[0125] In some embodiments, the third population of CD45+cells may be formulated for cryopreservation with a cryoprotectant that comprises DMSO, and that further comprises hetastarch. In some embodiments, the cryoprotectant comprises hetastarch in an amount that ranges from approximately44IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 0.1% weight by volume to approximately 5 % weight by volume. In some embodiments, the cryoprotectant comprises hetastarch in an amount that is approximately 0.1% weight by volume, approximately 0.2% weight by volume, approximately 0.3% weight by volume, approximately 0.4% weight by volume, approximately 0.5% weight by volume, approximately 0.6% weight by volume, approximately 0.7% weight by volume, approximately 0.8% weight by volume, approximately 0.9 weight by volume, approximately 1.0% weight by volume, approximately 1.1% weight by volume, approximately 1.2% weight by volume, approximately 1.3% weight by volume, approximately 1.4% weight by volume, approximately 1.5% weight by volume, approximately 1.6% weight by volume, approximately 1.7% weight by volume, approximately 1.8% weight by volume, approximately 1.9% weight by volume, approximately 2.0% weight by volume, approximately 2.1% weight by volume, approximately 2.2% weight by volume, approximately 2.3% weight by volume, approximately 2.4% weight by volume, approximately 2.5% weight by volume, approximately 2.6% weight by volume, approximately 2.7% weight by volume, approximately 2.8% weight by volume, approximately 2.9% weight by volume, approximately 3.0% weight by volume, approximately 3.1% weight by volume, approximately 3.2% weight by volume, approximately 3.3% weight by volume, approximately 3.4% weight by volume, approximately 3.5% weight by volume, approximately 3.6% weight by volume, approximately 3.7% weight by volume, approximately 3.8% weight by volume, approximately 3.9% weight by volume, approximately 4.0% weight by volume, approximately 4.1% weight by volume, approximately 4.2% weight by volume, approximately 4.3% weight by volume, approximately 4.4% weight by volume, approximately 4.5% weight by volume, approximately 4.6% weight by volume, approximately 4.7% weight by volume, approximately 4.8% weight by volume, approximately 4.9% weight by volume, or approximately 5.0% weight by volume.

[0126] Cryopreserved cells can be stored for periods of time ranging from hours to years at low temperatures. Cryopreserved cells can be stored at ultralow temperatures, for example, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -100 °C, -110 °C, -120 °C, -130 °C, -140 °C, -150 °C, -160 °C, -170 °C, -180 °C, -190 °C, -196 °C, or less. Cryopreserved cells can be stored in storage devices comprising liquid nitrogen.

[0127] Cells can be cryopreserved before or after certain steps in the methods of the disclosure, for example, before or after sorting steps, before or after characlcrizalion steps, such as determining cell viability or the concentration of cells of a particular type.

[0128] In some embodiments, whole blood can be cryopreserved. Whole blood can be cryopreserved without sorting or characlcrizalion. Whole blood can be cryopreserved after sorting but without characterization. Whole blood can be cryopreserved after characterization but without sorting. Whole blood can be cryopreserved after characterization and sori i ng. Whole blood can be cryopreserved after quantifying a cell type of the disclosure Whole blood can be cryopreserved after quantifying conventional T cells (Tcons, e.g., CD3+cells). Whole blood can be cryopreserved after quantifying viability of all cells or a population of cells of the disclosure (e.g., conventional T cells).

[0129] A peripheral blood apheresis product of the disclosure can be cryopreserved. A peripheral blood apheresis product can be cryopreserved without sorting or characterization. A peripheral blood45IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT apheresis product can be cryopreserved after sorting but without characterization. A peripheral blood apheresis product can be cryopreserved after characterization but without sorting. A peripheral blood apheresis product can be cryopreserved after characterization and sorting. A peripheral blood apheresis product can be cryopreserved after quantifying a cell type of the disclosure. A peripheral blood apheresis product can be cryopreserved after quantifying conventional T cells (Tcons, e.g., CD3+cells). A peripheral blood apheresis product can be cryopreserved after quantifying viability of all cells or a population of cells of the disclosure (e.g., conventional T cells).

[0130] A population of cells sorted or selected from another population of cells can be cryopreserved, for example, a population of CD45+cells, HSPCs, Tregs, Tcons, iNKTs, or Tmems can be cryopreserved.

[0131] A cell population of the disclosure can be cryopreserved for any amount of time. Cells of the disclosure may be cryopreserved for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours at least about 48 hours, at least about 50 hours, at least about 55 hours, at least about 60 hours, at least about 61 hours, at least about 62 hours, at least about 65 hours, at least about 70 hours, at least about 72 hours, at least about 80 hours, at least about 90 hours, at least about 96 hours, at least about 120 hours, at least about 150 hours, at least about 200 hours, at least about 300 hours, or more prior to thawing and administration to a subject.

[0132] In some embodiments, a cell population of the disclosure is cryopreserved for at most about 1 hour, at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 6 hours, at most about 7 hours, at most about 8 hours, at most about 9 hours, at most about 10 hours, at most about 11 hours, at most about 12 at most about 14 hours, at most about 16 hours, at most about 18 hours, at most about 20 hours, at most about 22 hours, at most about 24 hours, at most about 30 hours, at most about 36 hours at most about 48 hours, at most about 50 hours, at most about 55 hours, at most about 60 hours, at most about 61 hours, at most about 62 hours, at most about 65 hours, at most about 70 hours, at most about 72 hours, at most about 80 hours, at most about 90 hours, at most about 96 hours, at most about 120 hours, at most about 150 hours, at most about 200 hours, or at most about 300 hours prior to thawing and administration to a subject.

[0133] In some embodiments, a cell population of the disclosure is cryopreserved for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 28 days, at least about 50 days, at least about 60 days, or at least about 96 days, or more prior to thawing and administration to a subject.

[0134] In some embodiments, a cell population of the disclosure is cryopreserved for at most about 1 day, at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 10 days, at most about 14 days, at most about 21 days, at most46IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT about 28 days, at most about 50 days, at most about 60 days, or at most about 96 days prior to thawing and administration to a subject.

[0135] In some embodiments, once formulated, one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells, may be placed into one or more containers. In certain aspects, the present disclosure related to a system comprising one or more containers containing one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells. In some embodiments, the one or more containers are filled to an appropriate volume (e.g., 5 mL to 1 L) with one or more excipients of the present disclosure, such as one or more transport buffers, one or more human carrier proteins, and / or one or more cryoprotectants, at a suitable pH, e.g., a neutral pH. In some embodiments, a single container contains the first population of CD45+cells, the second population of CD45+cells, and the third population of CD45+cells. In some embodiments, a first container contains the first population of CD45+cells and the second population of CD45+cells, and a second container contains the third population of CD45+cells. In some embodiments, a first container contains the first population of CD45+cells and the third population of CD45+cells, and a second container contains the second population of CD45+cells. In some embodiments, a first container contains the second population of CD45+cells and the third population of CD45+cells, and a second container contains the first population of CD45+cells. In some embodiments, a first container contains the first population of CD45+cells, a second container contains the second population of CD45+cells, and a third container contains the third population of CD45+cells. In some embodiments, a container of the present disclosure includes, without limitation, a vial, a syringe, a bag (e.g., transfer bag), a cryoprotectant container, and any combinations thereof.

[0136] In some embodiments, the container is a bag, such as a transfer bag, that can be used, for example, for infusion. In some embodiments, a transfer bag of the present disclosure is a single dose transfer bag. In some embodiments, a transfer bag of the present disclosure is a polyvinyl chloride (PVC) transfer bag or an ethylene vinyl acetate (EVA) transfer bag.

[0137] Certain aspects of the present disclosure relate to systems, e.g., a cellular therapy or treatment systems that comprises one or more of the containers of the present disclosure. Other aspects of the present disclosure relate to articles of manufacture comprising one or more of the containers of the present disclosure. Other aspects of the present disclosure relate to kits comprising one or more of the containers of the present disclosure.Donors

[0138] In some embodiments, one or more cell populations of the present disclosure, e.g., a first population of CD45+cells of the present disclosure, a second population of CD45+cells of the present disclosure, and / or a third population of CD45+cells of the present disclosure, are derived from a single human blood donor. In embodiments, the respective cell populations are provided as separate cell populations and are derived from a single human blood donor.47IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0139] A cell population can comprise cells that are from one or more donors that have each been HLA typed, for example, to determine a degree of HLA matching to a subject that will receive the cell population. In some embodiments, the donor is unrelated to the recipient subject. In some embodiments, the donor is related to a recipient subject, for example, the donor is a parent, child, sibling, grandparent, grandchild, aunt, uncle, or cousin. In some embodiments, the donor is a first-degree blood relative of the recipient subject. In some embodiments, the donor is a second-degree blood relative of the recipient subject. In some embodiments, the related or unrelated donor is HLA matched to a recipient subject. In some embodiments, the related or unrelated donor is HLA mismatched to a recipient subject. In some embodiments, the related or unrelated donor is haploidentical to a recipient subject. In some embodiments, the related or unrelated donor is at least 16 years old. In some embodiments, the related or unrelated donor is at least 18 years old.

[0140] Human leukocyte antigens (HLA), also broadly referred to as Major histocompatibility complex (MHC) antigens, can be protein molecules expressed on the surface of a cell that can confer an antigenic identity to that cell. HLA / MHC antigens are target molecules that can be recognized by T cells and natural killer (NK) cells as being derived from the same source of hcinalopoiclic stem cells as the immune effector cells ("self"), or as being derived from another source of hcinalopoiclic cells ("non-self"). HLA class I antigens (A, B, and C in humans) can be expressed by the vast majority of cells, while HLA class II antigens (DR, DP, and DQ in humans) can be expressed primarily on professional antigen presenting cells. Both HLA classes can be implicated in GVHD.

[0141] HLA antigens are encoded by highly polymorphic genes; a range of alleles exist for each HLA class I and II gene. Allelic gene products can differ in one or more amino acids in the a and / or f> domain(s). Panels of specific antibodies or nucleic acid reagents can be used to determine HLA haplotypes of individuals, for example, using leukocytes that express class I and class II molecules. HLA alleles can be described at various levels of detail. Most designations begin with HLA- and the locus name, then * and some (even) number of digits specifying the allele. The first two digits can specify a group of alleles. The third through fourth digits, when present, can specify a synonymous allele. Digits five through six, when present, can denote any synonymous mutations within the coding frame of the gene. The seventh and eighth digits, when present, can distinguish mutations outside the coding region. Letters such as L, N, Q, or S may follow an allele's designation to specify an expression level or other non-genomic data known about it. Thus, a completely described allele may be up to 9 digits long, not including the HLA-prefix and locus notation.

[0142] The set of HLA alleles inherited from one parent forms a haplotype. HLA haploidentical can refer to a donor-recipient pair where one chromosome is matched at least at HLA-A; HLA-B, and HLA-DR between the donor and recipient. The haploidentical pair may or may not be matched at other alleles, e.g., other HLA genes on the other chromosome, or additional histocompatibility loci on either chromosome. Such donors can frequently occur in families, e.g., a parent can be haploidentical to a child; and siblings may be haploidentical.48IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0143] A cell population can be from a related or unrelated donor that has been HLA-typed at any number of HLA alleles. A donor and a subject can be HLA matched, e.g., matched at all typed HLA alleles. A donor and a subject can be HLA mismatched, e.g., at least one HLA antigen can be mismatched between the donor and recipient.

[0144] In some embodiments, a related or unrelated donor and a subject can be HLA-typed at six alleles, for example, HLA-A, HLA-B, and HLA-DR alleles. The donor and subject can be matched at, for example 3 / 64 / 6, 5 / 6, or 6 / 6 of the alleles. In some embodiments, the donor and subject are matched at least at 5 / 6 alleles. In some embodiments, the donor and subject are matched at 6 / 6 alleles.

[0145] In some embodiments, a related or unrelated donor and a subject can be HLA-typed at eight alleles, for example, HLA-A, HLA-B, HLA-C, and HLA-DR alleles (e.g., HLA-DRB1 alleles). The donor and subject can be matched at, for example 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 of the alleles. In some embodiments, the donor and subject are matched at least at 6 / 8 alleles. In some embodiments, the donor and subject are matched at least at 7 / 8 alleles. In some embodiments, the donor and subject are matched at 8 / 8 alleles.

[0146] In some embodiments, a related or unrelated donor and a subject can be HLA-typed at ten alleles, for example, HLA-A, HLA-B, HLA-C, and HLA-DR alleles (e.g., HLA-DRB1 alleles). The donor and subject can be matched at, for example 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 of the alleles. In some embodiments, the donor and subject are matched at least at 7 / 10 alleles. In some embodiments, the donor and subject are matched at least at 8 / 10 alleles. In some embodiments, the donor and subject are matched at least at 9 / 10 alleles. In some embodiments, the donor and subject are matched at 10 / 10 alleles.

[0147] In some embodiments, a related or unrelated donor and a subject can be HLA-typed at twelve alleles, for example, HLA-A, HLA-B, HLA-C, HLA-DR alleles (e.g., HLA-DRB1 alleles), and HLA-DP alleles (e.g., HLA-DPB1 alleles). The donor and subject can be matched at, for example 6 / 12, 7 / 12, 8 / 12, 9 / 12, 10 / 12, 11 / 12, or 12 / 12 of the alleles. In some embodiments, the donor and subject are matched at least at 9 / 12 alleles. In some embodiments, the donor and subject are matched at least at 10 / 12 alleles. In some embodiments, the donor and subject are matched at least at 11 / 12 alleles. In some embodiments, the donor and subject are matched at 12 / 12 alleles.

[0148] A cell population can be generated from a matched unrelated donor that is an 8 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 7 / 8 match for HLA-A, -B, -C, and -DRB 1 , all typed using DNA-based high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 10 / 10 match for HLA-A, -B, -C, -DQB1 and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1 and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB1 and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1 and -DRB1, all typed using DNA-based49IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched unrelated donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0149] A cell population can be generated from a matched related donor that is an 8 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched related donor that is an 7 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched related donor that is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched related donor that is an 10 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolulion methods. A cell population can be generated from a matched related donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched related donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched related donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched related donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched related donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched related donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched related donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0150] A cell population can be generated from a matched parent donor that is an 8 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched parent donor that is an 7 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched parent donor that is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched parent donor that is an 10 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched parent donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched parent donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based50IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT high-resolution methods. A cell population can be generated from a matched parent donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched parent donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched parent donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched parent donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched parent donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0151] A cell population can be generated from a matched child donor that is an 8 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 7 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 10 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0152] A cell population can be generated from a matched sibling donor that is an 8 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched sibling donor that is an 7 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched sibling donor that is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched sibling donor that is an 10 / 10 match for HLA-A, -B, -C, -DQB1 and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched sibling donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1 and -DRB1,51IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT all typed using DNA-based high-resolution methods. A cell population can be generated from a matched sibling donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB1 and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched sibling donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1 and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched child donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0153] A cell population can be generated from a matched grandparent donor that is an 8 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 7 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 10 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB 1 , and -DRB 1 , all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandparent donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0154] A cell population can be generated from a matched grandchild donor that is an 8 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 7 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 10 / 1052IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched grandchild donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0155] A cell population can be generated from a matched aunt donor that is an 8 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 7 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 10 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB 1 , -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB 1 , -DRB 1 , and -DPB 1 , all typed using DNA-based high-resolution methods. A cell population can be generated from a matched aunt donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0156] A cell population can be generated from a matched uncle donor that is an 8 / 8 match for HLA- A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that is an 7 / 8 match for HLA-A, -B , -C , and -DRB 1 , all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that53IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that is an 10 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched uncle donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0157] A cell population can be generated from a matched cousin donor that is an 8 / 8 match for HLA- A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched cousin donor that is an 7 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched cousin donor that is an 6 / 8 match for HLA-A, -B, -C, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched cousin donor that is an 10 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-rcsolulion methods. A cell population can be generated from a matched cousin donor that is an 9 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched cousin donor that is an 8 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched cousin donor that is an 7 / 10 match for HLA-A, -B, -C, -DQB1, and -DRB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched cousin donor that is an 12 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched cousin donor that is an 11 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched cousin donor that is an 10 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods. A cell population can be generated from a matched cousin donor that is an 9 / 12 match for HLA-A, -B, -C, -DQB1, -DRB1, and -DPB1, all typed using DNA-based high-resolution methods.

[0158] In some embodiments, the HLA mismatch occurs as the result of the allogeneic related or unrelated donor being homozygous for the HLA allele while the recipient subject is heterogeneous for54IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT the HLA allele. In some embodiments, the HLA mismatch occurs as the result of the allogeneic related or unrelated donor being heterogeneous for the HLA allele while the recipient subject is homozygous for the HLA allele. In some embodiments, the HLA mismatch occurs as the result of both the allogeneic related or unrelated donor and the recipient subject being heterozygous for the HLA allele.

[0159] In various embodiments, the first population of CD45+cells, the population of cells enriched for Tregs (e.g., the second population of CD45+cells), and / or the third population of CD45+cells is allogeneic relative to the human subject.

[0160] In some embodiments, the first population of CD45+cells, the population of cells enriched for Tregs (e.g., the second population of CD45+cells), and / or the third population of CD45+cells is obtained from a donor that is HLA-matched relative to the human subject.

[0161] In embodiments, the first population of CD45+cells, the population of cells enriched for Tregs (e.g., the second population of CD45+cells), and / or the third population of CD45+cells is obtained from a donor that is HLA-mismatched relative to the human subject.

[0162] In various embodiments, the first population of CD45+cells, the population of cells enriched for Tregs (e.g., the second population of CD45+cells), and / or the third population of CD45+cells is obtained from a donor that is haploidentical relative to the human subject.

[0163] A cell population can be derived from an allogeneic donor. A cell population can be generated from a donor that is a first-degree blood relative of the subject. A cell population can be generated from a donor that is a second-degree blood relali vc of the subject. A cell population can be generated from a donor that is not related to the subject. A cell population can be generated from a donor that is HLA matched to a recipient subject. A cell population can be generated from a donor that is HLA mismatched to a recipient subject. A cell population can be generated from a donor that is haploidenlical to a recipient subject. A cell population can be generated from a donor that is related to a recipient subject, for example, a parent, child, sibling, grandparent, grandchild, aunt, uncle, or cousin. A cell population can be generated from a donor that is at least 16 years old. A cell population can be generated from a donor that is at least 18 years old.

[0164] A cell population can be generated from a donor that meets eligibility criteria for donors of viable, leukocyte-rich cells or tissues as defined by 21 CFR § 1271 2018 and relevant FDA Guidance for Industry. For example, a cell population can be generated from a donor that meets eligibility criteria outlined in any one or more of the following: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products, 2007; Use of Donor Screening Tests to Test Donors of Human Cells, Tissues and Cellular and Tissue-Based Products for Infection with Treponema pallidum (Syphilis), 2015; Use of Nucleic Acid Tests to Reduce the Risk of Transmission of Hepatitis B Virus from Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products, 2016; Use of Nucleic Acid Tests to Reduce the Risk of Transmission of West Nile Virus from Living Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT / Ps), 2016; and Donor Screening Recommendations to Reduce the Risk of Transmission of Zika Virus by Human Cells, Tissues, and Cellular and Tissue-Based Products, 2018). A cell population can be generated from a donor that meets any criteria for donation as specified by standard NMDP guidelines (NMDP donors).55IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0165] A cell population can be generated from a donor that does not exhibit evidence of active infection. A cell population can be generated from a donor that is not seropositive for HIV-1 or -2, HTLV-1 or -2. A cell population can be generated from a donor that is not positive for anli-hcpalilis C (HCV) antibody or HCV NAT. A cell population can be generated from a donor that tests negative for chronic HBV infection. A cell population can be generated from a donor that does not have high potential for Zika virus infection as defined as any of the following: (i) Medical diagnosis of Zika virus infection in the past 6 months; (ii) Residence in, or travel to, an area with active Zika virus transmission within the past 6 months; (iii) Unprotected sex within the past 6 months with a person who is known to have either of the risk factors (i) or (ii). A cell population can be generated from a donor that does not have signs or symptoms consistent with active Zika virus infection.

[0166] One or more cell populations of the disclosure can be obtained from a single donor, for example, obtained from mobilized peripheral blood apheresis of a single donor. HSPCs, Tregs, Tcons, iNKTs, Tmems, or any combination thereof can be obtained from a single donor.

[0167] One or more cell populations of the disclosure can be obtained from one donor, and one or more additional cell populations of the disclosure can be obtained from a second donor. One cell population of the disclosure can be obtained from a single donor, and a second cell population of the disclosure can be obtained from multiple donors. Populations of the disclosure can be obtained from multiple donors, for example, obtained from mobilized peripheral blood apheresis of multiple donors. HSPCs can be obtained from multiple donors. Tregs can be obtained from multiple donors. Tcons can be obtained from multiple donors. iNKTs can be obtained from multiple donors. Tmems can be obtained from multiple donors. Doses of cell populations

[0168] Doses of cell populations administered to a subject e.g., human subject) may be based on the subject’s body weight. In some cases, a subject’s body weight may be used to determine a dose of one or more cell populations to be administered to the subject. In some cases, a cell dose may be based on the ideal body weight of the subject instead of their actual weight, e.g., actual body weight. Ideal body weight may be a preferable method of dose calculation to avoid erroneous cell doses due to excess body fat and / or muscle mass. A subject’s ideal body weight may be calculated using their height and sex. Other methods that calculate a subject’s ideal body weight may be used. For instance, other methods which determine a subject’s body fat percentage. A dose of one or more cell populations of the present disclosure, e.g., the first population of CD45+cells, the second population of CD45+cells, and / or the third population of CD45+cells, may be based on the subject’s adjusted body weight (ABW), if the subject’s actual body weight is greater than 120% of his / her ideal body weight (IBW).

[0169] In some embodiments the human subject has a body weight that ranges from approximately 4 kilograms (kg) to approximately 200 kilograms (kg). In some embodiments, the human subject has a body weight that is approximately 4 kilograms, approximately 5 kilograms, approximately 6 kilograms, approximately 7 kilograms, approximately 8 kilograms, approximately 9 kilograms, approximately 10 kilograms, approximately 11 kilograms, approximately 12 kilograms, approximately 13 kilograms, approximately 14 kilograms, approximately 15 kilograms, approximately 16 kilograms, approximately 1756IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT kilograms, approximately 18 kilograms, approximately 19 kilograms, approximately 20 kilograms, approximately 21 kilograms, approximately 22 kilograms, approximately 23 kilograms, approximately 24 kilograms, approximately 25 kilograms, approximately 26 kilograms, approximately 27 kilograms, approximately 28 kilograms, approximately 29 kilograms, approximately 30 kilograms, approximately 31 kilograms, approximately 32 kilograms, approximately 33 kilograms, approximately 34 kilograms, approximately 35 kilograms, approximately 36 kilograms, approximately 37 kilograms, approximately 38 kilograms, approximately 39 kilograms, approximately 40 kilograms, approximately 41 kilograms, approximately 42 kilograms, approximately 43 kilograms, approximately 44 kilograms, approximately 45 kilograms, approximately 46 kilograms, approximately 47 kilograms, approximately 48 kilograms, approximately 49 kilograms, approximately 50 kilograms, approximately 51 kilograms, approximately 52 kilograms, approximately 53 kilograms, approximately 54 kilograms, approximately 55 kilograms, approximately 56 kilograms, approximately 57 kilograms, approximately 58 kilograms, approximately 59 kilograms, approximately 60 kilograms, approximately 61 kilograms, approximately 62 kilograms, approximately 63 kilograms, approximately 64 kilograms, approximately 65 kilograms, approximately 66 kilograms, approximately 67 kilograms, approximately 68 kilograms, approximately 69 kilograms, approximately 70 kilograms, approximately 71 kilograms, approximately 72 kilograms, approximately 73 kilograms, approximately 74 kilograms, approximately 75 kilograms, approximately 76 kilograms, approximately 77 kilograms, approximately 78 kilograms, approximately 79 kilograms, approximately 80 kilograms, approximately 81 kilograms, approximately 82 kilograms, approximately 83 kilograms, approximately 84 kilograms, approximately 85 kilograms, approximately 86 kilograms, approximately 87 kilograms, approximately 88 kilograms, approximately 89 kilograms, approximately 90 kilograms, approximately 91 kilograms, approximately 92 kilograms, approximately 93 kilograms, approximately 94 kilograms, approximately 95 kilograms, approximately 96 kilograms, approximately 97 kilograms, approximately 98 kilograms, approximately 99 kilograms, approximately 100 kilograms, approximately 105 kilograms, approximately 110 kilograms, approximately 115 kilograms, approximately 120 kilograms, approximately 125 kilograms, approximately 130 kilograms, approximately 135 kilograms, approximately 140 kilograms, approximately 145 kilograms, approximately 150 kilograms, approximately 155 kilograms, approximately 160 kilograms, approximately 165 kilograms, approximately 170 kilograms, approximately 175 kilograms, approximately 180 kilograms, approximately 185 kilograms, approximately 190 kilograms, approximately 195 kilograms, or approximately 200 kilograms.HSPCs

[0170] A first population of CD45+cells which comprises, at least, HSPCs or at least one dose of HSPCs, can comprise at least about 1 x 104, at least about 2 x 104, at least about 3 x 104, at least about 4 x 104, at least about 5 x 104, at least about 6 x 104, at least about 7 x 104, at least about 8 x 104, at least about 9 x 104, at least about 1 x 105, at least about 2 x 105, at least about 3 x 105, at least about 4 x 105, at least about 5 x 105, at least about 6 x 105, at least about 7 x 105, at least about 8 x 105, at least about 9 x 105, at least about 1 x 106, at least about 1.1 x 106, at least about 1.2 x 106, at least about 1.3 x 106, at least about 1.4 x 106, at least about 1.5 x 106, at least about 1.6 x 106, at least about 1.7 x 106, at least about 1.8 x 106,57IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT at least about 1.9 x 106, at least about 2 x 106, at least about 2.1 x 106, at least about 2.2 x 106, at least about 2.3 x 106, at least about 2.4 x 106, at least about 2.5 x 106, at least about 2.6 x 106, at least about 2.7 x 106, at least about 2.8 x 106, at least about 2.9 x 106, at least about 3 x 106, at least about 3.1 x 106, at least about 3.2 x 106, at least about 3.3 x 106, at least about 3.4 x 106, at least about 3.5 x 106, at least about 3.6 x 106, at least about 3.7 x 106, at least about 3.8 x 106, at least about 3.9 x 106, at least about 4 x 106, at least about 4.1 x 106, at least about 4.2 x 106, at least about 4.3 x 106, at least about 4.4 x 106, at least about 4.5 x 106, at least about 4.6 x 106, at least about 4.7 x 106, at least about 4.8 x 106, at least about 4.9 x 106, at least about 5 x 106, at least about 5.1 x 106, at least about 5.2 x 106, at least about 5.3 x 106, at least about 5.4 x 106, at least about 5.5 x 106, at least about 5.6 x 106, at least about 5.7 x 106, at least about 5.8 x 106, at least about 5.9 x 106, at least about 6 x 106, at least about 6.1 x 106, at least about 6.2 x 106, at least about 6.3 x 106, at least about 6.4 x 106, at least about 6.5 x 106, at least about 6.6 x 106, at least about 6.7 x 106, at least about 6.8 x 106, at least about 6.9 x 106, at least about 7 x 106, at least about 7.1 x 106, at least about 7.2 x 106, at least about 7.3 x 106, at least about 7.4 x 106, at least about 7.5 x 106, at least about 7.6 x 106, at least about 7.7 x 106, at least about 7.8 x 106, at least about 7.9 x 106, at least about 8 x 106, at least about 8.1 x 106, at least about 8.2 x 106, at least about 8.3 x 106, at least about 8.4 x 106, at least about 8.5 x 106, at least about 8.6 x 106, at least about 8.7 x 106, at least about 8.8 x 106, at least about 8.9 x 106, at least about 9 x 106, at least about 9.1 x 106, at least about 9.2 x 106, at least about 9.3 x 106, at least about 9.4 x 106, at least about 9.5 x 106, at least about 9.6 x 106, at least about 9.7 x 106, at least about 9.8 x 106, at least about 9.9 x 106, at least about 1 x 107, at least about 1.5 x 107, at least about 2 x 107, at least about 2.5 x 107, at least about 3 x 107, at least about 3.5 x 107, at least about 4 x 107, at least about 4.5 x 107, at least about 5 x 107, at least about 5.5 x 107, at least about 6 x 107, at least about 6.5 x 107, at least about 7 x 107, at least about 7.5 x 107, at least about 8 x 107, at least about 8.5 x 107, at least about 9 x 107, at least about 9.5 x 107, at least about 1 x IO8, at least about 1.5 x IO8, at least about 2 x IO8, at least about 2.5 x IO8, at least about 3 x IO8, at least about 3.5 x IO8, at least about 4 x 107, at least about 4.5 x IO8, at least about 5 x IO8, at least about 5.5 x IO8, at least about 6 x IO8, at least about 6.5 x IO8, at least about 7 x IO8, at least about 7.5 x IO8, at least about 8 x IO8, at least about 8.5 x IO8, at least about 9 x IO8, at least about 9.5 x IO8, at least about 1 x IO9, or more cells of the first population of CD45+cells and / or HSPCs or doses of HSPCs (e.g., CD34+cells) per kilogram (kg) of recipient subject’s (e.g., human subject’s) actual body weight or ideal body weight.

[0171] For example, a first population of CD45+cells can comprise 1 x 104to 1 x 109, 1 x 105to 1 x 108, 1 x 105to 2 x 107, 5 x 105to 2 x 107, 5 x 105to 1.5 x 107, 5 x 105to 1 x 107, 5 x 105to 9 x 106, 5 x 105to 8 x 106, 5 x 105to 7 x 106, 5 x 105to 6 x 106, 5 x 105to 5 x 106, 5 x 105to 4 x 106, 5 x 105to 3 x 106, 5 x 105to 2 x 106, 5 x 105to 1 x 106, 1 x 106to 1.5 x 107, 1 x 106to 1 x 107, 1 x 106to 9 x 106, 1 x 106to 8 x 106, 1 x 106to 7 x 106, 1 x 106to 6 x 106, 1 x 106to 5 x 106, 1 x 106to 4 x 106, 1 x 106to 3 x 106, 1 x 106to 2 x 106, 1.5 x 106to 1.5 x 107, 1.5 x 106to 1 x 107, 1.5 x 106to 9 x 106, 1.5 x 106to 8 x 106, 1.5 x 106to 7 x 106, 1.5 x 106to 6 x 106, 1.5 x 106to 5 x 106, 1.5 x 106to 4 x 106, 1.5 x 106to 3 x 106, 1.5 x 106to 2 x 106, 2 x 106to 1.5 x 107, 2 x 106to 1 x 107, 2 x 106to 9 x 106, 2 x 106to 8 x 106, 2 x 106to 7 x 106, 2 x 106to 6 x 106, 2 x 106to 5 x 106, 2 x 106to 4 x 106, 2 x 106to 3 x 106, 2.5 x 106to 1.5 x 107, 2.5 x 106to 1 x 107, 2.5 x 10658IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT to 9 X 106, 2.5 X 106to 8 x 106, 2.5 x 106to 7 x 106, 2.5 x 106to 6 x 106, 2.5 x 106to 5 x 106, 2.5 x 106to 4 x 106, or 2.5 x 106to 3 x 106cells of the first population of CD45+cells and / or HSPCs or doses of HSPCs e.g., CD34+cells) per kg of recipient subject’s actual body weight or ideal body weight.

[0172] In some embodiments, the first population of CD45+cells comprising HSPCs, or comprising at least one dose of HSPCs, comprises from approximately 1.0 x 105to approximately 5.0 x IO10, from approximately 5.0 x 105to approximately 1.5 x IO10, from approximately 5.0 x 105to approximately 5.0 x 108, or from approximately 1.5 x 107to approximately 1.5 x IO10HSPCs, e.g., in the first population of CD45+cells. In some embodiments, the first population of CD45+cells comprising HSPCs, or comprising at least one dose of HSPCs, comprises approximately 1.0 x 105or more HSPCs, approximately 2.0 x 105or more HSPCs, approximately 3.0 x 105or more HSPCs, approximately 4.0 x 105or more HSPCs, approximately 5.0 x 105or more HSPCs, approximately 6.0 x 105or more HSPCs, approximately 7.0 x 105or more HSPCs, approximately 8.0 x 105or more HSPCs, approximately 9.0 x 105or more HSPCs, approximately 1.0 x 106or more HSPCs, approximately 1.0 x 106or more HSPCs, approximately 1.1 x 106or more HSPCs, approximately 1.2 x 106or more HSPCs, approximately 1.3 x 106or more HSPCs, approximately 1.4 x 106or more HSPCs, approximately 1.5 x 106or more HSPCs, approximately 1.6 x 106or more HSPCs, approximately 1.7 x 106or more HSPCs, approximately 1.8 x 106or more HSPCs, approximately 1.9 x 106or more HSPCs, approximately 2.0 x 106or more HSPCs, approximately 2.1 x 106or more HSPCs, approximately 2.2 x 106or more HSPCs, approximately 2.3 x 106or more HSPCs, approximately 2.4 x 106or more HSPCs, approximately 2.5 x 106or more HSPCs, approximately 2.6 x 106or more HSPCs, approximately 2.7 x 106or more HSPCs, approximately 2.8 x 106or more HSPCs, approximately 2.9 x 106or more HSPCs, approximately 3.0 x 106or more HSPCs, approximately 3.1 x 106or more HSPCs, approximately 3.2 x 106or more HSPCs, approximately 3.3 x 106or more HSPCs, approximately 3.4 x 106or more HSPCs, approximately 3.5 x 106or more HSPCs, approximately 3.6 x 106or more HSPCs, approximately 3.7 x 106or more HSPCs, approximately 3.8 x 106or more HSPCs, approximately 3.9 x 106or more HSPCs, approximately 4.0 x 106or more HSPCs, approximately 4.1 x 106or more HSPCs, approximately 4.2 x 106or more HSPCs, approximately 4.3 x 106or more HSPCs, approximately 4.4 x 106or more HSPCs, approximately 4.5 x 106or more HSPCs, approximately 4.6 x 106or more HSPCs, approximately 4.7 x 106or more HSPCs, approximately 4.8 x 106or more HSPCs, approximately 4.9 x 106or more HSPCs, approximately 5.0 x 106or more HSPCs, approximately 5.1 x 106or more HSPCs, approximately 5.2 x 106or more HSPCs, approximately 5.3 x 106or more HSPCs, approximately 5.4 x 106or more HSPCs, approximately 5.5 x 106or more HSPCs, approximately 5.6 x 106or more HSPCs, approximately 5.7 x 106or more HSPCs, approximately 5.8 x 106or more HSPCs, approximately 5.9 x 106or more HSPCs, approximately 6.0 x 106or more HSPCs, approximately 6.1 x 106or more HSPCs, approximately 6.2 x 106or more HSPCs, approximately 6.3 x 106or more HSPCs, approximately 6.4 x 106or more HSPCs, approximately 6.5 x 106or more HSPCs, approximately 6.6 x 106or more HSPCs, approximately 6.7 x 106or more HSPCs, approximately 6.8 x 106or more HSPCs, approximately 6.9 x 106or more HSPCs, approximately 7.0 x 106or more HSPCs, approximately 7.1 x 106or more HSPCs, approximately 7.2 x 106or more HSPCs, approximately 7.3 x 106or more HSPCs,59IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 7.4 x 106or more HSPCs, approximately 7.5 x 106or more HSPCs, approximately 7.6 x 106or more HSPCs, approximately 7.7 x 106or more HSPCs, approximately 7.8 x 106or more HSPCs, approximately 7.9 x 106or more HSPCs, approximately 8.0 x 106or more HSPCs, approximately 8.1 x 106or more HSPCs, approximately 8.2 x 106or more HSPCs, approximately 8.3 x 106or more HSPCs, approximately 8.4 x 106or more HSPCs, approximately 8.5 x 106or more HSPCs, approximately 8.6 x 106or more HSPCs, approximately 8.7 x 106or more HSPCs, approximately 8.8 x 106or more HSPCs, approximately 8.9 x 106or more HSPCs, approximately 9.0 x 106or more HSPCs, approximately 9.1 x 106or more HSPCs, approximately 9.2 x 106or more HSPCs, approximately 9.3 x 106or more HSPCs, approximately 9.4 x 106or more HSPCs, approximately 9.5 x 106or more HSPCs, approximately 9.6 x 106or more HSPCs, approximately 9.7 x 106or more HSPCs, approximately 9.8 x 106or more HSPCs, approximately 9.9 x 106or more HSPCs, approximately 1.0 x 107or more HSPCs, approximately 1.1 x 107or more HSPCs, approximately 1.2 x 107or more HSPCs, approximately 1.3 x 107or more HSPCs, approximately 1.4 x 107or more HSPCs, approximately 1.5 x 107or more HSPCs, approximately 1.6 x 107or more HSPCs, approximately 1.7 x 107or more HSPCs, approximately 1.8 x 107or more HSPCs, approximately 1.9 x 107or more HSPCs, approximately 2.0 x 107or more HSPCs, approximately 2.1 x 107or more HSPCs, approximately 2.2 x 107or more HSPCs, approximately 2.3 x 107or more HSPCs, approximately 2.4 x 107or more HSPCs, approximately 2.5 x 107or more HSPCs, approximately 2.6 x 107or more HSPCs, approximately 2.7 x 107or more HSPCs, approximately 2.8 x 107or more HSPCs, approximately 2.9 x 107or more HSPCs, approximately 3.0 x 107or more HSPCs, approximately 3.1 x 107or more HSPCs, approximately 3.2 x 107or more HSPCs, approximately 3.3 x 107or more HSPCs, approximately 3.4 x 107or more HSPCs, approximately 3.5 x 107or more HSPCs, approximately 3.6 x 107or more HSPCs, approximately 3.7 x 107or more HSPCs, approximately 3.8 x 107or more HSPCs, approximately 3.9 x 107or more HSPCs, approximately 4.0 x 107or more HSPCs, approximately 4.1 x 107or more HSPCs, approximately 4.2 x 107or more HSPCs, approximately 4.3 x 107or more HSPCs, approximately 4.4 x 107or more HSPCs, approximately 4.5 x 107or more HSPCs, approximately 4.6 x 107or more HSPCs, approximately 4.7 x 107or more HSPCs, approximately 4.8 x 107or more HSPCs, approximately 4.9 x 107or more HSPCs, approximately 5.0 x 107or more HSPCs, approximately 5.1 x 107or more HSPCs, approximately 5.2 x 107or more HSPCs, approximately 5.3 x 107or more HSPCs, approximately 5.4 x 107or more HSPCs, approximately 5.5 x 107or more HSPCs, approximately 5.6 x 107or more HSPCs, approximately 5.7 x 107or more HSPCs, approximately 5.8 x 107or more HSPCs, approximately 5.9 x 107or more HSPCs, approximately 6.0 x 107or more HSPCs, approximately 6.1 x 107or more HSPCs, approximately 6.2 x 107or more HSPCs, approximately 6.3 x 107or more HSPCs, approximately 6.4 x 107or more HSPCs, approximately 6.5 x 107or more HSPCs, approximately 6.6 x 107or more HSPCs, approximately 6.7 x 107or more HSPCs, approximately 6.8 x 107or more HSPCs, approximately 6.9 x 107or more HSPCs, approximately 7.0 x 107or more HSPCs, approximately 7.1 x 107or more HSPCs, approximately 7.2 x 107or more HSPCs, approximately 7.3 x 107or more HSPCs, approximately 7.4 x 107or more HSPCs, approximately 7.5 x 107or more HSPCs, approximately 7.6 x 107or more HSPCs, approximately 7.7 x 107or more HSPCs, approximately 7.8 x 107or more HSPCs,60IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 7.9 x 107or more HSPCs, approximately 8.0 x 107or more HSPCs, approximately 8.1 x 107or more HSPCs, approximately 8.2 x 107or more HSPCs, approximately 8.3 x 107or more HSPCs, approximately 8.4 x 107or more HSPCs, approximately 8.5 x 107or more HSPCs, approximately 8.6 x 107or more HSPCs, approximately 8.7 x 107or more HSPCs, approximately 8.8 x 107or more HSPCs, approximately 8.9 x 107or more HSPCs, approximately 9.0 x 107or more HSPCs, approximately 9.1 x 107or more HSPCs, approximately 9.2 x 107or more HSPCs, approximately 9.3 x 107or more HSPCs, approximately 9.4 x 107or more HSPCs, approximately 9.5 x 107or more HSPCs, approximately 9.6 x 107or more HSPCs, approximately 9.7 x 107or more HSPCs, approximately 9.8 x 107or more HSPCs, approximately 9.9 x 107or more HSPCs, approximately 1.0 x 108or more HSPCs, approximately 1.1 x 108or more HSPCs, approximately 1.2 x 108or more HSPCs, approximately 1.3 x 108or more HSPCs, approximately 1.4 x 108or more HSPCs, approximately 1.5 x 108or more HSPCs, approximately 1.6 x 108or more HSPCs, approximately 1.7 x 108or more HSPCs, approximately 1.8 x 108or more HSPCs, approximately 1.9 x 108or more HSPCs, approximately 2.0 x 108or more HSPCs, approximately 2.1 x 108or more HSPCs, approximately 2.2 x 108or more HSPCs, approximately 2.3 x 108or more HSPCs, approximately 2.4 x 108or more HSPCs, approximately 2.5 x 108or more HSPCs, approximately 2.6 x 108or more HSPCs, approximately 2.7 x 108or more HSPCs, approximately 2.8 x 108or more HSPCs, approximately 2.9 x 108or more HSPCs, approximately 3.0 x 108or more HSPCs, approximately 3.1 x 108or more HSPCs, approximately 3.2 x 108or more HSPCs, approximately 3.3 x 108or more HSPCs, approximately 3.4 x 108or more HSPCs, approximately 3.5 x 108or more HSPCs, approximately 3.6 x 108or more HSPCs, approximately 3.7 x 108or more HSPCs, approximately 3.8 x 108or more HSPCs, approximately 3.9 x 108or more HSPCs, approximately 4.0 x 108or more HSPCs, approximately 4.1 x 108or more HSPCs, approximately 4.2 x 108or more HSPCs, approximately 4.3 x 108or more HSPCs, approximately 4.4 x 108or more HSPCs, approximately 4.5 x 108or more HSPCs, approximately 4.6 x 108or more HSPCs, approximately 4.7 x 108or more HSPCs, approximately 4.8 x 108or more HSPCs, approximately 4.9 x 108or more HSPCs, approximately 5.0 x 108or more HSPCs, approximately 5.1 x 108or more HSPCs, approximately 5.2 x 108or more HSPCs, approximately 5.3 x 108or more HSPCs, approximately 5.4 x 108or more HSPCs, approximately 5.5 x 108or more HSPCs, approximately 5.6 x 108or more HSPCs, approximately 5.7 x 108or more HSPCs, approximately 5.8 x 108or more HSPCs, approximately 5.9 x 108or more HSPCs, approximately 6.0 x 108or more HSPCs, approximately 6.1 x 108or more HSPCs, approximately 6.2 x 108or more HSPCs, approximately 6.3 x 108or more HSPCs, approximately 6.4 x 108or more HSPCs, approximately 6.5 x 108or more HSPCs, approximately 6.6 x 108or more HSPCs, approximately 6.7 x 108or more HSPCs, approximately 6.8 x 108or more HSPCs, approximately 6.9 x 108or more HSPCs, approximately 7.0 x 108or more HSPCs, approximately 7.1 x 108or more HSPCs, approximately 7.2 x 108or more HSPCs, approximately 7.3 x 108or more HSPCs, approximately 7.4 x 108or more HSPCs, approximately 7.5 x 108or more HSPCs, approximately 7.6 x 108or more HSPCs, approximately 7.7 x 108or more HSPCs, approximately 7.8 x 108or more HSPCs, approximately 7.9 x 108or more HSPCs, approximately 8.0 x 108or more HSPCs, approximately 8.1 x 108or more HSPCs, approximately 8.2 x 108or more HSPCs, approximately 8.3 x 108or more HSPCs,61IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 8.4 x 108or more HSPCs, approximately 8.5 x 108or more HSPCs, approximately 8.6 x 108or more HSPCs, approximately 8.7 x 108or more HSPCs, approximately 8.8 x 108or more HSPCs, approximately 8.9 x 108or more HSPCs, approximately 9.0 x 108or more HSPCs, approximately 9.1 x 108or more HSPCs, approximately 9.2 x 108or more HSPCs, approximately 9.3 x 108or more HSPCs, approximately 9.4 x 108or more HSPCs, approximately 9.5 x 108or more HSPCs, approximately 9.6 x 108or more HSPCs, approximately 9.7 x 108or more HSPCs, approximately 9.8 x 108or more HSPCs, approximately 9.9 x 108or more HSPCs, approximately 1.0 x 109or more HSPCs, approximately 1.1 x 109or more HSPCs, approximately 1.2 x 109or more HSPCs, approximately 1.3 x 109or more HSPCs, approximately 1.4 x 109or more HSPCs, approximately 1.5 x 109or more HSPCs, approximately 1.6 x 109or more HSPCs, approximately 1.7 x 109or more HSPCs, approximately 1.8 x 109or more HSPCs, approximately 1.9 x 109or more HSPCs, approximately 2.0 x 109or more HSPCs, approximately 2.1 x 109or more HSPCs, approximately 2.2 x 109or more HSPCs, approximately 2.3 x 109or more HSPCs, approximately 2.4 x 109or more HSPCs, approximately 2.5 x 109or more HSPCs, approximately 2.6 x 109or more HSPCs, approximately 2.7 x 109or more HSPCs, approximately 2.8 x 109or more HSPCs, approximately 2.9 x 109or more HSPCs, approximately 3.0 x 109or more HSPCs, approximately 3.1 x 109or more HSPCs, approximately 3.2 x 109or more HSPCs, approximately 3.3 x 109or more HSPCs, approximately 3.4 x 109or more HSPCs, approximately 3.5 x 109or more HSPCs, approximately 3.6 x 109or more HSPCs, approximately 3.7 x 109or more HSPCs, approximately 3.8 x 109or more HSPCs, approximately 3.9 x 109or more HSPCs, approximately 4.0 x 109or more HSPCs, approximately 4.1 x 109or more HSPCs, approximately 4.2 x 109or more HSPCs, approximately 4.3 x 109or more HSPCs, approximately 4.4 x 109or more HSPCs, approximately 4.5 x 109or more HSPCs, approximately 4.6 x 109or more HSPCs, approximately 4.7 x 109or more HSPCs, approximately 4.8 x 109or more HSPCs, approximately 4.9 x 109or more HSPCs, approximately 5.0 x 109or more HSPCs, approximately 5.1 x 109or more HSPCs, approximately 5.2 x 109or more HSPCs, approximately 5.3 x 109or more HSPCs, approximately 5.4 x 109or more HSPCs, approximately 5.5 x 109or more HSPCs, approximately 5.6 x 109or more HSPCs, approximately 5.7 x 109or more HSPCs, approximately 5.8 x 109or more HSPCs, approximately 5.9 x 109or more HSPCs, approximately 6.0 x 109or more HSPCs, approximately 6.1 x 109or more HSPCs, approximately 6.2 x 109or more HSPCs, approximately 6.3 x 109or more HSPCs, approximately 6.4 x 109or more HSPCs, approximately 6.5 x 109or more HSPCs, approximately 6.6 x 109or more HSPCs, approximately 6.7 x 109or more HSPCs, approximately 6.8 x 109or more HSPCs, approximately 6.9 x 109or more HSPCs, approximately 7.0 x 109or more HSPCs, approximately 7.1 x 109or more HSPCs, approximately 7.2 x 109or more HSPCs, approximately 7.3 x 109or more HSPCs, approximately 7.4 x 109or more HSPCs, approximately 7.5 x 109or more HSPCs, approximately 7.6 x 109or more HSPCs, approximately 7.7 x 109or more HSPCs, approximately 7.8 x 109or more HSPCs, approximately 7.9 x 109or more HSPCs, approximately 8.0 x 109or more HSPCs, approximately 8.1 x 109or more HSPCs, approximately 8.2 x 109or more HSPCs, approximately 8.3 x 109or more HSPCs, approximately 8.4 x 109or more HSPCs, approximately 8.5 x 109or more HSPCs, approximately 8.6 x 109or more HSPCs, approximately 8.7 x 109or more HSPCs, approximately 8.8 x 109or more HSPCs,62IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 8.9 x 109or more HSPCs, approximately 9.0 x 109or more HSPCs, approximately 9.1 x 109or more HSPCs, approximately 9.2 x 109or more HSPCs, approximately 9.3 x 109or more HSPCs, approximately 9.4 x 109or more HSPCs, approximately 9.5 x 109or more HSPCs, approximately 9.6 x 109or more HSPCs, approximately 9.7 x 109or more HSPCs, approximately 9.8 x 109or more HSPCs, approximately 9.9 x 109or more HSPCs, approximately 1.0 x IO10or more HSPCs, approximately 1.1 x IO10or more HSPCs, approximately 1.2 x IO10or more HSPCs, approximately 1.3 x IO10or more HSPCs, approximately 1.4 x IO10or more HSPCs, approximately 1.5 x IO10or more HSPCs, approximately 1.6 x IO10or more HSPCs, approximately 1.7 x IO10or more HSPCs, approximately 1.8 x IO10or more HSPCs, approximately 1.9 x IO10or more HSPCs, approximately 2.0 x IO10or more HSPCs, approximately 2.1 x IO10or more HSPCs, approximately 2.2 x IO10or more HSPCs, approximately 2.3 x IO10or more HSPCs, approximately 2.4 x IO10or more HSPCs, approximately 2.5 x IO10or more HSPCs, approximately 2.6 x IO10or more HSPCs, approximately 2.7 x IO10or more HSPCs, approximately 2.8 x IO10or more HSPCs, approximately 2.9 x IO10or more HSPCs, approximately 3.0 x IO10or more HSPCs, approximately 3.1 x IO10or more HSPCs, approximately 3.2 x IO10or more HSPCs, approximately 3.3 x IO10or more HSPCs, approximately 3.4 x IO10or more HSPCs, approximately 3.5 x IO10or more HSPCs, approximately 3.6 x IO10or more HSPCs, approximately 3.7 x IO10or more HSPCs, approximately 3.8 x IO10or more HSPCs, approximately 3.9 x IO10or more HSPCs, approximately 4.0 x IO10or more HSPCs, approximately 4.1 x IO10or more HSPCs, approximately 4.2 x IO10or more HSPCs, approximately 4.3 x IO10or more HSPCs, approximately 4.4 x IO10or more HSPCs, approximately 4.5 x IO10or more HSPCs, approximately 4.6 x IO10or more HSPCs, approximately 4.7 x IO10or more HSPCs, approximately 4.8 x 1 IO10or more HSPCs, approximately 4.9 x IO10or more HSPCs, or approximately 5.0 x IO10or more HSPCs.

[0173] A first population of CD45+cells can have a defined level of purity for CD34+cells. For example, a first population of CD45+cells can comprise at least about 5%, at least about at least about 10%, at least about at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or more CD34+cells as a percentage of total cells, as a percentage of nucleated cells, or as a percentage of CD45+cells.

[0174] A first population of CD45+cells can have a defined level of contaminating CD3+cells. In some embodiments, at most about 1 x 102, at most about 2 x 102, at most about 3 x 102, at most about 4 x63IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 102, at most about 5 x 102, at most about 6 x 102, at most about 7 x 102, at most about 8 x 102, at most about 9 x 102, at most about 1 x 103, at most about 2 x 103, at most about 3 x 103, at most about 4 x 103, at most about 5 x 103, at most about 6 x 103, at most about 7 x 103, at most about 8 x 103, at most about 9 x 103, at most about 1 x 104, at most about 2 x 104, at most about 3 x 104, at most about 4 x 104, at most about 5 x 104, at most about 6 x 104, at most about 7 x 104, at most about 8 x 104, at most about 9 x 104, or at most about 1 x 105CD3+cells per kg of a recipient subject’s body weight are present in a first population of CD45+cells or ideal body weight.

[0175] In some embodiments, a first population of CD45+cells comprises at most about 0.001%, at most about 0.002%, at most about 0.003%, at most about 0.004%, at most about 0.005%, at most about 0.006%, at most about 0.007%, at most about 0.008% 0.009%, at most about 0.01%, at most about 0.02%, at most about 0.03%, at most about 0.04%, at most about 0.05%, at most about 0.06%, at most about 0.07%, at most about 0.08%, at most about 0.09%, at most about 0.1%, at most about 0.2%, at most about 0.3%, at most about 0.4%, at most about 0.5%, at most about 0.6%, at most about 0.7%, at most about 0.8%, at most about 0.9%, at most about 1%, at most about 1.1%, at most about 1.2%, at most about 1.3%, at most about 1.4%, at most about 1.5%, at most about 1.6%, at most about 1.7%, at most about 1.8%, at most about 1.9%, at most about 2%, at most about 2.1%, at most about 2.2%, at most about 2.3%, at most about 2.4%, at most about 2.5%, at most about 2.6%, at most about 2.7%, at most about 2.8%, at most about 2.9%, at most about 3%, at most about 3.1%, at most about 3.2%, at most about 3.3%, at most about 3.4%, at most about 3.5%, at most about 3.6%, at most about 3.7%, at most about 3.8%, at most about 3.9%, at most about 4%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, or at most about 10% CD3+cells as a percentage of total cells, as a percentage of nucleated cells, or as a percentage of CD45+cells.

[0176] In some embodiments, the first population of CD45+cells comprises less than about 5 EU of endotoxins per ml of the solution, less than about 1 EU of endotoxins per ml of the solution, and / or less than about 0.5 EU of endotoxins per ml of the solution.

[0177] In embodiments, at least one of the cell populations comprise less than about 5 EU of endotoxins / ml of respective suspension liquid.

[0178] A first population of CD45+cells can comprise 0.5 EU / ml endotoxins to 10 EU / ml endotoxins. A first population of CD45+cells can comprise at least 0.5 EU / ml endotoxins. A first population of CD45+cells can comprise at most 10 EU / ml endotoxins. A first population of CD45+cells can comprise 10 EU / ml endotoxins to 8 EU / ml endotoxins, 10 EU / ml endotoxins to 6 EU / ml endotoxins, 10 EU / ml endotoxins to 5 EU / ml endotoxins, 10 EU / ml endotoxins to 4 EU / ml endotoxins, 10 EU / ml endotoxins to 2 EU / ml endotoxins, 10 EU / ml endotoxins to 1 EU / ml endotoxins, 10 EU / ml endotoxins to 0.5 EU / ml endotoxins, 8 EU / ml endotoxins to 6 EU / ml endotoxins, 8 EU / ml endotoxins to 5 EU / ml endotoxins, 8 EU / ml endotoxins to 4 EU / ml endotoxins, 8 EU / ml endotoxins to 2 EU / ml endotoxins, 8 EU / ml endotoxins to 1 EU / ml endotoxins, 8 EU / ml endotoxins to 0.5 EU / ml endotoxins, 6 EU / ml endotoxins to 5 EU / ml endotoxins, 6 EU / ml endotoxins to 4 EU / ml endotoxins, 6 EU / ml endotoxins to 2 EU / ml endotoxins, 6 EU / ml endotoxins to 1 EU / ml endotoxins, 6 EU / ml endotoxins to 0.5 EU / ml endotoxins, 5 EU / ml64IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT endotoxins to 4 EU / ml endotoxins, 5 EU / ml endotoxins to 2 EU / ml endotoxins, 5 EU / ml endotoxins to 1 EU / ml endotoxins, 5 EU / ml endotoxins to 0.5 EU / ml endotoxins, 4 EU / ml endotoxins to 2 EU / ml endotoxins, 4 EU / ml endotoxins to 1 EU / ml endotoxins, 4 EU / ml endotoxins to 0.5 EU / ml endotoxins, 2 EU / ml endotoxins to 1 EU / ml endotoxins, 2 EU / ml endotoxins to 0.5 EU / ml endotoxins, or 1 EU / ml endotoxins to 0.5 EU / ml endotoxins. A first population of CD45+cells can comprise 10 EU / ml endotoxins, 8 EU / ml endotoxins, 6 EU / ml endotoxins, 5 EU / ml endotoxins, 4 EU / ml endotoxins, 2 EU / ml endotoxins, 1 EU / ml endotoxins, or 0.5 EU / ml endotoxins. A first population of CD45+cells can comprise at least 8 EU / ml endotoxins, 6 EU / ml endotoxins, 5 EU / ml endotoxins, 4 EU / ml endotoxins, 2 EU / ml endotoxins, 1 EU / ml endotoxins, or 0.5 EU / ml endotoxins. A first population of CD45+cells can comprise at most 10 EU / ml endotoxins, 8 EU / ml endotoxins, 6 EU / ml endotoxins, 5 EU / ml endotoxins, 4 EU / ml endotoxins, 2 EU / ml endotoxins or 1 EU / ml endotoxins.

[0179] A first population of CD45+cells can comprise 0.5% w / w to 10% w / w unbound reagents. These unbound reagents may include any affinity reagents used for the sorting of HSPCs, for instance, antibodies, or purification particles or magnetic particles. A first population of CD45+cells can comprise at least 0.5% w / w unbound reagents. A first population of CD45+cells can comprise at most 10% w / w unbound reagents. A first population of CD45+cells can comprise 10% w / w to 8% w / w, 10% w / w to 6% w / w, 10% w / w to 5% w / w, 10% w / w to 4% w / w, 10% w / w to 2% w / w, 10% w / w to 1% w / w, 10% w / w to 0.5% w / w, 8% w / w to 6% w / w, 8% w / w to 5% w / w, 8% w / w to 4% w / w, 8% w / w to 2% w / w, 8% w / w to 1% w / w, 8% w / w to 0.5% w / w, 6% w / w to 5% w / w, 6% w / w to 4% w / w, 6% w / w to 2% w / w, 6% w / w to 1% w / w, 6% w / w to 0.5% w / w, 5% w / w to 4% w / w, 5% w / w to 2% w / w, 5% w / w to 1% w / w, 5% w / w to 0.5% w / w, 4% w / w to 2% w / w, 4% w / w to 1% w / w, 4% w / w to 0.5% w / w, 2% w / w to 1% w / w, 2% w / w to 0.5% w / w, or 1% w / w to 0.5% w / w unbound reagents. A first population of CD45+cells can comprise 10% w / w, 8% w / w, 6% w / w, 5% w / w, 4% w / w, 2% w / w, 1% w / w, or 0.5% w / w unbound reagents. A first population of CD45+cells can comprise at least 8% w / w, 6% w / w, 5% w / w, 4% w / w, 2% w / w, 1% w / w, or 0.5% w / w unbound reagents. A first population of CD45+cells can comprise at most 10% w / w, 8% w / w, 6% w / w, 5% w / w, 4% w / w, 2% w / w or 1% w / w unbound reagents.

[0180] A first population of CD45+cells can comprise 5 xlO3to 90 xlO3microbeads per cell. These microbeads may comprise microbeads used to purify the HSPC population, for instance, an anti-CD34 antibody comprising microbead used to sort the HSPC population. A first population of CD45+cells can comprise at least 5 xlO3microbeads per cell. A first population of CD45+cells can comprise at most 90 xlO3microbeads per cell. A first population of CD45+cells can comprise 90 xlO3to 70 xlO3, 90 xlO3to 50 xlO3, 90 xlO3to 40 xlO3, 90 xlO3to 30 xlO3, 90 xlO3to 20 xlO3, 90 xlO3to 10 xlO3, 90 xlO3to 5 xlO3, 70 xlO3to 50 xlO3, 70 xlO3to 40 xlO3, 70 xlO3to 30 xlO3, 70 xlO3to 20 xlO3, 70 xlO3to 10 xlO3, 70 xlO3to 5 xlO3, 50 xlO3to 40 xlO3, 50 xlO3to 30 xlO3, 50 xlO3to 20 xlO3, 50 xlO3to 10 xlO3, 50 xlO3to 5 xlO3, 40 xlO3to 30 xlO3, 40 xlO3to 20 xlO3, 40 xlO3to 10 xlO3, 40 xlO3to 5 xlO3, 30 xlO3to 20 xlO3, 30 xlO3to 10 xlO3, 30 xlO3to 5 xlO3, 20 xlO3to 10 xlO3, 20 xlO3to 5 xlO3, or 10 xlO3to 5 xlO3microbeads per cell. A first population of CD45+cells can comprise 90 xlO3, 70 xlO3, 50 xlO3, 40 xlO3, 30 xlO3, 20 xlO3, 10 xlO3, or 5 xlO3microbeads per cell. A first population of CD45+cells can comprise at least 70 xlO3, 5065IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT xlO3, 40 xlO3, 30 xlO3, 20 xlO3, 10 xlO3, or 5 xlO3microbeads per cell. A first population of CD45+cells can comprise at most 90 xlO3, 70 xlO3, 50 xlO3, 40 xlO3, 30 xlO3, 20 xlO3, or 10 xlO3microbeads per cell.Tregs

[0181] A population of cells enriched for Tregs or comprising at least one dose of Tregs, e.g., a second population of CD45+cells comprising at least one dose of Tregs, can comprise at least about 1 x 104, at least about 1 x 105, at least about 2 x 104, at least about 3 x 104, at least about 4 x 104, at least about 5 x 104, at least about 6 x 104, at least about 7 x 104, at least about 8 x 104, least about 9 x 104, at least about 1 x 105, at least about 2 x 105, at least about 3 x 105, at least about 4 x 105, at least about 5 x 105, at least about 6 x 105, at least about 7 x 105, at least about 8 x 105, at least about 9 x 105, at least about 1 x 106, at least about 1.1 x 106, at least about 1.2 x 106, at least about 1.3 x 106, at least about 1.4 x 106, at least about 1.5 x 106, at least about 1.6 x 106, at least about 1.7 x 106, at least about 1.8 x 106, at least about 1.9 x 106, at least about 2 x 106, at least about 2.1 x 106, at least about 2.2 x 106, at least about 2.3 x 106, at least about 2.4 x 106, at least about 2.5 x 106, at least about 2.6 x 106, at least about 2.7 x 106, at least about 2.8 x 106, at least about 2.9 x 106, at least about 3 x 106, at least about 3.1 x 106, at least about 3.2 x 106, at least about 3.3 x 106, at least about 3.4 x 106, at least about 3.5 x 106, at least about 3.6 x 106, at least about 3.7 x 106, at least about 3.8 x 106, at least about 3.9 x 106, at least about 4 x 106, at least about 4.1 x 106, at least about 4.2 x 106, at least about 4.3 x 106, at least about 4.4 x 106, at least about 4.5 x 106, at least about 4.6 x 106, at least about 4.7 x 106, at least about 4.8 x 106, at least about 4.9 x 106, at least about 5 x 106, at least about 5.1 x 106, at least about 5.2 x 106, at least about 5.3 x 106, at least about 5.4 x 106, at least about 5.5 x 106, at least about 5.6 x 106, at least about 5.7 x 106, at least about 5.8 x 106, at least about 5.9 x 106, at least about 6 x 106, at least about 6.1 x 106, at least about 6.2 x 106, at least about 6.3 x 106, at least about 6.4 x 106, at least about 6.5 x 106, at least about 6.6 x 106, at least about 6.7 x 106, at least about 6.8 x 106, at least about 6.9 x 106, at least about 7 x 106, at least about 7.1 x 106, at least about 7.2 x 106, at least about 7.3 x 106, at least about 7.4 x 106, at least about 7.5 x 106, at least about 7.6 x 106, at least about 7.7 x 106, at least about 7.8 x 106, at least about 7.9 x 106, at least about 8 x 106, at least about 8.1 x 106, at least about 8.2 x 106, at least about 8.3 x 106, at least about 8.4 x 106, at least about 8.5 x 106, at least about 8.6 x 106, at least about 8.7 x 106, at least about 8.8 x 106, at least about 8.9 x 106, at least about 9 x 106, at least about 9.1 x 106, at least about 9.2 x 106, at least about 9.3 x 106, at least about 9.4 x 106, at least about 9.5 x 106, at least about 9.6 x 106, at least about 9.7 x 106, at least about 9.8 x 106, at least about 9.9 x 106, at least about 1 x 107, at least about 1.5 x 107, at least about 2 x 107, at least about 2.5 x 107, at least about 3 x 107, at least about 3.5 x 107, at least about 4 x 107, at least about 4.5 x 107, at least about 5 x 107, at least about 5.5 x 107, at least about 6 x 107, at least about 6.5 x 107, at least about 7 x 107, at least about 7.5 x 107, at least about 8 x 107, at least about 8.5 x 107, at least about 9 x 107, at least about 9.5 x 107, at least about 1 x IO8, at least about 1.5 x IO8, at least about 2 x IO8, at least about 2.5 x IO8, at least about 3 x IO8, at least about 3.5 x IO8, at least about 4 x 107, at least about 4.5 x IO8, at least about 5 x IO8, at least about 5.5 x IO8, at least about 6 x IO8, at least about 6.5 x IO8, at least about 7 x IO8, at least about 7.5 x IO8, at least about 8 x IO8, at least about 8.5 x IO8, at least about 9 x IO8, at least about 9.5 x IO8, at least about 1 x IO9, or more cells of the cell population enriched for Tregs e.g., the second population of CD45+cells) and / or Tregs or66IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT doses of Tregs per kg of recipient subject’s actual body weight or ideal body weight (e.g., where Tregs are CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+CD4+CD25+CD127dimFOXP3+, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim).

[0182] For example, a population of cells enriched for Tregs or comprising at least one dose of Tregs, e.g., a second population of CD45+cells comprising at least one dose of Tregs, can comprise 1 x 104to 1 x 109, 1 x 105to 1 x 108, 1 x 105to 2 x 107, 5 x 105to 2 x 107, 5 x 105to 1.5 x 107, 5 x 105to 1 x 107, 5 x 105to 9 x 106, 5 x 105to 8 x 106, 5 x 105to 7 x 106, 5 x 105to 6 x 106, 5 x 105to 5 x 106, 5 x 105to 4 x 106, 5 x 105to 3 x 106, 5 x 105to 2 x 106, 5 x 105to 1 x 106, 1 x 106to 1.5 x 107, 1 x 106to 1 x 107, 1 x 106to 9 x 106, 1 x 106to 8 x 106, 1 x 106to 7 x 106, 1 x 106to 6 x 106, 1 x 106to 5 x 106, 1 x 106to 4 x 106, 1 x 106to 3 x 106, 1 x 106to 2 x 106, 1.5 x 106to 1.5 x 107, 1.5 x 106to 1 x 107, 1.5 x 106to 9 x 106, 1.5 x 106to 8 x 106, 1.5 x 106to 7 x 106, 1.5 x 106to 6 x 106, 1.5 x 106to 5 x 106, 1.5 x 106to 4 x 106, 1.5 x 106to 3 x 106, 1.5 x 106to 2 x 106, 2 x 106to 1.5 x 107, 2 x 106to 1 x 107, 2 x 106to 9 x 106, 2 x 106to 8 x 106, 2 x 106to 7 x 106, 2 x 106to 6 x 106, 2 x 106to 5 x 106, 2 x 106to 4 x 106, 2 x 106to 3 x 106, 2.5 x 106to 1.5 x 107, 2.5 x 106to 1 x 107, 2.5 x 106to 9 x 106, 2.5 x 106to 8 x 106, 2.5 x 106to 7 x 106, 2.5 x 106to 6 x 106, 2.5 x 106to 5 x 106, 2.5 x 106to 4 x 106, or 2.5 x 106to 3 x 106cells of the cell population enriched for Tregs e.g., the second population of CD45+cells) and / or Tregs or doses of Tregs per kg of recipient subject’s actual body weight or ideal body weight e.g., where Tregs are CD4+CD25+CD127dlm, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+CD4+CD25+CD127dimFOXP3+, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim).

[0183] In some embodiments, the population of cells enriched for Tregs or comprising at least one dose of Tregs, e.g., a second population of CD45+cells comprising at least one dose of Tregs, comprises from approximately 1.0 x 105to approximately 5.0 x 109, from approximately 5.0 x 105to approximately 3.0 x 109, from approximately 1.5 x 107to approximately 3.0 x 109, or from approximately 5.0 x 105to approximately 1.0 x 108Tregs, e.g., fresh isolated Tregs in the population of cells enriched for Tregs. In some embodiments, the population of cells enriched for Tregs or comprising at least one dose of Tregs, e.g., a second population of CD45+ cells comprising at least one dose of Tregs, comprises approximately 1.0 x 105or more Tregs, approximately 2.0 x 105or more Tregs, approximately 3.0 x 105or more Tregs, approximately 4.0 x 105or more Tregs, approximately 5.0 x 105or more Tregs, approximately 6.0 x 105or more Tregs, approximately 7.0 x 105or more Tregs, approximately 8.0 x 105or more Tregs, approximately 9.0 x 105or more Tregs, approximately 1.0 x 106or more Tregs, approximately 1.1 x 106or more Tregs, approximately 1.2 x 106or more Tregs, approximately 1.3 x 106or more Tregs, approximately 1.4 x 106or more Tregs, approximately 1.5 x 106or more Tregs, approximately 1.6 x 106or more Tregs, approximately 1.7 x 106or more Tregs, approximately 1.8 x 106or more Tregs, approximately 1.9 x 106or more Tregs, approximately 2.0 x 106or more Tregs, approximately 2.1 x 106or more Tregs, approximately 2.2 x 106or67IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT more Tregs, approximately 2.3 x 106or more Tregs, approximately 2.4 x 106or more Tregs, approximately 2.5 x 106or more Tregs, approximately 2.6 x 106or more Tregs, approximately 2.7 x 106or more Tregs, approximately 2.8 x 106or more Tregs, approximately 2.9 x 106or more Tregs, approximately 3.0 x 106or more Tregs, approximately 3.1 x 106or more Tregs, approximately 3.2 x 106or more Tregs, approximately 3.3 x 106or more Tregs, approximately 3.4 x 106or more Tregs, approximately 3.5 x 106or more Tregs, approximately 3.6 x 106or more Tregs, approximately 3.7 x 106or more Tregs, approximately 3.8 x 106or more Tregs, approximately 3.9 x 106or more Tregs, approximately 4.0 x 106or more Tregs, approximately 4.1 x 106or more Tregs, approximately 4.2 x 106or more Tregs, approximately 4.3 x 106or more Tregs, approximately 4.4 x 106or more Tregs, approximately 4.5 x 106or more Tregs, approximately 4.6 x 106or more Tregs, approximately 4.7 x 106or more Tregs, approximately 4.8 x 106or more Tregs, approximately 4.9 x 106or more Tregs, approximately 5.0 x 106or more Tregs, approximately 5.1 x 106or more Tregs, approximately 5.2 x 106or more Tregs, approximately 5.3 x 106or more Tregs, approximately 5.4 x 106or more Tregs, approximately 5.5 x 106or more Tregs, approximately 5.6 x 106or more Tregs, approximately 5.7 x 106or more Tregs, approximately 5.8 x 106or more Tregs, approximately 5.9 x 106or more Tregs, approximately 6.0 x 106or more Tregs, approximately 6.1 x 106or more Tregs, approximately 6.2 x 106or more Tregs, approximately 6.3 x 106or more Tregs, approximately 6.4 x 106or more Tregs, approximately 6.5 x 106or more Tregs, approximately 6.6 x 106or more Tregs, approximately 6.7 x 106or more Tregs, approximately 6.8 x 106or more Tregs, approximately 6.9 x 106or more Tregs, approximately 7.0 x 106or more Tregs, approximately 7.1 x 106or more Tregs, approximately 7.2 x 106or more Tregs, approximately 7.3 x 106or more Tregs, approximately 7.4 x 106or more Tregs, approximately 7.5 x 106or more Tregs, approximately 7.6 x 106or more Tregs, approximately 7.7 x 106or more Tregs, approximately 7.8 x 106or more Tregs, approximately 7.9 x 106or more Tregs, approximately 8.0 x 106or more Tregs, approximately 8.1 x 106or more Tregs, approximately 8.2 x 106or more Tregs, approximately 8.3 x 106or more Tregs, approximately 8.4 x 106or more Tregs, approximately 8.5 x 106or more Tregs, approximately 8.6 x 106or more Tregs, approximately 8.7 x 106or more Tregs, approximately 8.8 x 106or more Tregs, approximately 8.9 x 106or more Tregs, approximately 9.0 x 106or more Tregs, approximately 9.1 x 106or more Tregs, approximately 9.2 x 106or more Tregs, approximately 9.3 x 106or more Tregs, approximately 9.4 x 106or more Tregs, approximately 9.5 x 106or more Tregs, approximately 9.6 x 106or more Tregs, approximately 9.7 x 106or more Tregs, approximately 9.8 x 106or more Tregs, approximately 9.9 x 106or more Tregs, approximately 1.0 x 107or more Tregs, approximately 1.1 x 107or more Tregs, approximately 1.2 x 107or more Tregs, approximately 1.3 x 107or more Tregs, approximately 1.4 x 107or more Tregs, approximately 1.5 x 107or more Tregs, approximately 1.6 x 107or more Tregs, approximately 1.7 x 107or more Tregs, approximately 1.8 x 107or more Tregs, approximately 1.9 x 107or more Tregs, approximately 2.0 x 107or more Tregs, approximately 2.1 x 107or more Tregs, approximately 2.2 x 107or more Tregs, approximately 2.3 x 107or more Tregs, approximately 2.4 x 107or more Tregs, approximately 2.5 x 107or more Tregs, approximately 2.6 x 107or more Tregs, approximately 2.7 x 107or more Tregs, approximately 2.8 x 107or more Tregs, approximately 2.9 x 107or more Tregs, approximately 3.0 x 107or more Tregs, approximately 3.1 x 107or more Tregs, approximately 3.2 x 107or more Tregs, approximately 3.3 x 107or more Tregs,68IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 3.4 x 107or more Tregs, approximately 3.5 x 107or more Tregs, approximately 3.6 x 107or more Tregs, approximately 3.7 x 107or more Tregs, approximately 3.8 x 107or more Tregs, approximately 3.9 x 107or more Tregs, approximately 4.0 x 107or more Tregs, approximately 4.1 x 107or more Tregs, approximately 4.2 x 107or more Tregs, approximately 4.3 x 107or more Tregs, approximately 4.4 x 107or more Tregs, approximately 4.5 x 107or more Tregs, approximately 4.6 x 107or more Tregs, approximately 4.7 x 107or more Tregs, approximately 4.8 x 107or more Tregs, approximately 4.9 x 107or more Tregs, approximately 5.0 x 107or more Tregs, approximately 5.1 x 107or more Tregs, approximately 5.2 x 107or more Tregs, approximately 5.3 x 107or more Tregs, approximately 5.4 x 107or more Tregs, approximately 5.5 x 107or more Tregs, approximately 5.6 x 107or more Tregs, approximately 5.7 x 107or more Tregs, approximately 5.8 x 107or more Tregs, approximately 5.9 x 107or more Tregs, approximately 6.0 x 107or more Tregs, approximately 6.1 x 107or more Tregs, approximately 6.2 x 107or more Tregs, approximately 6.3 x 107or more Tregs, approximately 6.4 x 107or more Tregs, approximately 6.5 x 107or more Tregs, approximately 6.6 x 107or more Tregs, approximately 6.7 x 107or more Tregs, approximately 6.8 x 107or more Tregs, approximately 6.9 x 107or more Tregs, approximately 7.0 x 107or more Tregs, approximately 7.1 x 107or more Tregs, approximately 7.2 x 107or more Tregs, approximately 7.3 x 107or more Tregs, approximately 7.4 x 107or more Tregs, approximately 7.5 x 107or more Tregs, approximately 7.6 x 107or more Tregs, approximately 7.7 x 107or more Tregs, approximately 7.8 x 107or more Tregs, approximately 7.9 x 107or more Tregs, approximately 8.0 x 107or more Tregs, approximately 8.1 x 107or more Tregs, approximately 8.2 x 107or more Tregs, approximately 8.3 x 107or more Tregs, approximately 8.4 x 107or more Tregs, approximately 8.5 x 107or more Tregs, approximately 8.6 x 107or more Tregs, approximately 8.7 x 107or more Tregs, approximately 8.8 x 107or more Tregs, approximately 8.9 x 107or more Tregs, approximately 9.0 x 107or more Tregs, approximately 9.1 x 107or more Tregs, approximately 9.2 x 107or more Tregs, approximately 9.3 x 107or more Tregs, approximately 9.4 x 107or more Tregs, approximately 9.5 x 107or more Tregs, approximately 9.6 x 107or more Tregs, approximately 9.7 x 107or more Tregs, approximately 9.8 x 107or more Tregs, approximately 9.9 x 107or more Tregs, approximately 1.0 x 108or more Tregs, approximately 1.1 x 108or more Tregs, approximately 1.2 x 108or more Tregs, approximately 1.3 x 108or more Tregs, approximately 1.4 x 108or more Tregs, approximately 1.5 x 108or more Tregs, approximately 1.6 x 108or more Tregs, approximately 1.7 x 108or more Tregs, approximately 1.8 x 108or more Tregs, approximately 1.9 x 108or more Tregs, approximately 2.0 x 108or more Tregs, approximately 2.1 x 108or more Tregs, approximately 2.2 x 108or more Tregs, approximately 2.3 x 108or more Tregs, approximately 2.4 x 108or more Tregs, approximately 2.5 x 108or more Tregs, approximately 2.6 x 108or more Tregs, approximately 2.7 x 108or more Tregs, approximately 2.8 x 108or more Tregs, approximately 2.9 x 108or more Tregs, approximately 3.0 x 108or more Tregs, approximately 3.1 x 108or more Tregs, approximately 3.2 x 108or more Tregs, approximately 3.3 x 108or more Tregs, approximately 3.4 x 108or more Tregs, approximately 3.5 x 108or more Tregs, approximately 3.6 x 108or more Tregs, approximately 3.7 x 108or more Tregs, approximately 3.8 x 108or more Tregs, approximately 3.9 x 108or more Tregs, approximately 4.0 x 108or more Tregs, approximately 4.1 x 108or more Tregs, approximately 4.2 x 108or more Tregs, approximately 4.3 x 108or more Tregs, approximately 4.4 x 108or more Tregs, approximately69IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 4.5 x IO8or more Tregs, approximately 4.6 x 108or more Tregs, approximately 4.7 x 108or more Tregs, approximately 4.8 x 108or more Tregs, approximately 4.9 x 108or more Tregs, approximately 5.0 x 108or more Tregs, approximately 5.1 x 108or more Tregs, approximately 5.2 x 108or more Tregs, approximately 5.3 x 108or more Tregs, approximately 5.4 x 108or more Tregs, approximately 5.5 x 108or more Tregs, approximately 5.6 x 108or more Tregs, approximately 5.7 x 108or more Tregs, approximately 5.8 x 108or more Tregs, approximately 5.9 x 108or more Tregs, approximately 6.0 x 108or more Tregs, approximately 6.1 x 108or more Tregs, approximately 6.2 x 108or more Tregs, approximately 6.3 x 108or more Tregs, approximately 6.4 x 108or more Tregs, approximately 6.5 x 108or more Tregs, approximately 6.6 x 108or more Tregs, approximately 6.7 x 108or more Tregs, approximately 6.8 x 108or more Tregs, approximately 6.9 x 108or more Tregs, approximately 7.0 x 108or more Tregs, approximately 7.1 x 108or more Tregs, approximately 7.2 x 108or more Tregs, approximately 7.3 x 108or more Tregs, approximately 7.4 x 108or more Tregs, approximately 7.5 x 108or more Tregs, approximately 7.6 x 108or more Tregs, approximately 7.7 x 108or more Tregs, approximately 7.8 x 108or more Tregs, approximately 7.9 x 108or more Tregs, approximately 8.0 x 108or more Tregs, approximately 8.1 x 108or more Tregs, approximately 8.2 x 108or more Tregs, approximately 8.3 x 108or more Tregs, approximately 8.4 x 108or more Tregs, approximately 8.5 x 108or more Tregs, approximately 8.6 x 108or more Tregs, approximately 8.7 x 108or more Tregs, approximately 8.8 x 108or more Tregs, approximately 8.9 x 108or more Tregs, approximately 9.0 x 108or more Tregs, approximately 9.1 x 108or more Tregs, approximately 9.2 x 108or more Tregs, approximately 9.3 x 108or more Tregs, approximately 9.4 x 108or more Tregs, approximately 9.5 x 108or more Tregs, approximately 9.6 x 108or more Tregs, approximately 9.7 x 108or more Tregs, approximately 9.8 x 108or more Tregs, approximately 9.9 x 108or more Tregs, approximately 1.0 x 109or more Tregs, approximately 1.1 x 109or more Tregs, approximately 1.2 x 109or more Tregs, approximately 1.3 x 109or more Tregs, approximately 1.4 x 109or more Tregs, approximately 1.5 x 109or more Tregs, approximately 1.6 x 109or more Tregs, approximately 1.7 x 109or more Tregs, approximately 1.8 x 109or more Tregs, approximately 1.9 x 109or more Tregs, approximately 2.0 x 109or more Tregs, approximately 2.1 x 109or more Tregs, approximately 2.2 x 109or more Tregs, approximately 2.3 x 109or more Tregs, approximately 2.4 x 109or more Tregs, approximately 2.5 x 109or more Tregs, approximately 2.6 x 109or more Tregs, approximately 2.7 x 109or more Tregs, approximately 2.8 x 109or more Tregs, approximately 2.9 x 109or more Tregs, approximately 3.0 x 109or more Tregs, approximately 3.1 x 109or more Tregs, approximately 3.2 x 109or more Tregs, approximately 3.3 x 109or more Tregs, approximately 3.4 x 109or more Tregs, approximately 3.5 x 109or more Tregs, approximately 3.6 x 109or more Tregs, approximately 3.7 x 109or more Tregs, approximately 3.8 x 109or more Tregs, approximately 3.9 x 109or more Tregs, approximately 4.0 x 109or more Tregs, approximately 4.1 x 109or more Tregs, approximately 4.2 x 109or more Tregs, approximately 4.3 x 109or more Tregs, approximately 4.4 x 109or more Tregs, approximately 4.5 x 109or more Tregs, approximately 4.6 x 109or more Tregs, approximately 4.7 x 109or more Tregs, approximately 4.8 x 109or more Tregs, approximately 4.9 x 109or more Tregs, or approximately 5.0 x 109or more Tregs. In some embodiments, the Tregs are CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+CD4+CD25+CD127dimFOXP3+, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+,70IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim.

[0184] A population of cells enriched for Tregs (e.g., the second population of CD45+cells) can have a defined level of purity for Treg cells. For example, a population of cells enriched for Tregs of the disclosure can comprise at least about 5%, at least about at least about 10%, at least about at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or more Treg cells as a percentage of total cells, as a percentage of nucleated cells, or as a percentage of CD45+cells (e.g., where Tregs are CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+CD4+CD25+CD127dimFOXP3+, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim).

[0185] A population of cells enriched for Tregs e.g., the second population of CD45+cells) of the disclosure can comprise 50% to 100%, 60% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 81% to 100%, 82% to 100%, 83% to 100%, 84% to 100%, 84% to 100%, 86% to 100%, 87% to 100%, 88% to 100%, 89% to 100%, 90% to 91%, 92% to 100%, 93% to 100%, 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 99.5% to 100%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, 81% to 99%, 82% to 99%, 83% to 99%, 84% to 99%, 85% to 99%, 86% to 99%, 87% to 99%, 88% to 99%, 89% to 99%, 90% to 99%, 91% to 99%, 92% to 99%, 94% to 99%, 95% to 99%, 96% to 97%, 98% to 99%, 50% to 98%, 60% to 98%, 70% to 98%, 80% to 98%, 81% to 98%, 82% to 98%, 83% to 98%, 84% to 98%, 85% to 98%, 86% to 98%, 87% to 98%, 88% to 98%, 89% to 98%, 90% to 98%, 91% to 98%, 92% to 98%, 94% to 98%, 95% to 98%, 96% to 97%, 98% to 98%, 50% to 97%, 60% to 97%, 70% to 97%, 80% to 97%, 81% to 97%, 82% to 97%, 83% to 97%, 84% to 97%, 85% to 97%, 86% to 97%, 87% to 97%, 88% to 97%, 89% to 97%, 90% to 97%, 91% to 97%, 92% to 97%, 94% to 97%, 95% to 97%, 96% to 97%, 50% to 96%, 60% to 96%, 70% to 96%, 80% to 96%, 81% to 96%, 82% to 96%, 83% to 96%, 84% to 96%, 85% to 96%, 86% to 96%, 87% to 96%, 88% to 96%, 89% to 96%, 90% to 96%, 91% to 96%, 92% to 96%, 94% to 96%, 95% to 96%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, 81% to 95%, 82% to 95%, 83% to 95%, 84% to 95%, 85% to 95%, 86% to 95%, 87% to 95%, 88% to 95%, 89% to 95%, 90% to 95%, 91% to 95%, 92% to 95%, or 94% to 95% Tregs as71IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT a percentage of total cells, nucleated cells, or CD45+cells (e.g., where the Tregs are CD4+CD25+CD127dlm, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+CD4+CD25+CD127dimFOXP3+, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim).

[0186] A population of cells enriched for Tregs of the disclosure e.g., the second population of CD45+cells) can have a defined level of contaminating non-Treg cells. In some embodiments, at most about 1 x 102, at most about 2 x 102, at most about 3 x 102, at most about 4 x 102, at most about 5 x 102, at most about 6 x 102, at most about 7 x 102, at most about 8 x 102, at most about 9 x 102, at most about 1 x 103, at most about 2 x 103, at most about 3 x 103, at most about 4 x 103, at most about 5 x 103, at most about 6 x 103, at most about 7 x 103, at most about 8 x 103, at most about 9 x 103, at most about 1 x 104, at most about 2 x 104, at most about 3 x 104, at most about 4 x 104, at most about 5 x 104, at most about 6 x 104, at most about 7 x 104, at most about 8 x 104, at most about 9 x 104, or at most about 1 x 105non-Treg cells per kg of body weight or ideal body weight are present in a population of cells enriched for Tregs of the disclosure, where non-Treg cells are FOXP3" or CD127+ / brlght.

[0187] In some embodiments, a population of cells enriched for Tregs of the disclosure e.g., the second population of CD45+cells) comprises at most about 0.001%, at most about 0.002%, at most about 0.003%, at most about 0.004%, at most about 0.005%, at most about 0.006%, at most about 0.007%, at most about 0.008% 0.009%, at most about 0.01%, at most about 0.02%, at most about 0.03%, at most about 0.04%, at most about 0.05%, at most about 0.06%, at most about 0.07%, at most about 0.08%, at most about 0.09%, at most about 0.1%, at most about 0.2%, at most about 0.3%, at most about 0.4%, at most about 0.5%, at most about 0.6%, at most about 0.7%, at most about 0.8%, at most about 0.9%, at most about 1%, at most about 1.1%, at most about 1.2%, at most about 1.3%, at most about 1.4%, at most about 1.5%, at most about 1.6%, at most about 1.7%, at most about 1.8%, at most about 1.9%, at most about 2%, at most about 2.1%, at most about 2.2%, at most about 2.3%, at most about 2.4%, at most about 2.5%, at most about 2.6%, at most about 2.7%, at most about 2.8%, at most about 2.9%, at most about 3%, at most about 3.1%, at most about 3.2%, at most about 3.3%, at most about 3.4%, at most about 3.5%, at most about 3.6%, at most about 3.7%, at most about 3.8%, at most about 3.9%, at most about 4%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, or at most about 10% non-Treg cells, where non-Treg cells are FOXP3" or CD1277brlght.

[0188] In various embodiments, the population of cells enriched for Tregs (e.g., the second population of CD45+cells) comprises less than about 5 EU of endotoxins per ml of the solution, less than about 1 EU of endotoxins per ml of the solution, and / or less than about 0.5 EU of endotoxins per ml of the soludon.

[0189] In some embodiments, at least one of the cell populations comprise less than about 5 EU of endotoxins / ml of respective suspension liquid.

[0190] A population of cells enriched for Tregs (e.g., the second population of CD45+cells) can comprise 0.5 EU / ml endotoxins to 10 EU / ml endotoxins. A population of cells enriched for Tregs can72IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT comprise at least 0.5 EU / ml endotoxins. A population of cells enriched for Tregs can comprise at most 10 EU / ml endotoxins. A population of cells enriched for Tregs can comprise 10 EU / ml endotoxins to 8 EU / ml endotoxins, 10 EU / ml endotoxins to 6 EU / ml endotoxins, 10 EU / ml endotoxins to 5 EU / ml endotoxins, 10 EU / ml endotoxins to 4 EU / ml endotoxins, 10 EU / ml endotoxins to 2 EU / ml endotoxins, 10 EU / ml endotoxins to 1 EU / ml endotoxins, 10 EU / ml endotoxins to 0.5 EU / ml endotoxins, 8 EU / ml endotoxins to 6 EU / ml endotoxins, 8 EU / ml endotoxins to 5 EU / ml endotoxins, 8 EU / ml endotoxins to 4 EU / ml endotoxins, 8 EU / ml endotoxins to 2 EU / ml endotoxins, 8 EU / ml endotoxins to 1 EU / ml endotoxins, 8 EU / ml endotoxins to 0.5 EU / ml endotoxins, 6 EU / ml endotoxins to 5 EU / ml endotoxins, 6 EU / ml endotoxins to 4 EU / ml endotoxins, 6 EU / ml endotoxins to 2 EU / ml endotoxins, 6 EU / ml endotoxins to 1 EU / ml endotoxins, 6 EU / ml endotoxins to 0.5 EU / ml endotoxins, 5 EU / ml endotoxins to 4 EU / ml endotoxins, 5 EU / ml endotoxins to 2 EU / ml endotoxins, 5 EU / ml endotoxins to 1 EU / ml endotoxins, 5 EU / ml endotoxins to 0.5 EU / ml endotoxins, 4 EU / ml endotoxins to 2 EU / ml endotoxins, 4 EU / ml endotoxins to 1 EU / ml endotoxins, 4 EU / ml endotoxins to 0.5 EU / ml endotoxins, 2 EU / ml endotoxins to 1 EU / ml endotoxins, 2 EU / ml endotoxins to 0.5 EU / ml endotoxins, or 1 EU / ml endotoxins to 0.5 EU / ml endotoxins. A population of cells enriched for Tregs can comprise 10 EU / ml endotoxins, 8 EU / ml endotoxins, 6 EU / ml endotoxins, 5 EU / ml endotoxins, 4 EU / ml endotoxins, 2 EU / ml endotoxins, 1 EU / ml endotoxins, or 0.5 EU / ml endotoxins. A population of cells enriched for Tregs can comprise at least 8 EU / ml endotoxins, 6 EU / ml endotoxins, 5 EU / ml endotoxins, 4 EU / ml endotoxins, 2 EU / ml endotoxins, 1 EU / ml endotoxins, or 0.5 EU / ml endotoxins. A population of cells enriched for Tregs can comprise at most 10 EU / ml endotoxins, 8 EU / ml endotoxins, 6 EU / ml endotoxins, 5 EU / ml endotoxins, 4 EU / ml endotoxins, 2 EU / ml endotoxins or 1 EU / ml endotoxins.

[0191] A population of cells enriched for Tregs (e.g., the second population of CD45+cells) can comprise 0.5% w / w to 10% w / w unbound reagents. These unbound reagents may include any affinity reagents used for the sorting of Tregs, for instance, antibodies, or purification panicles or magnetic particles. A population of cells enriched for Tregs can comprise at least 0.5% w / w unbound reagents. A population of cells enriched for Tregs can comprise at most 10% w / w unbound reagents. A population of cells enriched for Tregs can comprise 10% w / w to 8% w / w, 10% w / w to 6% w / w, 10% w / w to 5% w / w, 10% w / w to 4% w / w, 10% w / w to 2% w / w, 10% w / w to 1% w / w, 10% w / w to 0.5% w / w, 8% w / w to 6% w / w, 8% w / w to 5% w / w, 8% w / w to 4% w / w, 8% w / w to 2% w / w, 8% w / w to 1% w / w, 8% w / w to 0.5% w / w, 6% w / w to 5% w / w, 6% w / w to 4% w / w, 6% w / w to 2% w / w, 6% w / w to 1% w / w, 6% w / w to 0.5% w / w, 5% w / w to 4% w / w, 5% w / w to 2% w / w, 5% w / w to 1% w / w, 5% w / w to 0.5% w / w, 4% w / w to 2% w / w, 4% w / w to 1% w / w, 4% w / w to 0.5% w / w, 2% w / w to 1% w / w, 2% w / w to 0.5% w / w, or 1% w / w to 0.5% w / w unbound reagents. A population of cells enriched for Tregs can comprise 10% w / w, 8% w / w, 6% w / w, 5% w / w, 4% w / w, 2% w / w, 1% w / w, or 0.5% w / w unbound reagents. A population of cells enriched for Tregs can comprise at least 8% w / w, 6% w / w, 5% w / w, 4% w / w, 2% w / w, 1% w / w, or 0.5% w / w unbound reagents. A population of cells enriched for Tregs can comprise at most 10% w / w, 8% w / w, 6% w / w, 5% w / w, 4% w / w, 2% w / w or 1% w / w unbound reagents.73IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT

[0192] A population of cells enriched for Tregs (e.g., the second population of CD45+cells) can comprise 1 xlO3to 50 xlO3microbeads per cell. These microbeads may comprise microbeads used to purify the Treg population, for instance, an anti-CD25 antibody comprising microbead used to sort the Treg population, or an anti-CD4 antibody comprising microbead used to sort the Treg population, and / or an anti-CD127 antibody comprising microbead used to sort the Treg population. A population of cells enriched for Tregs can comprise at least 1 xlO3microbeads per cell. A population of cells enriched for Tregs can comprise at most 50 xlO3microbeads per cell. A population of cells enriched for Tregs can comprise 50 xlO3to 40 xlO3, 50 xlO3to 30 xlO3, 50 xlO3to 20 xlO3, 50 xlO3to 10 xlO3, 50 xlO3to 5 xlO3, 50 xlO3to 4 xlO3, 50 xlO3to 2 xlO3, 50 xlO3to 1 xlO3, 40 xlO3to 30 xlO3, 40 xlO3to 20 xlO3, 40 xlO3to 10 xlO3, 40 xlO3to 5 xlO3, 40 xlO3to 4 xlO3, 40 xlO3to 2 xlO3, 40 xlO3to 1 xlO3, 30 xlO3to 20 xlO3, 30 xlO3to 10 xlO3, 30 xlO3to 5 xlO3, 30 xlO3to 4 xlO3, 30 xlO3to 2 xlO3, 30 xlO3to 1 xlO3, 20 xlO3to 10 xlO3, 20 xlO3to 5 xlO3, 20 xlO3to 4 xlO3, 20 xlO3to 2 xlO3, 20 xlO3to 1 xlO3, 10 xlO3to 5 xlO3, 10 xlO3to 4 xlO3, 10 xlO3to 2 xlO3, 10 xlO3to 1 xlO3, 5 xlO3to 4 xlO3, 5 xlO3to 2 xlO3, 5 xlO3to 1 xlO3, 4 xlO3to 2 xlO3, 4 xlO3to 1 xlO3, or 2 xlO3to 1 xlO3microbeads per cell. A population of cells enriched for Tregs can comprise 50 xlO3, 40 xlO3, 30 xlO3, 20 xlO3, 10 xlO3, 5 xlO3, 4 xlO3, 2 xlO3, or 1 xlO3microbeads per cell.Icons

[0193] A third population of CD45+cells that comprises, at least, Tcons or at least one dose of Tcons, can comprise at least about 1 x 104, at least about 2 x 104, at least about 3 x 104, at least about 4 x 104, at least about 5 x 104, at least about 6 x 104, at least about 7 x 104, at least about 8 x 104, at least about 9 x 104, at least about 1 x 105, at least about 2 x 105, at least about 3 x 105, at least about 4 x 105, at least about 5 x 105, at least about 6 x 105, at least about 7 x 105, at least about 8 x 105, at least about 9 x 105, at least about 1 x 106, at least about 1.1 x 106, at least about 1.2 x 106, at least about 1.3 x 106, at least about 1.4 x 106, at least about 1.5 x 106, at least about 1.6 x 106, at least about 1.7 x 106, at least about 1.8 x 106, at least about 1.9 x 106, at least about 2 x 106, at least about 2.1 x 106, at least about 2.2 x 106, at least about 2.3 x 106, at least about 2.4 x 106, at least about 2.5 x 106, at least about 2.6 x 106, at least about 2.7 x 106, at least about 2.8 x 106, at least about 2.9 x 106, at least about 3 x 106, at least about 3.1 x 106, at least about 3.2 x 106, at least about 3.3 x 106, at least about 3.4 x 106, at least about 3.5 x 106, at least about 3.6 x 106, at least about 3.7 x 106, at least about 3.8 x 106, at least about 3.9 x 106, at least about 4 x 106, at least about 4.1 x 106, at least about 4.2 x 106, at least about 4.3 x 106, at least about 4.4 x 106, at least about 4.5 x 106, at least about 4.6 x 106, at least about 4.7 x 106, at least about 4.8 x 106, at least about 4.9 x 106, at least about 5 x 106, at least about 5.1 x 106, at least about 5.2 x 106, at least about 5.3 x 106, at least about 5.4 x 106, at least about 5.5 x 106, at least about 5.6 x 106, at least about 5.7 x 106, at least about 5.8 x 106, at least about 5.9 x 106, at least about 6 x 106, at least about 6.1 x 106, at least about 6.2 x 106, at least about 6.3 x 106, at least about 6.4 x 106, at least about 6.5 x 106, at least about 6.6 x 106, at least about 6.7 x 106, at least about 6.8 x 106, at least about 6.9 x 106, at least about 7 x 106, at least about 7.1 x 106, at least about 7.2 x 106, at least about 7.3 x 106, at least about 7.4 x 106, at least about 7.5 x 106, at least about 7.6 x 106, at least about 7.7 x 106, at least about 7.8 x 106, at least about 7.9 x 106, at least about 8 x 106, at least about 8.1 x 106, at least74IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT about 8.2 x 106, at least about 8.3 x 106, at least about 8.4 x 106, at least about 8.5 x 106, at least about 8.6 x 106, at least about 8.7 x 106, at least about 8.8 x 106, at least about 8.9 x 106, at least about 9 x 106, at least about 9.1 x 106, at least about 9.2 x 106, at least about 9.3 x 106, at least about 9.4 x 106, at least about 9.5 x 106, at least about 9.6 x 106, at least about 9.7 x 106, at least about 9.8 x 106, at least about 9.9 x 106, at least about 1 x 107, at least about 1.5 x 107, at least about 2 x 107, at least about 2.5 x 107, at least about 3 x 107, at least about 3.5 x 107, at least about 4 x 107, at least about 4.5 x 107, at least about 5 x 107, at least about 5.5 x 107, at least about 6 x 107, at least about 6.5 x 107, at least about 7 x 107, at least about 7.5 x 107, at least about 8 x 107, at least about 8.5 x 107, at least about 9 x 107, at least about 9.5 x 107, at least about 1 x IO8, at least about 1.5 x IO8, at least about 2 x IO8, at least about 2.5 x IO8, at least about 3 x IO8, at least about 3.5 x IO8, at least about 4 x 107, at least about 4.5 x IO8, at least about 5 x IO8, at least about 5.5 x IO8, at least about 6 x IO8, at least about 6.5 x IO8, at least about 7 x IO8, at least about 7.5 x IO8, at least about 8 x IO8, at least about 8.5 x IO8, at least about 9 x IO8, at least about 9.5 x IO8, at least about 1 x IO9, or more cells of the third population of CD45+cells and / or Tcons or doses of Tcons per kg of recipient subject’s actual body weight or ideal body weight (e.g., where Tcons are CD3+or CD3+CD127+ / brlght).

[0194] For example, a third population of CD45+cells that comprises, at least, Tcons or at least one dose of Tcons, can comprise 1 x 104to 1 x 109, 1 x 105to 1 x 108, 1 x 105to 2 x 107, 5 x 105to 2 x 107, 5 x 105to 1.5 x 107, 5 x 105to 1 x 107, 5 x 105to 9 x 106, 5 x 105to 8 x 106, 5 x 105to 7 x 106, 5 x 105to 6 x 106, 5 x 105to 5 x 106, 5 x 105to 4 x 106, 5 x 105to 3 x 106, 5 x 105to 2 x 106, 5 x 105to 1 x 106, 1 x 106to 1.5 x 107, 1 x 106to 1 x 107, 1 x 106to 9 x 106, 1 x 106to 8 x 106, 1 x 106to 7 x 106, 1 x 106to 6 x 106, 1 x 106to 5 x 106, 1 x 106to 4 x 106, 1 x 106to 3 x 106, 1 x 106to 2 x 106, 1.5 x 106to 1.5 x 107, 1.5 x 106to 1 x 107, 1.5 x 106to 9 x 106, 1.5 x 106to 8 x 106, 1.5 x 106to 7 x 106, 1.5 x 106to 6 x 106, 1.5 x 106to 5 x 106, 1.5 x 106to 4 x 106, 1.5 x 106to 3 x 106, 1.5 x 106to 2 x 106, 2 x 106to 1.5 x 107, 2 x 106to 1 x 107, 2 x 106to 9 x 106, 2 x 106to 8 x 106, 2 x 106to 7 x 106, 2 x 106to 6 x 106, 2 x 106to 5 x 106, 2 x 106to 4 x 106, 2 x 106to 3 x 106, 2.5 x 106to 1.5 x 107, 2.5 x 106to 1 x 107, 2.5 x 106to 9 x 106, 2.5 x 106to 8 x 106, 2.5 x 106to 7 x 106, 2.5 x 106to 6 x 106, 2.5 x 106to 5 x 106, 2.5 x 106to 4 x 106, or 2.5 x 106to 3 x 106cells of the third population of CD45+cells and / or Tcons or doses of Tcons per kg of recipient subject’s actual body weight or ideal body weight (e.g., where Tcons are CD3+or CD3+CD127+ / brlght).

[0195] In some embodiments, the third population of CD45+cells comprising Tcons, or comprising at least one does of Tcons, comprises from approximately 1.0 x 105to approximately 1.0 x IO10, from approximately 5.0 x 105to approximately 6.0 x 109, from approximately 5.0 x 105to approximately 1.0 x 108, or from approximately 1.5 x 107to approximately 6.0 x 109Tcons, e.g., in the third population of CD45+cells. In some embodiments, the third population of CD45+cells comprising Tcons, or comprising at least one dose of Tcons, comprises approximately 1.0 x 105or more Tcons, approximately 2.0 x 105or more Tcons, approximately 3.0 x 105or more Tcons, approximately 4.0 x 105or more Tcons, approximately 5.0 x 105or more Tcons, approximately 6.0 x 105or more Tcons, approximately 7.0 x 105or more Tcons, approximately 8.0 x 105or more Tcons, approximately 9.0 x 105or more Tcons, approximately 1.0 x 106or more Tcons, approximately 1.0 x 106or more Tcons, approximately 1.1 x 106or more Tcons, approximately 1.2 x 106or more Tcons, approximately 1.3 x 106or more Tcons,75IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 1.4 x 106or more Tcons, approximately 1.5 x 106or more Tcons, approximately 1.6 x 106or more Tcons, approximately 1.7 x 106or more Tcons, approximately 1.8 x 106or more Tcons, approximately 1.9 x 106or more Tcons, approximately 2.0 x 106or more Tcons, approximately 2.1 x 106or more Tcons, approximately 2.2 x 106or more Tcons, approximately 2.3 x 106or more Tcons, approximately 2.4 x 106or more Tcons, approximately 2.5 x 106or more Tcons, approximately 2.6 x 106or more Tcons, approximately 2.7 x 106or more Tcons, approximately 2.8 x 106or more Tcons, approximately 2.9 x 106or more Tcons, approximately 3.0 x 106or more Tcons, approximately 3.1 x 106or more Tcons, approximately 3.2 x 106or more Tcons, approximately 3.3 x 106or more Tcons, approximately 3.4 x 106or more Tcons, approximately 3.5 x 106or more Tcons, approximately 3.6 x 106or more Tcons, approximately 3.7 x 106or more Tcons, approximately 3.8 x 106or more Tcons, approximately 3.9 x 106or more Tcons, approximately 4.0 x 106or more Tcons, approximately 4.1 x 106or more Tcons, approximately 4.2 x 106or more Tcons, approximately 4.3 x 106or more Tcons, approximately 4.4 x 106or more Tcons, approximately 4.5 x 106or more Tcons, approximately 4.6 x 106or more Tcons, approximately 4.7 x 106or more Tcons, approximately 4.8 x 106or more Tcons, approximately 4.9 x 106or more Tcons, approximately 5.0 x 106or more Tcons, approximately 5.1 x 106or more Tcons, approximately 5.2 x 106or more Tcons, approximately 5.3 x 106or more Tcons, approximately 5.4 x 106or more Tcons, approximately 5.5 x 106or more Tcons, approximately 5.6 x 106or more Tcons, approximately 5.7 x 106or more Tcons, approximately 5.8 x 106or more Tcons, approximately 5.9 x 106or more Tcons, approximately 6.0 x 106or more Tcons, approximately 6.1 x 106or more Tcons, approximately 6.2 x 106or more Tcons, approximately 6.3 x 106or more Tcons, approximately 6.4 x 106or more Tcons, approximately 6.5 x 106or more Tcons, approximately 6.6 x 106or more Tcons, approximately 6.7 x 106or more Tcons, approximately 6.8 x 106or more Tcons, approximately 6.9 x 106or more Tcons, approximately 7.0 x 106or more Tcons, approximately 7.1 x 106or more Tcons, approximately 7.2 x 106or more Tcons, approximately 7.3 x 106or more Tcons, approximately 7.4 x 106or more Tcons, approximately 7.5 x 106or more Tcons, approximately 7.6 x 106or more Tcons, approximately 7.7 x 106or more Tcons, approximately 7.8 x 106or more Tcons, approximately 7.9 x 106or more Tcons, approximately 8.0 x 106or more Tcons, approximately 8.1 x 106or more Tcons, approximately 8.2 x 106or more Tcons, approximately 8.3 x 106or more Tcons, approximately 8.4 x 106or more Tcons, approximately 8.5 x 106or more Tcons, approximately 8.6 x 106or more Tcons, approximately 8.7 x 106or more Tcons, approximately 8.8 x 106or more Tcons, approximately 8.9 x 106or more Tcons, approximately 9.0 x 106or more Tcons, approximately 9.1 x 106or more Tcons, approximately 9.2 x 106or more Tcons, approximately 9.3 x 106or more Tcons, approximately 9.4 x 106or more Tcons, approximately 9.5 x 106or more Tcons, approximately 9.6 x 106or more Tcons, approximately 9.7 x 106or more Tcons, approximately 9.8 x 106or more Tcons, approximately 9.9 x 106or more Tcons, approximately 1.0 x 107or more Tcons, approximately 1.1 x 107or more Tcons, approximately 1.2 x 107or more Tcons, approximately 1.3 x 107or more Tcons, approximately 1.4 x 107or more Tcons, approximately 1.5 x 107or more Tcons, approximately 1.6 x 107or more Tcons, approximately 1.7 x 107or more Tcons, approximately 1.8 x 107or more Tcons,76IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 1.9 x 107or more Tcons, approximately 2.0 x 107or more Tcons, approximately 2.1 x 107or more Tcons, approximately 2.2 x 107or more Tcons, approximately 2.3 x 107or more Tcons, approximately 2.4 x 107or more Tcons, approximately 2.5 x 107or more Tcons, approximately 2.6 x 107or more Tcons, approximately 2.7 x 107or more Tcons, approximately 2.8 x 107or more Tcons, approximately 2.9 x 107or more Tcons, approximately 3.0 x 107or more Tcons, approximately 3.1 x 107or more Tcons, approximately 3.2 x 107or more Tcons, approximately 3.3 x 107or more Tcons, approximately 3.4 x 107or more Tcons, approximately 3.5 x 107or more Tcons, approximately 3.6 x 107or more Tcons, approximately 3.7 x 107or more Tcons, approximately 3.8 x 107or more Tcons, approximately 3.9 x 107or more Tcons, approximately 4.0 x 107or more Tcons, approximately 4.1 x 107or more Tcons, approximately 4.2 x 107or more Tcons, approximately 4.3 x 107or more Tcons, approximately 4.4 x 107or more Tcons, approximately 4.5 x 107or more Tcons, approximately 4.6 x 107or more Tcons, approximately 4.7 x 107or more Tcons, approximately 4.8 x 107or more Tcons, approximately 4.9 x 107or more Tcons, approximately 5.0 x 107or more Tcons, approximately 5.1 x 107or more Tcons, approximately 5.2 x 107or more Tcons, approximately 5.3 x 107or more Tcons, approximately 5.4 x 107or more Tcons, approximately 5.5 x 107or more Tcons, approximately 5.6 x 107or more Tcons, approximately 5.7 x 107or more Tcons, approximately 5.8 x 107or more Tcons, approximately 5.9 x 107or more Tcons, approximately 6.0 x 107or more Tcons, approximately 6.1 x 107or more Tcons, approximately 6.2 x 107or more Tcons, approximately 6.3 x 107or more Tcons, approximately 6.4 x 107or more Tcons, approximately 6.5 x 107or more Tcons, approximately 6.6 x 107or more Tcons, approximately 6.7 x 107or more Tcons, approximately 6.8 x 107or more Tcons, approximately 6.9 x 107or more Tcons, approximately 7.0 x 107or more Tcons, approximately 7.1 x 107or more Tcons, approximately 7.2 x 107or more Tcons, approximately 7.3 x 107or more Tcons, approximately 7.4 x 107or more Tcons, approximately 7.5 x 107or more Tcons, approximately 7.6 x 107or more Tcons, approximately 7.7 x 107or more Tcons, approximately 7.8 x 107or more Tcons, approximately 7.9 x 107or more Tcons, approximately 8.0 x 107or more Tcons, approximately 8.1 x 107or more Tcons, approximately 8.2 x 107or more Tcons, approximately 8.3 x 107or more Tcons, approximately 8.4 x 107or more Tcons, approximately 8.5 x 107or more Tcons, approximately 8.6 x 107or more Tcons, approximately 8.7 x 107or more Tcons, approximately 8.8 x 107or more Tcons, approximately 8.9 x 107or more Tcons, approximately 9.0 x 107or more Tcons, approximately 9.1 x 107or more Tcons, approximately 9.2 x 107or more Tcons, approximately 9.3 x 107or more Tcons, approximately 9.4 x 107or more Tcons, approximately 9.5 x 107or more Tcons, approximately 9.6 x 107or more Tcons, approximately 9.7 x 107or more Tcons, approximately 9.8 x 107or more Tcons, approximately 9.9 x 107or more Tcons, approximately 1.0 x 108or more Tcons, approximately 1.1 x 108or more Tcons, approximately 1.2 x 108or more Tcons, approximately 1.3 x 108or more Tcons, approximately 1.4 x 108or more Tcons, approximately 1.5 x 108or more Tcons, approximately 1.6 x 108or more Tcons, approximately 1.7 x 108or more Tcons, approximately 1.8 x 108or more Tcons, approximately 1.9 x 108or more Tcons, approximately 2.0 x 108or more Tcons, approximately 2.1 x 108or more Tcons, approximately 2.2 x 108or more Tcons, approximately 2.3 x 108or more Tcons,77IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 2.4 x 108or more Tcons, approximately 2.5 x 108or more Tcons, approximately 2.6 x 108or more Tcons, approximately 2.7 x 108or more Tcons, approximately 2.8 x 108or more Tcons, approximately 2.9 x 108or more Tcons, approximately 3.0 x 108or more Tcons, approximately 3.1 x 108or more Tcons, approximately 3.2 x 108or more Tcons, approximately 3.3 x 108or more Tcons, approximately 3.4 x 108or more Tcons, approximately 3.5 x 108or more Tcons, approximately 3.6 x 108or more Tcons, approximately 3.7 x 108or more Tcons, approximately 3.8 x 108or more Tcons, approximately 3.9 x 108or more Tcons, approximately 4.0 x 108or more Tcons, approximately 4.1 x 108or more Tcons, approximately 4.2 x 108or more Tcons, approximately 4.3 x 108or more Tcons, approximately 4.4 x 108or more Tcons, approximately 4.5 x 108or more Tcons, approximately 4.6 x 108or more Tcons, approximately 4.7 x 108or more Tcons, approximately 4.8 x 108or more Tcons, approximately 4.9 x 108or more Tcons, approximately 5.0 x 108or more Tcons, approximately 5.1 x 108or more Tcons, approximately 5.2 x 108or more Tcons, approximately 5.3 x 108or more Tcons, approximately 5.4 x 108or more Tcons, approximately 5.5 x 108or more Tcons, approximately 5.6 x 108or more Tcons, approximately 5.7 x 108or more Tcons, approximately 5.8 x 108or more Tcons, approximately 5.9 x 108or more Tcons, approximately 6.0 x 108or more Tcons, approximately 6.1 x 108or more Tcons, approximately 6.2 x 108or more Tcons, approximately 6.3 x 108or more Tcons, approximately 6.4 x 108or more Tcons, approximately 6.5 x 108or more Tcons, approximately 6.6 x 108or more Tcons, approximately 6.7 x 108or more Tcons, approximately 6.8 x 108or more Tcons, approximately 6.9 x 108or more Tcons, approximately 7.0 x 108or more Tcons, approximately 7.1 x 108or more Tcons, approximately 7.2 x 108or more Tcons, approximately 7.3 x 108or more Tcons, approximately 7.4 x 108or more Tcons, approximately 7.5 x 108or more Tcons, approximately 7.6 x 108or more Tcons, approximately 7.7 x 108or more Tcons, approximately 7.8 x 108or more Tcons, approximately 7.9 x 108or more Tcons, approximately 8.0 x 108or more Tcons, approximately 8.1 x 108or more Tcons, approximately 8.2 x 108or more Tcons, approximately 8.3 x 108or more Tcons, approximately 8.4 x 108or more Tcons, approximately 8.5 x 108or more Tcons, approximately 8.6 x 108or more Tcons, approximately 8.7 x 108or more Tcons, approximately 8.8 x 108or more Tcons, approximately 8.9 x 108or more Tcons, approximately 9.0 x 108or more Tcons, approximately 9.1 x 108or more Tcons, approximately 9.2 x 108or more Tcons, approximately 9.3 x 108or more Tcons, approximately 9.4 x 108or more Tcons, approximately 9.5 x 108or more Tcons, approximately 9.6 x 108or more Tcons, approximately 9.7 x 108or more Tcons, approximately 9.8 x 108or more Tcons, approximately 9.9 x 108or more Tcons, approximately 1.0 x 109or more Tcons, approximately 1.1 x 109or more Tcons, approximately 1.2 x 109or more Tcons, approximately 1.3 x 109or more Tcons, approximately 1.4 x 109or more Tcons, approximately 1.5 x 109or more Tcons, approximately 1.6 x 109or more Tcons, approximately 1.7 x 109or more Tcons, approximately 1.8 x 109or more Tcons, approximately 1.9 x 109or more Tcons, approximately 2.0 x 109or more Tcons, approximately 2.1 x 109or more Tcons, approximately 2.2 x 109or more Tcons, approximately 2.3 x 109or more Tcons, approximately 2.4 x 109or more Tcons, approximately 2.5 x 109or more Tcons, approximately 2.6 x 109or more Tcons, approximately 2.7 x 109or more Tcons, approximately 2.8 x 109or more Tcons,78IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT approximately 2.9 x 109or more Tcons, approximately 3.0 x 109or more Tcons, approximately 3.1 x 109or more Tcons, approximately 3.2 x 109or more Tcons, approximately 3.3 x 109or more Tcons, approximately 3.4 x 109or more Tcons, approximately 3.5 x 109or more Tcons, approximately 3.6 x 109or more Tcons, approximately 3.7 x 109or more Tcons, approximately 3.8 x 109or more Tcons, approximately 3.9 x 109or more Tcons, approximately 4.0 x 109or more Tcons, approximately 4.1 x 109or more Tcons, approximately 4.2 x 109or more Tcons, approximately 4.3 x 109or more Tcons, approximately 4.4 x 109or more Tcons, approximately 4.5 x 109or more Tcons, approximately 4.6 x 109or more Tcons, approximately 4.7 x 109or more Tcons, approximately 4.8 x 109or more Tcons, approximately 4.9 x 109or more Tcons, approximately 5.0 x 109or more Tcons, approximately 5.1 x 109or more Tcons, approximately 5.2 x 109or more Tcons, approximately 5.3 x 109or more Tcons, approximately 5.4 x 109or more Tcons, approximately 5.5 x 109or more Tcons, approximately 5.6 x 109or more Tcons, approximately 5.7 x 109or more Tcons, approximately 5.8 x 109or more Tcons, approximately 5.9 x 109or more Tcons, approximately 6.0 x 109or more Tcons, approximately 6.1 x 109or more Tcons, approximately 6.2 x 109or more Tcons, approximately 6.3 x 109or more Tcons, approximately 6.4 x 109or more Tcons, approximately 6.5 x 109or more Tcons, approximately 6.6 x 109or more Tcons, approximately 6.7 x 109or more Tcons, approximately 6.8 x 109or more Tcons, approximately 6.9 x 109or more Tcons, approximately 7.0 x 109or more Tcons, approximately 7.1 x 109or more Tcons, approximately 7.2 x 109or more Tcons, approximately 7.3 x 109or more Tcons, approximately 7.4 x 109or more Tcons, approximately 7.5 x 109or more Tcons, approximately 7.6 x 109or more Tcons, approximately 7.7 x 109or more Tcons, approximately 7.8 x 109or more Tcons, approximately 7.9 x 109or more Tcons, approximately 8.0 x 109or more Tcons, approximately 8.1 x 109or more Tcons, approximately 8.2 x 109or more Tcons, approximately 8.3 x 109or more Tcons, approximately 8.4 x 109or more Tcons, approximately 8.5 x 109or more Tcons, approximately 8.6 x 109or more Tcons, approximately 8.7 x 109or more Tcons, approximately 8.8 x 109or more Tcons, approximately 8.9 x 109or more Tcons, approximately 9.0 x 109or more Tcons, approximately 9.1 x 109or more Tcons, approximately 9.2 x 109or more Tcons, approximately 9.3 x 109or more Tcons, approximately 9.4 x 109or more Tcons, approximately 9.5 x 109or more Tcons, approximately 9.6 x 109or more Tcons, approximately 9.7 x 109or more Tcons, approximately 9.8 x 109or more Tcons, approximately 9.9 x 109or more Tcons, or approximately 1.0 x 1010or more Tcons. In some embodiments, the Tcons are CD3+or CD3+CD127+ / bright.

[0196] A third population of CD45+cells of the disclosure can have a defined level of purity for Tcon cells. For example, a third population of CD45+cells of the disclosure can comprise at least about 5%, at least about at least about 10%, at least about at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about79IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or more Tcon cells as a percentage of total cells, as a percentage of nucleated cells, or as a percentage of CD45+cells (e.g., where Tcons are CD3+or CD3+CD127+ / brlght).

[0197] A third population of CD45+cells of the disclosure can comprise 50% to 100%, 60% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 81% to 100%, 82% to 100%, 83% to 100%, 84% to 100%, 84% to 100%, 86% to 100%, 87% to 100%, 88% to 100%, 89% to 100%, 90% to 91%, 92% to 100%, 93% to 100%, 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 99.5% to 100%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, 81% to 99%, 82% to 99%, 83% to 99%, 84% to 99%, 85% to 99%, 86% to 99%, 87% to 99%, 88% to 99%, 89% to 99%, 90% to 99%, 91% to 99%, 92% to 99%, 94% to 99%, 95% to 99%, 96% to 97%, 98% to 99%, 50% to 98%, 60% to 98%, 70% to 98%, 80% to 98%, 81% to 98%, 82% to 98%, 83% to 98%, 84% to 98%, 85% to 98%, 86% to 98%, 87% to 98%, 88% to 98%, 89% to 98%, 90% to 98%, 91% to 98%, 92% to 98%, 94% to 98%, 95% to 98%, 96% to 97%, 98% to 98%, 50% to 97%, 60% to 97%, 70% to 97%, 80% to 97%, 81% to 97%, 82% to 97%, 83% to 97%, 84% to 97%, 85% to 97%, 86% to 97%, 87% to 97%, 88% to 97%, 89% to 97%, 90% to 97%, 91% to 97%, 92% to 97%, 94% to 97%, 95% to 97%, 96% to 97%, 50% to 96%, 60% to 96%, 70% to 96%, 80% to 96%, 81% to 96%, 82% to 96%, 83% to 96%, 84% to 96%, 85% to 96%, 86% to 96%, 87% to 96%, 88% to 96%, 89% to 96%, 90% to 96%, 91% to 96%, 92% to 96%, 94% to 96%, 95% to 96%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, 81% to 95%, 82% to 95%, 83% to 95%, 84% to 95%, 85% to 95%, 86% to 95%, 87% to 95%, 88% to 95%, 89% to 95%, 90% to 95%, 91% to 95%, 92% to 95%, or 94% to 95%, Tcons as a percentage of total cells, nucleated cells, or CD45+cells (e.g., where Tcons are CD3+or CD3+CD127+ / bright).

[0198] A third population of CD45+cells of the disclosure can have a defined level of contaminating non-Tcon cells. In some embodiments, at most about 1 x 102, at most about 2 x 102, at most about 3 x 102, at most about 4 x 102, at most about 5 x 102, at most about 6 x 102, at most about 7 x 102, at most about 8 x 102, at most about 9 x 102, at most about 1 x 103, at most about 2 x 103, at most about 3 x 103, at most about 4 x 103, at most about 5 x 103, at most about 6 x 103, at most about 7 x 103, at most about 8 x 103, at most about 9 x 103, at most about 1 x 104, at most about 2 x 104, at most about 3 x 104, at most about 4 x 104, at most about 5 x 104, at most about 6 x 104, at most about 7 x 104, at most about 8 x 104, at most about 9 x 104, or at most about 1 x 105non-Tcon cells per kg of recipient subject’s actual body weight or ideal body weight are present in a third population of CD45+cells of the disclosure, e.g., where non-Tcon cells are CD3 or CD3+CD127dim).

[0199] In some embodiments, a third population of CD45+cells of the disclosure comprises at most about 0.001%, at most about 0.002%, at most about 0.003%, at most about 0.004%, at most about 0.005%, at most about 0.006%, at most about 0.007%, at most about 0.008% 0.009%, at most about 0.01%, at most80IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT about 0.02%, at most about 0.03%, at most about 0.04%, at most about 0.05%, at most about 0.06%, at most about 0.07%, at most about 0.08%, at most about 0.09%, at most about 0.1%, at most about 0.2%, at most about 0.3%, at most about 0.4%, at most about 0.5%, at most about 0.6%, at most about 0.7%, at most about 0.8%, at most about 0.9%, at most about 1%, at most about 1.1%, at most about 1.2%, at most about 1.3%, at most about 1.4%, at most about 1.5%, at most about 1.6%, at most about 1.7%, at most about 1.8%, at most about 1.9%, at most about 2%, at most about 2.1%, at most about 2.2%, at most about 2.3%, at most about 2.4%, at most about 2.5%, at most about 2.6%, at most about 2.7%, at most about 2.8%, at most about 2.9%, at most about 3%, at most about 3.1%, at most about 3.2%, at most about 3.3%, at most about 3.4%, at most about 3.5%, at most about 3.6%, at most about 3.7%, at most about 3.8%, at most about 3.9%, at most about 4%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, or at most about 10% non-Tcon cells, (e.g., where non-Tcon cells are CD3 or CD3+CD127dim).

[0200] In some cases, the population of cells enriched for Tregs comprises less than about 5 EU of endotoxins per ml of the solution, less than about 1 EU of endotoxins per ml of the solution, and / or less than about 0.5 EU of endotoxins per ml of the solution.

[0201] In embo...

Claims

1. Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT CLAIMSWhat is claimed is:

1. A multi-component cellular therapy product comprising:a) a first population of isolated CD45+ cells comprising CD34+ hematopoietic stem and progenitor cells (HSPCs);b) a second population of isolated CD45+ cells comprising isolated fresh regulatory T cells (Tregs);c) a third population of isolated CD45+ cells comprising conventional CD3+ T cells (Tcons); andd) a pharmaceutical composition comprising a Janus kinase (JAK) inhibitor, optionally a JAK1 / 2 inhibitor.

2. The multi-component cellular therapy product of claim 1, wherein the first population of isolated CD45+ cells comprises a dose of CD34+ HSPCs that ranges from approximately 1.0 x 105or more CD34+ HSPCs per kilogram of body weight of a human subject receiving the product to approximately 1.0 x 108or more CD34+ HSPCs per kilogram of body weight of the human subject receiving the product.

3. The multi-component cellular therapy product of claim 1 or claim 2, wherein the first population of isolated CD45+ cells comprises a dose of CD34+ HSPCs that ranges from approximately 5.0 x 105or more CD34+ HSPCs to approximately 1.5 x 1010or more CD34+ HSPCs, optionally wherein the dose of CD34+ HSPCs comprises at least 1.0 x 106cells per patient kilogram.

4. The multi-component cellular therapy product of any one of claims 1-3, wherein the second population of isolated CD45+ cells comprises a dose of isolated fresh Tregs that ranges from approximately 1.0 x 105or more isolated fresh Tregs per kilogram of body weight of a human subject receiving the product to approximately 2.0 x 107or more isolated fresh Tregs per kilogram of body weight of the human subject receiving the product, optionally wherein the dose of isolated fresh Tregs is from 0.9 x 106to 3.5 x 106cells per patient kilogram.

5. The multi-component cellular therapy product of any one of claims 1-4, wherein the second population of isolated CD45+ cells comprises a dose of isolated fresh Tregs that ranges from approximately 5.0 x 105or more isolated fresh Tregs to approximately 3.0 x 109or more isolated fresh Tregs.

6. The multi-component cellular therapy product of any one of claims 1-5, wherein the Tregs are CD4+CD25+CD127dim or CD4+FOXP3+.

7. The multi-component cellular therapy product of any one of claims 1-6, wherein the third population of isolated CD45+ cells comprises a dose of CD3+ Tcons that ranges from approximately 1.0 x 105or more CD3+ Tcons per kilogram of body weight of a human subject receiving the product to approximately 4.0 x 107or more CD3+ Tcons per kilogram of body weight of the human subject 210IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT receiving the product, optionally wherein the dose of CD3+ Tcons is from 0.9 x 106to 6.9 x 106cells per patient kilogram.

8. The multi-component cellular therapy product of any one of claims 1-7, wherein the third population of isolated CD45+ cells comprises a dose of CD3+ Tcons that ranges from approximately 5.0 x 105or more CD3+ Tcons to approximately 6.0 x 109or more CD3+ Tcons.

9. The multi-component cellular therapy product of any one of claims 1-8, wherein the pharmaceutical composition comprises the JAK inhibitor at a dose that ranges from approximately 2 mg to approximately 20 mg twice per day, optionally 5mg twice per day.

10. The multi-component cellular therapy product of any one of claims 1-9, wherein the JAK inhibitor dosage comprises approximately 5mg twice per day and tapers to approximately 5mg once per day after approximately 4 weeks and to zero after approximately 8 weeks.

11. The multi-component cellular therapy product of any one of claims 1-10, wherein the JAK inhibitor is a JAK1 / 2 inhibitor, a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor, a JAK1 / 3 inhibitor, a JAK2 / 3 inhibitor, a JAK1 / 2 / 3 inhibitor, a TYK2 inhibitor, or any combination thereof.

12. The multi-component cellular therapy product of any one of claims 1-10, wherein the JAK inhibitor is selected from the group consisting of: ruxolilinib, tofacitinib, oclacitinib, baricilinib, pcl'icilinib, upadacitinib, fedratinib, filgotinib, abrocilinib, pacritinib, dcucravacilinib, rillecilinib, momelotinib, cerdulatinib, gandotinib, leslaurlinib, , decernotinib, solcitinib, itacitinib, SHR0302, delgocitinib, cucurbitacin I, CHZ868, XL019, AZDI 480, BMS911543, and ilginatinib.

13. The multi-component cellular therapy product of any one of claims 1-12, wherein the human subject receiving the product has received, is receiving, or scheduled to receive a graft vs host disease (GVHD) prophylactic agent.

14. The multi-component cellular therapy product of claim 13, wherein the GVHD prophylactic agent is selected from the group consisting of: tacrolimus, cyclosporine A, sirolimus, basiliximab, daclizumab, methotrexate, muromonab-CD3, mycophenolate, anti-thymocyte globulin, corticosteroids, azathioprine, and mycophenolate mofetil.

15. The multi-component cellular therapy product of claim 13 or claim 14, wherein the dose of the GVHD prophylactic agent is sufficient to maintain a trough blood level of approximately 5 ng / mL to approximately 10 ng / mL in a human subject receiving the product.

16. The multi-component cellular therapy product of any one of claims 13-15, wherein the second pharmaceutical composition comprises the GVHD prophylactic agent at a dose that ranges from approximately 0.01 mg per kilogram of body weight of a human subject receiving the product to approximately 0.50 mg per kilogram of body weight of a human subject receiving the product twice per day, optionally wherein the GVHD prophylactic comprises tacrolimus at 0.03 mg / kg per day, further optionally 0.015 mg / kg per day administered twice a day IV.211IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 17. The multi-component cellular therapy product of any one of claims 1-16, wherein the first population of isolated CD45+ cells, the second population of isolated CD45+ cells, and / or the third population of isolated CD45+ cells is formulated at a volume that ranges from approximately 5 mL to approximately 1 L.

18. The multi-component cellular therapy product of any one of claims 1-17, wherein the first population of isolated CD45+ cells, the second population of isolated CD45+ cells, and / or the third population of isolated CD45+ cells is formulated with an excipient at a neutral pH.

19. The multi-component cellular therapy product of claim 18, wherein the neutral pH ranges from approximately 6.8 to approximately 7.6.

20. The multi-component cellular therapy product of claim 18 or claim 19, wherein the excipient comprises a transport buffer.

21. The multi-component cellular therapy product of claim 20, wherein the transport buffer comprises approximately 120 to approximately 160 mEq sodium.

22. The multi-component cellular therapy product of claim 20 or claim 21, wherein the transport buffer comprises approximately 270 to approximately 320 mOsmol / L total.

23. The multi-component cellular therapy product of any one of claims 20-22, wherein the transport buffer is selected from the group consisting of: phosphate-buffered saline (PBS), human serum, PlasmaLyte, and any combination thereof.

24. The multi-component cellular therapy product of any one of claims 20-23, wherein the transport buffer further comprises approximately 0.1% weight by volume to approximately 10% weight by volume of a human carrier protein.

25. The multi-component cellular therapy product of claim 24, wherein the human carrier protein is selected from the group consisting of: human serum albumin (HSA), intravenous immune globulin (IVIG), AB serum, and any combination thereof.

26. The multi-component cellular therapy product of any one of claims 1-25, the first population of isolated CD45+ cells, the second population of isolated CD45+ cells, and / or the third population of isolated CD45+ cells is formulated in a single dose transfer bag.

27. The multi-component cellular therapy product of claim 26, wherein the single dose transfer bag is a polyvinyl chloride (PVC) transfer bag or an ethylene vinyl acetate (EVA) transfer bag.

28. The multi-component cellular therapy product of any one of claims 1-27, wherein the third population of isolated CD45+ cells is formulated with an excipient comprising one more cryoprotectants.

29. The multi-component cellular therapy product of claim 28, wherein the one or more cryoprotectants are selected from the group consisting of: sorbitol, dimethyl sulfoxide (DMSO),212IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT propylene glycol, glycerol, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG), serum, HSA, hetastarch, CRYOSTOR CS2, CRYOSTOR CS5, and CRYOSTOR CS10.

30. The multi-component cellular therapy product of any one of claims 1-29, wherein the first population of isolated CD45+ cells, the second population of isolated CD45+ cells, and the third population of isolated CD45+ cells are from an allogeneic donor having at least one HLA mismatch relative to a human subject receiving the product.

31. The multi-component cellular therapy product of claim 30, wherein the HLA-mismatched donor is unrelated to a human subject receiving the product.

32. The multi-component cellular therapy product of claim 30, wherein the HLA-mismatched donor is related to a human subject receiving the product.

33. The multi-component cellular therapy product of any one of claims 30-32, wherein the at least one HLA mismatch is at an allele selected from the group consisting of: HLA- A, HLA-B, HLA-C, HLA-DRB 1 , and any combination thereof.

34. The multi-component cellular therapy product of any one of claims 30-33, wherein the cells having at least one HLA mismatch are from a donor that is 6 / 8 HLA-mismatched relative to a human subject receiving the product or is 7 / 8 HLA-mismatched relative to a human subject receiving the product.

35. The multi-component cellular therapy product of claim 34, wherein the cells having at least one HLA mismatch are from a donor that is 7 / 8 HLA-mismatched relative to a human subject receiving the product.

36. The multi-component cellular therapy product of claim 35, wherein the donor that is 7 / 8 HLA-mismatched relative to a human subject receiving the product has a mismatch in HLA-A.

37. The multi-component cellular therapy product of claim 35, wherein the donor that is 7 / 8 HLA-mismatched relative to a human subject receiving the product has a mismatch in HLA-B.

38. The multi-component cellular therapy product of claim 35, wherein the donor that is 7 / 8 HLA-mismatched relative to a human subject receiving the product has a mismatch in HLA-C.

39. The multi-component cellular therapy product of claim 35, wherein the donor that is 7 / 8 HLA-mismatched relative to a human subject receiving the product has a mismatch in HLA-DRB1.

40. The multi-component cellular therapy product of any one of claims 30-39, wherein the donor that has the at least one HLA mismatch relative to a human subject receiving the product has a mismatched HLA allele as a result of the donor being homozygous for the HLA allele while a human subject receiving the product is heterogeneous for the HLA allele.

41. The multi-component cellular therapy product of any one of claims 30-39, wherein the donor that has at least one HLA mismatch relative to a human subject receiving the product has a mismatched HLA213IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT allele as a result of the donor being heterozygous for the HL A allele while a human subject receiving the product is homozygous for the HLA allele.

42. The multi-component cellular therapy product of any one of claims 30-39, wherein the donor that has at least one HLA mismatch relative to a human subject receiving the product has a mismatched HLA allele as a result of both the donor and a human subject receiving the product being heterozygous for the HLA allele.

43. The multi-component cellular therapy product of any one of claims 1-42, wherein upon administration the first population of isolated CD45+ cells, the second population of isolated CD45+ cells, and the third population of isolated CD45+ cells decrease incidence of non-relapse mortality in a human subject receiving the product and / or increase overall survival in a human subject receiving the product compared to incidence of non-relapse mortality and / or over survival in a corresponding human subject that has been administered a standard myeloablative allogeneic hematopoietic stem cell transplant (alloHSCT) that is 7 / 8 HLA-mismatched relative to the corresponding human subject.

44. The multi-component cellular therapy product of any one of claims 1-43, wherein the human subject receiving the product has received, is receiving, or scheduled to receive tacrolimus at 0.03 mg / kg per day, further optionally 0.015 mg / kg per day administered twice a day IV, and has received, is receiving, or scheduled to receive ruxolitinib at 5mg twice per day.

45. The multi-component cellular therapy product of any one of claims 1-44, wherein the body weight of a human subject receiving the product is actual body weight.

46. The multi-component cellular therapy product of any one of claims 1-44, wherein the body weight of a human subject receiving the product is ideal body weight.

47. A method of treating a human subject having or suspected of having a hematologic malignancy, the method comprising administering to a human subject the multi-component cellular therapy product of any one of claims 1-46.

48. A method of treating a human subject having or suspected of having a hematologic malignancy, the method comprising administering to a human subject a multi-component pharmaceutical treatment comprising:a) a soludon comprising a first population of isolated CD45+ cells comprising hematopoietic stem and progenitor cells (HSPCs);b) a solution comprising a second population of isolated CD45+ cells comprising regulatory T cells (Tregs);c) a solution comprising a third population of isolated CD45+ cells wherein the third population of isolated CD45+ cells comprise CD3+ conventional T cells (Tcons); andd) a solution comprising one or more doses of a Janus kinase (JAK) inhibitor.

49. The method of claim 47 or claim 48, wherein the hematologic malignancy is selected from the group consisting of: leukemia, acute leukemia, acute myeloid leukemia (AML), acute lymphoid leukemia 214IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT (ALL), mixed phenotype acute leukemia (MP AL), chronic myelogenous leukemia (CML), multiple myeloma, lymphoma, Hodgkin’s lymphoma, non-Hodgkin lymphoma, myelodysplastic syndrome, myeloproliferative syndrome, myelofibrosis, blastic plasmacytoid dendritic cell neoplasm (BPDCN), and any combinations thereof.

50. The method of any one of claims 47-49, wherein the administering comprises infusing into a human subject the first population of isolated CD45+ cells, the second population of isolated CD45+ cells, and the third population of isolated CD45+ cells.

51. The method of any one of claims 47-50, wherein the third population of isolated CD45+ cells is administered from approximately 12 hours to approximately 120 hours after the first population of isolated CD45+ cells.

52. The method of any one of claims 47-51, wherein the third population of isolated CD45+ cells is administered from approximately 36 hours to approximately 72 hours after the first population of isolated CD45+ cells.

53. The method of any one of claims 47-52, wherein the third population of isolated CD45+ cells is administered from approximately 12 hours to approximately 120 hours after the second population of isolated CD45+ cells.

54. The method of any one of claims 47-53, wherein the third population of isolated CD45+ cells is administered from approximately 36 to approximately 72 hours after the second population of isolated CD45+ cells.

55. The method of any one of claims 47-54, wherein the HSPCs are CD34+.

56. The method of any one of claims 47-55, wherein the first population of isolated CD45+ cells comprises from approximately 5 x 105HSPCs per kilogram of actual or ideal body weight of a human subject to approximately 2 x 107HSPCs per kilogram of actual or ideal body weight of the human subject.

57. The method of any one of claims 47-56, wherein the Tregs are CD4+CD25+CD127dim or CD4+FOXP3+.

58. The method of any one of claims 47-57, wherein in the second population of isolated CD45+ cells more than approximately 90% of the CD45+ cells are Tregs.

59. The method of any one of claims 47-58, wherein the second population of isolated CD45+ cells comprises from approximately 1 x 105Tregs per kilogram of actual or ideal body weight of the human subject to approximately 1 x 107Tregs per kilogram of actual or ideal body weight of the human subject.

60. The method of any one of claims 47-59, wherein the third population of isolated CD45+ cells comprises from approximately 1 x 105Tcons per kilogram of actual or ideal body weight of the human subject to approximately 1 x 107Tcons per kilogram of actual or ideal body weight of the human subject.215IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 61. The method of any one of claims 47-60, wherein the human subject does not develop higher than stage 2 GVHD within approximately 100 days of the administering of the third population of isolated CD45+ cells.

62. The method of any one of claims 47-61, wherein the human subject does not develop higher than stage 2 GVHD within approximately 180 days or within approximately 200 days of the administering of the third population of isolated CD45+ cells.

63. The method of any one of claims 47-62, wherein the human subject does not develop higher than stage 2 GVHD within approximately 1 year of the administering of the third population of isolated CD45+ cells.

64. The method of any one of claims 47-63, wherein the human subject has previously been or is concurrently being treated for the hematologic malignancy.

65. The method of any one of claims 47-64, wherein the JAK inhibitor is a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor, a TYK2 inhibitor, or any combination thereof.

66. The method of any one of claims 47-64, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricilinib, peficitinib, upadacitinib, Icdralinib, filgotinib, abrocilinib, pacritinib, deucravacitinib, ritlecitinib, momelotinib, cerdulatinib, gandolinib, and lestaurtinib.

67. The method of any one of claims 47-66, wherein the JAK inhibitor is initially administered to the human subject at a dose that ranges from approximately 2 mg to approximately 20 mg twice per day.

68. The method of any one of claims 47-67, wherein the JAK inhibitor dosage comprises 5 mg twice per day.

69. The method of any one of claims 47-68, wherein the JAK inhibitor is initially administered from approximately 2 days to approximately 5 days after the administering of the third population of isolated CD45+ cells.

70. The method of any one of claims 47-69, wherein the JAK inhibitor is administered for at least approximately 15 days, at least approximately 30 days, at least approximately 45 days, at least approximately 60 days, or at least approximately 90 days after administering the third population of isolated CD45+ cells.

71. The method of any one of claims 47-70, wherein administration of the JAK inhibitor is tapered starting at approximately 30 days after initial administration of the JAK inhibitor.

72. The method of claim 71, wherein administration of the JAK inhibitor is tapered to 5mg once per day starting at approximately 30 days after inilial administration of the JAK inhibitor.

73. The method of claim 71 or claim 72, wherein administration of the JAK inhibitor is tapered to zero at approximately 60 days after initial administration of the JAK inhibitor.216IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 74. The method of any one of claims 47-73, wherein the method further comprises administering a graft vs host disease (GVHD) prophylactic agent.

75. The method of claim 74, wherein the GVHD prophylactic agent is selected from the group consisting of: tacrolimus, cyclosporine A, sirolimus, basiliximab, daclizumab, methotrexate, muromonab-CD3, mycophenolate, anti-thymocyte globulin, corticosteroids, azathioprine, and mycophenolate mofetil.

76. The method of claim 74 or claim 75, wherein the GVHD prophylactic agent is administered at a dose sufficient to maintain a trough blood level of approximately 5 ng / mL to approximately 10 ng / mL in the human subject.

77. The method of any one of claims 74-76, wherein the GVHD prophylactic agent is administered at a dose that ranges from approximately 0.01 mg per kilogram of body weight of the human subject to approximately 0.50 mg per kilogram of body weight of the human subject.

78. The method of any one of claims 74-77, wherein the GVHD prophylactic agent is initially administered from approximately 12 hours to approximately 2 days after the administering of the third population of isolated CD45+ cells.

79. The method of any one of claims 74-78, wherein the GVHD prophylactic agent is administered for at least approximately 60 days, at least approximately 90 days, at least approximately 120 days, or at most approximately 160 days after administering the third population of isolated CD45+ cells.

80. The method of any one of claims 74-79, wherein administration of the GVHD prophylactic agent is tapered starting at approximately 60 days, approximately 70 days, approximately 80 days, approximately 90 days, approximately 100 days, approximately 110 days, or approximately 1200 days after initial administration of the GVHD prophylactic agent.

81. The method of any one of claims 74-80, wherein the GVHD prophylactic agent is initially administered to the human subject at a dose that ranges from approximately 0.01 mg to approximately 0.50 mg per kilogram of body weight of the human subject twice per day.

82. The method of any one of claims 74-81, wherein the dose of the GVHD prophylactic agent is sufficient to maintain a trough blood level of approximately 1 ng / mL to approximately 10 ng / mL in the human subject.

83. The method of any one of claims 74-82, wherein the GVHD prophylactic agent is administered approximately 1 day prior to adminislralion of the JAK inhibitor.

84. The method of any one of claims 47-83, wherein the method further comprises administering tacrolimus at 0.03 mg / kg per day, further optionally 0.015 mg / kg per day administered twice a day IV, and administering ruxolitinib at 5mg twice per day..

85. The method of claim 84, wherein the MMF is initially administered from approximately 12 hours to approximately 24 hours after the administering of the third population of isolated CD45+ cells.217IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 86. The method of claim 84 or claim 85, wherein administration of the MMF is tapered starting at approximately 30 days, at approximately 35 days, at approximately 40 days, at approximately 41 days, at approximately 42 days, at approximately 43 days, at approximately 44 days, at approximately 45 days, at approximately 46 days, at approximately 47 days, at approximately 48 days, at approximately 49 days, or at approximately 50 days after initial administration of the MMF.

87. The method of any one of claims 47-86, wherein the single allogeneic donor that has at least one HLA mismatch is unrelated to the human subject.

88. The method of any one of claims 47-87, wherein the single allogeneic donor that has at least one HLA mismatch is related to the human subject.

89. The method of any one of claims 47-88, wherein the method further comprises collecting one or more, or two or more mobilized peripheral blood donations from the donor.

90. The method of any one of claims 47-89, wherein the method further comprises collecting at most two mobilized peripheral blood donations from the donor.

91. The method of claim 89 or claim 90, wherein the peripheral blood donations are mobilized by granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), plerixafor, or any combination thereof.

92. The method of any one of claims 47-91, wherein the at least one HLA mismatch is at an allele selected from the group consisting of: HLA-A, HLA-B, HLA-C, HLA-DRB1, and any combination thereof.

93. The method of any one of claims 47-92, wherein the allogeneic donor that has at least one HLA mismatch is 6 / 8 HLA-mismatched relative to the human subject or is 7 / 8 HLA-mismatched relative to the human subject.

94. The method of claim 93, wherein the allogeneic donor is 7 / 8 HLA-mismatched relative to the human subject.

95. The method of claim 94, wherein the allogeneic donor that is 7 / 8 HLA-mismatched relative to the human subject has a mismatch in HLA-A.

96. The method of claim 94, wherein the allogeneic donor that is 7 / 8 HLA-mismatched relative to the human subject has a mismatch in HLA-B.

97. The method of claim 94, wherein the allogeneic donor that is 7 / 8 HLA-mismatched relative to the human subject has a mismatch in HLA-C.

98. The method of claim 94, wherein the allogeneic donor that is 7 / 8 HLA-mismatched relative to the human subject has a mismatch in HLA-DRB 1.218IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 99. The method of any one of claims 47-98, wherein the allogeneic donor that has at least one HLA mismatch relative to the human subject has a mismatched HLA allele as a result of the allogeneic donor being homozygous for the HLA allele while the human subject is heterogeneous for the HLA allele.

100. The method of any one of claims 47-99, wherein the allogeneic donor that has at least one HLA mismatch relative to the human subject has a mismatched HLA allele as a result of the allogeneic donor being heterozygous for the HLA allele while the human subject is homozygous for the HLA allele.

101. The method of any one of claims 47-100, wherein the allogeneic donor that has at least one HLA mismatch relative to the human subject has a mismatched HLA allele as a result of both the allogeneic donor and the human subject being heterozygous for the HLA allele.

102. The method of any one of claims 47-101, wherein the method further comprises a conditioning regimen, wherein the conditioning regimen is administered before administration of the first population of isolated CD45+ cells, the second population of isolated CD45+ cells, and / or the third population of isolated CD45+ cells.

103. The method of claim 102, wherein the conditioning regimen is administered from approximately two days to approximately ten days before administration of the first population of isolated CD45+ cells, the second population of isolated CD45+ cells, and / or the third population of isolated CD45+ cells.

104. The method of any one of claims 48-103, wherein the method further comprises a conditioning regimen, wherein the conditioning regimen is administered before any of (a) to (d).

105. The method of claim 104, wherein the conditioning regimen is administered from approximately two days to approximately ten days before any of (a) to (d).

106. The method of any one of claims 102-105, wherein the conditioning regimen is a myeloablative conditioning regimen.

107. The method of claim 106, wherein the myeloablative conditioning regimen comprises at least three conditioning reagents, wherein at least one conditioning reagent comprises thiotepa.

108. The method of claim 106, wherein the myeloablative conditioning regimen comprises one or more doses of busulfan, fludarabine and thiotepa.

109. The method of claim 108, wherein the one or more doses of busulfan, fludarabine and thiotepa comprise from approximately 5 to approximately 12 mg of thiotepa per kg human subject’s actual or ideal body weight, from approximately 7 to approximately 11 mg of busulfan per kg human subject actual or ideal body weight, and from approximately 100 to approximately 200 mg of fludarabine per meter2 body surface area respectively.

110. The method of claim 106, wherein the myeloablative conditioning regimen comprises a total body irradiation-based (TBI-based) regimen.219IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT 111. The method of claim 110, wherein the TBI-based regimen comprises fractionated total body irradiation (fTBI).

112. The method of claim 111, wherein the fTBI comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more doses.

113. The method of claim 111 or claim 112, wherein the fTBI comprises a total dose that ranges from approximately 500 cGy to approximately 1600 cGy.

114. The method of any one of claims 111-113, wherein the fTBI-based regimen comprises 4 doses of TBI.

115. The method of any one of claims 110-114, wherein the TBI-based regimen further comprises one or more conditioning reagents.

116. The method of claim 115, wherein the one or more conditioning reagents are selected from the group consisting of: cyclophosphamide, etoposide, thiotepa, and any combination thereof.

117. The method of claim 115 or claim 116, wherein the TBI-based regimen further comprises one or more doses of cyclophosphamide.

118. The method of claim 117, wherein each of the one or more doses of cyclophosphamide comprise from approximately 10 mg to approximately 100 mg of cyclophosphamide per kilogram of body weight of the human subject.

119. A cellular therapy kit comprising the multi-component cellular therapy product of any one of claims 1-46.

120. The cellular therapy kit of claim 119, wherein the kit further comprises written instructions for using the cellular therapy for treating a hematologic malignancy in a human subject.

121. A unit dose comprising the multi-component cellular therapy product of any one of claims 1-46.

122. An article of manufacture comprising the multi-component cellular therapy product of any one of claims 1-46.

123. The method of any one of the above claims, wherein the subject does not receive fluconazole at a dose of greater than 200 mg daily.

124. A multi-component cellular therapy product comprising:a) a first population of isolated CD45+ cells comprising CD34+ hematopoietic stem and progenitor cells (HSPCs);b) a second population of isolated CD45+ cells comprising isolated fresh regulatory T cells (Tregs);c) a third population of isolated CD45+ cells comprising conventional CD3+ T cells (Tcons); and220IPTS / 200310417.2Attorney Docket No. ORCA-024WO Client Ref. No. ORCA-0024-PCT d) one or more pharmaceutical compositions separately or combined comprising Mycophenolate mofetil (MMF) and tacrolimus, optionally 1000 mg of MMF, further optionally wherein the MMF is administered twice a day and / or for 8 weeks.

125. A method of treating a human subject in need of an allogeneic hematopoietic cell transplant optionally in the treatment of a hematologic malignancy, wherein the human subject has received, is receiving, or scheduled to receive a JAK inhibitor, optionally wherein the JAK inhibitor comprises a JAK1 / 2 inhibitor, further optionally wherein the JAK inhibitor comprises ruxolitinib, the method comprising administering to a human subject a multi-component cellular therapy product comprising:a) a first population of isolated CD45+ cells comprising CD34+ hematopoietic stem and progenitor cells (HSPCs);b) a second population of isolated CD45+ cells comprising isolated fresh regulatory T cells (Tregs); andc) a third population of isolated CD45+ cells comprising conventional CD3+ T cells (Tcons).

126. The method of claim 125, wherein the human subject receiving the product has received, is receiving, or scheduled to receive tacrolimus at 0.03 mg / kg per day, further optionally 0.015 mg / kg per day administered twice a day IV; and wherein the JAK inhibitor comprises ruxolitinib and the human subject has received, is receiving, or scheduled to receive ruxolitinib at 5mg twice per day.

127. A method of treating a human subject in need of an allogeneic hematopoietic cell transplant optionally in the treatment of a hematologic malignancy, wherein the human subject has received, is receiving, or scheduled to receive one or more pharmaceutical compositions separately or combined comprising Mycophenolate mofetil (MMF) and tacrolimus, the method comprising administering to a human subject a multi-component cellular therapy product comprising:a) a first population of isolated CD45+ cells comprising CD34+ hematopoietic stem and progenitor cells (HSPCs);b) a second population of isolated CD45+ cells comprising isolated fresh regulatory T cells (Tregs); andc) a third population of isolated CD45+ cells comprising conventional CD3+ T cells (Tcons).

128. The composition or method of any one of the above claims, wherein:a) the dose of CD34+ HSPCs comprises at least 1.0 x 106cells per padent kilogram;b) the dose of isolated fresh Tregs is from 0.9 x 106to 3.5 x 106cells per patient kilogram; and c) the dose of CD3+ Tcons is from 0.9 x 106to 6.9 x 106cells per patient kilogram.221IPTS / 200310417.2

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