CN117177748A

CN117177748A - Method for allogeneic hematopoietic stem cell transplantation

Info

Publication number: CN117177748A
Application number: CN202180089306.7A
Authority: CN
Inventors: 埃弗里特·赫托·梅耶; 罗伯特·S·内格林; 纳撒尼尔·弗恩霍夫; 斯科特·麦克莱伦; 斯科特·基利安
Original assignee: Orca Biosystems Inc
Current assignee: Orca Biosystems Inc
Priority date: 2020-11-04
Filing date: 2021-11-04
Publication date: 2023-12-05

Abstract

Various embodiments of the present application provide therapeutic compositions and associated methods for improved hematopoietic stem cell transplantation, including methods of enhancing protection against graft versus host disease while maintaining an effective immune response, such as a graft versus tumor immune response. Embodiments of the application are particularly useful for treating hematological cancers (e.g., leukemia, lymphoma, GVHD, and other diseases).

Description

Method for allogeneic hematopoietic stem cell transplantation

Statement regarding federally sponsored research

The present application was completed with the government support under HL114591 awarded by the national institutes of health. The government has certain rights in the application.

Cross reference

The present application claims the benefit of priority from U.S. provisional application No. 63/109,811, filed on 4 months 11 and 2020, and U.S. provisional application nos. 63/121,453, 63/121,742, and 63/121,534, respectively, filed on 4 months 12 and 2020, all of which are incorporated herein by reference in their entirety for all purposes.

Background

Patients with hematological malignancies (e.g., leukemia and lymphoma that exceed first remission) or refractory recurrence are rarely cured with standard chemotherapy. Myeloablative allogeneic hematopoietic cell transplantation (alloHCT) is associated with improved survival in these patients, but the morbidity and mortality associated with Graft Versus Host Disease (GVHD) are major factors limiting the success of alloHCT.

Disclosure of Invention

In a first aspect, embodiments of the present invention provide a multi-component drug therapy (multi-component pharmaceutical treatment) to be administered to a human subject in need thereof. According to one embodiment, the multicomponent treatment comprises: (a) A solution comprising a first cd45+ cell population comprising Hematopoietic Stem and Progenitor Cells (HSPCs) and granulocytes, wherein up to about 10% of the first cd45+ cell population comprises granulocytes; (b) A solution comprising a population of cells enriched for regulatory T cells (tregs); (c) A solution comprising a second cd45+ cell population, wherein the second cd45+ cell population comprises at least about 20% cd3+ normal T cells (Tcon), at least about 10% monocytes and at least about 10% granulocytes; and (d) a solution comprising one or more doses of a Graft Versus Host Disease (GVHD) prophylaxis agent.

In many embodiments, the GVHD prophylaxis agent comprises tacrolimus (tacrolimus) and/or analogs and derivatives thereof, which may be formulated for oral administration or intravenous administration to human subjects or other methods of administration or delivery known in the pharmaceutical arts, including, for example, intramuscular, transdermal, nasal, buccal and vaginal administration, according to various embodiments.

In various embodiments, tacrolimus may be administered in an amount to maintain a target blood level of at least about 3ng/ml of blood in a human subject for at least about 20 days after administration of the second cd45+ cell population, at least about 40 days after administration of the second cd45+ cell population in an amount to maintain a target blood level of about 4ng/ml or more, and/or at least about 40 days after administration of the second cd45+ cell population in an amount to maintain a target blood level of about 4ng/ml or more. In some cases, tacrolimus is administered in an amount to maintain a target blood level of up to about 10ng/ml for at least 30 days after administration of the second cd45+ cell population.

In some embodiments, tacrolimus is administered for at least about 60 days after administration of the second cd45+ cell population, for at least about 90 days after administration of the second cd45+ cell population, for up to about 150 days after administration of the second cd45+ cell population, for up to about 120 days after administration of the second cd45+ cell population.

In some embodiments, the first cd45+ cell population comprises at least about 0.5% granulocytes, at least about 1% granulocytes, at most about 5% 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 some embodiments, the second population of cd45+ cells comprises at least about 0.1% cd34+ cells or from about 0.2% to about 20% cd34+ cells and/or at least about 0.1% tregs.

In various embodiments, the multicomponent drug treatment further comprises a conditioning regimen (conditioning regimen), wherein the conditioning regimen is administered prior to any of components (a) to (d) listed above, e.g., the conditioning regimen is administered about two days to about ten days prior to any of (a) to (d). In some embodiments, the conditioning regimen is a myeloablative conditioning regimen. In some cases, the conditioning regimen comprises at least three conditioning agents, wherein at least one conditioning agent is thiotepa. In embodiments, the myeloablative conditioning regimen comprises at least one dose of thiotepa, e.g., at least about 5 milligrams of thiotepa per kg of the actual or ideal body weight of the human subject, or at least about 10 milligrams of thiotepa per kg of the actual or ideal body weight of the human subject. In various embodiments, the conditioning regimen comprises one or more doses of busulfan, fludarabine, and thiotepa. In some embodiments, the one or more doses comprise about 5 to about 12mg thiotepa/kg of the actual or ideal body weight of the human subject, about 7 to about 11mg busulfan/kg of the actual or ideal body weight of the human subject, and about 100 to about 200mg fludarabine/m, respectively ² Body surface area.

In some embodiments, the first cd45+ cell population comprises less than about 5EU endotoxin/ml solution, less than about 1EU endotoxin/ml solution, and/or less than about 0.5EU endotoxin/ml solution. In some cases, the Treg-enriched cell population comprises less than about 5EU endotoxin/ml solution, less than about 1EU endotoxin/ml solution, and/or less than about 0.5EU endotoxin/ml solution. In various embodiments, the second cd45+ cell population comprises less than about 5EU endotoxin/ml solution, less than about 1EU endotoxin/ml solution, and/or less than about 0.5EU endotoxin/ml solution.

In some embodiments, the HSPCs are cd34+.

In some embodiments, treg is cd4+cd25+cd127dim or cd4+foxp3+. In some cases, the population of Treg-enriched cells comprises cd45+ cells, e.g., more than about 90% of the cd45+ cells are tregs. In various embodiments, the Treg-enriched cell population comprises about 1 x 10 ⁵ Up to about 1X 10 ⁷ Individual tregs/kg of the actual or ideal body weight of the human subject, or about 5 x 10 ⁵ Up to about 4X 10 ⁶ Individual tregs/kg the actual or ideal body weight of the human subject.

In some embodiments, the first cd45+ cell population comprising HSPCs comprises about 5 x 10 ⁵ Up to about 2X 10 ⁷ Individual HSPCs/kg of the ideal body of the human subject. In some cases, the second population of cd45+ cells comprises about 1×10 ⁵ Up to about 1X 10 ⁷ The actual or ideal body weight of the human subject, or the second CD45+ cell population, comprises about 5X 10 cells per kg of Tcon ⁵ Up to about 5X 10 ⁶ Tcon/kg the actual or ideal body weight of the human subject.

In some embodiments, the first cd45+ cell population and the Treg-enriched cell population are administered before the second cd45+ cell population.

In various embodiments, the first dose of the one or more doses of the GVHD preventing agent is administered after the administration of the second cd45+ cell population.

In another aspect, a method of treating a human subject diagnosed with a hematological malignancy is provided. According to one embodiment, the method comprises administering to a human subject a solution comprising a first cd45+ cell population, a solution comprising a cell population enriched for regulatory tregs, a solution comprising a second cd45+ cell population, and a solution comprising one or more doses of a GVHD preventing agent. In this aspect, the solution comprising the first cd45+ cell population, the solution comprising the cell population enriched for regulatory tregs, the solution comprising the second cd45+ cell population, and the solution comprising one or more doses of a GVHD prophylaxis agent are as defined according to any of the multicomponent drug therapies disclosed herein.

Hematological malignancies may correspond to one or more of acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, multiple myeloma, lymphoma, hodgkin's lymphoma, and non-hodgkin's lymphoma.

In some embodiments, administering comprises infusing the first cd45+ cell population, the Treg-enriched cell population, and the second cd45+ cell population into a human subject.

In some embodiments, the second cd45+ cell population may be administered at least about 12 hours after the first cd45+ cell population, the second cd45+ cell population is administered about 24 to about 96 hours after the first cd45+ cell population, the second cd45+ cell population is administered about 36 to about 60 hours after the first cd45+ cell population, the second cd45+ cell population is administered at least about 12 hours after the Treg-enriched cell population, the second cd45+ cell population is administered about 24 to about 96 hours after the Treg-enriched cell population, and/or the second cd45+ cell population is administered about 36 to about 60 hours after the Treg-enriched cell population.

In various embodiments, the human subject does not develop stage 2 GVHD within about 100 days of the administration of the second cd45+ cell population, the human subject does not develop stage 2 GVHD within about 180 days or about 200 days of the administration of the second cd45+ cell population, and the human subject does not develop stage 2 GVHD within about 1 year of the administration of the second cd45+ cell population.

In some embodiments, the human subject has been previously or is being treated for hematological malignancy.

In some embodiments, the GVHD prophylaxis agent is tacrolimus (and/or analogs and derivatives thereof) and is initially administered to the human subject at about 0.03mg/kg of the actual or ideal body weight of the human subject per day, or tacrolimus is initially administered from about 12 hours to about 24 hours after said administration of said second cd45+ cell population.

In various embodiments, the dose of tacrolimus may begin to taper about 90 days after administration of the first dose to the human subject, or the dose of tacrolimus may begin to taper about 45 days after administration of the first dose to the human subject.

In some embodiments, the first cd45+ cell population, the Treg-enriched cell population, and the second cd45+ cell population are obtained from a single donor within one or more days.

In some embodiments, at least one mobilized peripheral blood donation is collected from the donor, or at most two mobilized peripheral blood donations are collected from the donor.

In various embodiments, at least one of the mobilized peripheral blood donations is processed and sorted to enrich for cd34+ cells and tregs. In some embodiments, the one or more of the mobilized peripheral blood donations has a processing and sorting time of less than about 35 hours, the one or more of the mobilized peripheral blood donations has a processing and sorting time of less than about 30 hours, the one or more of the mobilized peripheral blood donations has a processing and sorting time of less than about 25 hours, the one or more of the mobilized peripheral blood donations has a processing and sorting time of at most about 35 hours, and/or the one or more of the mobilized peripheral blood donations has a processing and sorting time of at most about 25 hours.

In various embodiments, one or more of the mobilized peripheral blood donations are processed and sorted using one or more immunoseparation particles (ISPs), e.g., ISPs include affinity reagents, such as immunomagnetic separation particles, which may be antibodies each conjugated to an iron-containing particle. In some embodiments, the affinity agent comprises a plurality of CD34 agents (e.g., anti-CD 34 antibodies) that bind to one or more CD34 receptors on HSPCs. In some cases, the average number of ISPs for each HSPC in the HSPC cell population is less than about 20,000, the average number of ISPs for each HSPC in the HSPC cell population is equal to or less than about 10,000, and/or the average number of ISPs for each HSPC in the HSPC cell population is from about 1500 to about 20,000. In embodiments, the affinity agent comprises a plurality of CD25 agents (e.g., anti-CD 25 antibodies) that bind to one or more CD25 receptors on tregs. In some cases, the average number of ISPs per T-reg cell in the Treg population is equal to or less than about 4000, or the average number of ISPs per T-reg cell in the Treg population is about 1500 to about 2500.

In various embodiments, the mobilized peripheral blood donated cells are sorted such that the first cd45+ cell population comprises up to about 10% granulocytes. In some cases, the mobilized peripheral blood donated cells are sorted such that the first cd45+ cell population comprises up to about 7% granulocytes.

In some embodiments, the mobilized donor peripheral blood donated cells are sorted such that the first cd45+ cell population comprises up to about 4% monocytes. In some cases, the mobilized donor peripheral blood donated cells are sorted such that the first cd45+ cell population comprises at least about 0.1% monocytes.

In embodiments, mobilized donor peripheral blood-donated cells are sorted such that the Treg-enriched population comprises up to about 10% CD25 cells.

In various embodiments, the first cd45+ cell population, the Treg-enriched cell population, and/or the second cd45+ cell population is allogeneic with respect to the human subject.

In some embodiments, the first cd45+ cell population, the Treg-enriched cell population, and/or the second cd45+ cell population are obtained from a donor that is HLA-matched relative to the human subject.

In embodiments, the first cd45+ cell population, the Treg-enriched cell population, and/or the second cd45+ cell population are obtained from a donor that is HLA-mismatched relative to the human subject.

In various embodiments, the first cd45+ cell population, the Treg-enriched cell population, and/or the second cd45+ cell population are obtained from a donor that is haploid matched relative to the human subject.

In some embodiments, the second population of cd45+ cells comprises a population of constant natural killer T cells (inkts), e.g., inkts that are cd3+vα24jα18+.

In embodiments, the iNKT population comprises more than about 5 x 10 ² Individual iNKT/kg ideal physical reality or ideal body weight of the human subject.

In various embodiments, the iNKT population comprises about 5 x 10 ² Up to about 1X 10 ⁷ Individual iNKT/kg ideal physical reality or ideal body weight of the human subject.

In some embodiments, the second population of cd45+ cells comprises a population of memory T cells (Tmem), e.g., tmem that is cd3+cd45ra-cd45ro+.

In embodiments, the Tmem population comprises greater than about 3 x 10 ⁵ Tmem/kg of the ideal physical reality or ideal weight of the human subject.

In various embodiments, the Tmem population comprises about 3 x 10 ⁵ Up to about 1X 10 ⁹ Tmem/kg of the ideal physical reality or ideal weight of the human subject.

Another aspect provides a multi-component drug treatment wherein the risk and/or severity of adverse events associated with the multi-component drug treatment is reduced compared to a similar drug treatment wherein a human subject received Tcon but did not receive Treg, or any of the methods disclosed herein wherein the risk and/or severity of adverse events associated with the method is reduced compared to a similar method wherein a human subject received Tcon but did not receive Treg.

In some embodiments, the adverse event is acute GVHD (aGVHD), which may include aGVHD in the second or higher phase.

In various embodiments, the adverse event is chronic GVHD (cGVHD), which may be moderate to severe cGVHD.

In some embodiments, the human subject has no cGVHD about one year after administration of the cell population.

In various embodiments, the adverse event is a recurrence of a malignancy in a human subject.

In some embodiments, the human subject has no recurrence of its malignancy about one year after administration of the drug dosing regimen.

In embodiments, the human subject has undergone a myeloablative conditioning regimen prior to administration of any cell population, and the adverse event is associated with myeloablative conditioning.

In various embodiments, the method further comprises providing instructions for use (IFU) comprising instructions for administering the cell population to the patient. In some cases, the IFU further comprises instructions for administering one or more pharmaceutical agents or compositions to the patient.

A further aspect provides a method of transplanting a population of conventional T cells (Tcon) as part of a treatment regimen for hematological malignancies, wherein the method reduces the risk and/or severity of adverse events associated with the treatment regimen. The method comprises administering to the patient a population of regulatory T cells (tregs) comprising tregs and a fluid that suspends the tregs; administering to a patient a heterogeneous population of cells comprising lymphocytes, granulocytes, monocytes and a liquid suspending said cells. In this aspect, at least about 30% of the lymphocytes comprise Tcon, and after administration of the population of cells, the patient has a reduced risk and/or severity of adverse events compared to hematological malignancy patients who received Tcon but did not receive Treg.

A further aspect provides a method of transplanting a population of cells into a human patient as part of a treatment regimen for a hematological malignancy, wherein the method reduces the risk and/or severity of adverse events associated with the treatment regimen. The method comprises providing a population of Hematopoietic Stem and Progenitor Cells (HSPCs) to be administered to a patient, the HSPC population comprising HSPCs and a liquid suspending the HSPCs; providing a population of regulatory T cells (tregs) to be administered to a patient, the population of tregs comprising tregs and a fluid that suspends the tregs; and providing a heterologous cell population to be administered to the patient, the heterologous cell population comprising lymphocytes, granulocytes, monocytes and a liquid suspending said cells. In this aspect, at least about 30% of the lymphocytes comprise conventional T cells (Tcon), and after administration of the cell population, the patient has a reduced risk and/or severity of adverse events compared to a hematologic malignancy patient who receives a population of Tcon cells but does not receive a population of T-reg cells.

In some embodiments, the population of cells is administered to the patient by intravenous infusion.

In embodiments, the corresponding cell populations are provided as separate cell populations and are derived from a single human blood donor.

In various embodiments, the adverse event is acute graft versus host disease (aGVHD), e.g., aGVHD in the second phase or higher. In some cases, the patient has no second-phase or higher aGVHD about 180 days after administration of the cell population.

In some embodiments, the adverse event is chronic graft versus host disease (cGVHD). In some cases, the patient has no cGVHD about one year after administration of the cell population.

In embodiments, the adverse event is moderate to severe cGVHD. In some cases, the patient has no moderate to severe cGVHD about one year after administration of the cell population.

In various embodiments, the adverse event is a recurrence of a malignancy in the patient. In some cases, the patient has no recurrence of his malignancy about one year after administration of the cell population.

In some embodiments, adverse events include Graft Versus Host Disease (GVHD) and recurrence of malignancy in the patient. In some cases, the patient has no recurrence of GHVD or malignancy thereof one year after administration of the cell population.

In embodiments, at least one of the cell populations comprises less than about 5EU endotoxin/ml of the corresponding suspension.

In various embodiments, the patient has undergone a myeloablative conditioning regimen prior to administration of the cell population, and the adverse event is associated with myeloablative conditioning. In some cases, the adverse event includes recurrence or infection of a malignancy in the patient.

In some embodiments, the heterologous cell population comprises from about 0.2% to about 2.0% hematopoietic stem cells and progenitor cells.

In embodiments, hematological malignancies are acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, multiple myeloma, lymphoma, hodgkin's lymphoma, and non-hodgkin's lymphoma.

In various embodiments, the gene expression level of the T-reg cells is correlated with cells harvested from the donor within about 60 hours prior to administration to the patient.

In some embodiments, the number of T-reg cells in the T-reg population is about equal to the number of T-con cells in the heterologous cell population. In some cases, the T-reg cells in the T-reg population inhibit activation of conventional T cells in the heterologous cell population by an amount up to about 20% of the healthy tissue of the patient.

In embodiments, the peripheral blood of the patient exhibits an elevated ratio of Treg to cd4+ T cells up to about 100 days after administration of the cell population as compared to a healthy human subject not administered the cell population.

In various embodiments, at least about 50% of the cells in the HSPC cell population are colony forming units.

In some embodiments, at least one of the cell populations has an elevated amount of granulocyte colony stimulating factor as compared to the non-mobilized blood. In some cases, the at least one cell population is a heterologous cell population.

In embodiments, at least one of the cell populations has a plurality of immunoisolatory particles (ISPs) attached to receptors on cells of the cell populations. In some cases, the plurality of ISPs are immunomagnetically isolated particles. In various embodiments, the plurality of ISPs comprises antibodies conjugated to iron-containing particles.

In some embodiments, the Tcon population is administered at least about 12 hours after the HSPC population, e.g., the Tcon population is administered about 24 to about 96 hours after the HSPC population, or the Tcon population is administered about 36 to about 60 hours after the HSPC population.

In some embodiments, the population of Tcon is administered at least about 12 hours after the population of cells comprising tregs, e.g., the population of Tcon is administered about 24 to about 96 hours after the population of cells comprising tregs, or the population of Tcon is administered about 36 to about 60 hours after the population of cells comprising tregs.

In various embodiments, the methods disclosed herein further comprise administering to the patient a single Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) comprising tacrolimus (tacrolimus GHVDPA) over a period of up to about 180 days; wherein tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the blood of the patient above a threshold level during said period of time; and wherein the risk and/or severity of GHVD is significantly reduced.

In some embodiments, the threshold level is above about 4ng tacrolimus/ml patient blood, or the threshold level is above about 5ng tacrolimus/ml patient blood.

In embodiments, tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the blood of a patient below an upper threshold level during said period of time. In some cases, the upper threshold level is below about 10ng tacrolimus/ml patient blood.

In various embodiments, the patient has a reduced risk of at least one of malignancy recurrence, infection, or renal failure.

In some embodiments, the patient does not develop GVHD within about 30 days of administering a Tcon, does not develop GVHD within about 100 days of administering a Tcon, does not develop GVHD within about 180 days of administering a Tcon, and/or does not develop GVHD within about one year of administering a Tcon.

In various embodiments, the tacrolimus Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) may be administered to the patient intravenously or orally. In various embodiments, the administration of the tacrolimus Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) begins about 12 to about 24 hours after administration of T-con. In some cases, tacrolimus GHVDPA is administered for a period of up to about 90 days, for a period of up to about 60 days. In some embodiments, tacrolimus GHVDPA is initially administered to a patient at about 0.03mg/kg of the patient's actual or ideal body weight per day. In some cases, the dose of tacrolimus gvhpa administered to the patient begins to taper about 90 days after the first dose is administered to the patient, or about 45 days after the first dose is administered to the patient.

In some embodiments, the method further comprises administering to the patient, prior to administering any cell population, a myeloablative conditioning regimen comprising administering to the patient at least one conditioning agent.

In embodiments, the patient does not receive any radiation as part of the myeloablative conditioning regimen.

In various embodiments, the at least one conditioning agent is administered from about two days to about ten days prior to administration of any of the cell populations. In some cases, the at least one conditioning agent is administered about five days prior to administration of any of the cell populations.

In some embodiments, the at least one conditioning agent comprises thiotepa. In some cases, the dosage of thiotepa administered to the patient is in the range of about 5 to about 10mg/kg of actual or ideal body weight.

In embodiments, the at least one conditioning agent comprises busulfan and fludarabine. In some cases, the dosages of thiotepa, busulfan and fludarabine administered to the patient include about 10mg/kg of the patient's actual or ideal body weight, about 9.6mg/kg of the patient's actual or ideal body weight, and about 150mg/m, respectively ² Body surface area.

In another aspect, a method of transplanting a population of cells into a human patient as part of a treatment regimen for a hematological malignancy is provided. The method comprises administering to a patient a population of Hematopoietic Stem and Progenitor Cells (HSPCs) comprising HSPCs and a liquid suspending the HSPCs; administering to a patient a population of regulatory T cells (tregs) to be administered to the patient, the population of tregs comprising tregs and a liquid suspending the tregs; and administering to the patient a heterologous cell population to be administered to the patient, the heterologous cell population comprising lymphocytes, granulocytes, monocytes and a liquid suspending said cells, wherein at least about 30% of said lymphocytes comprise conventional T cells (Tcon); and administering to the patient a single Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) comprising tacrolimus (tacrolimus GHVDPA) over a period of up to about 180 days, wherein tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the patient's blood above a threshold level during said period of time; and wherein the risk and/or severity of GHVD associated with a treatment regimen for hematological malignancy is significantly reduced.

In another aspect there is provided a kit comprising a solution, the kit comprising:

A first container comprising a first cd45+ cell population, a second container comprising a solution comprising a second cd45+ cell population, and a third container comprising a solution comprising a cell population enriched for regulatory T cells (tregs). In this aspect, the solution comprising the first cd45+ cell population, the solution comprising the second cd45+ cell population, and the solution comprising the Treg-enriched cell population are as defined according to any multicomponent drug treatment or method disclosed herein. In some embodiments, the kit further comprises a fourth container comprising a GVHD preventing agent. In some cases, instructions for performing any of the methods disclosed herein are also included.

In another aspect, a kit is provided, comprising: (a) One or more agents that sort cd34+ cells from the mobilized peripheral blood composition; (b) One or more agents that sort regulatory T cells (tregs) from the mobilized peripheral blood composition; (c) One or more reagents for detecting the number of cd3+ conventional T cells in mobilized peripheral blood; and (d) a solution comprising one or more doses of a Graft Versus Host Disease (GVHD) prophylaxis agent. In embodiments, the kit further comprises instructions for performing any of the methods disclosed herein.

One aspect provides a method of transplanting a population of conventional T cells (Tcon) into a human subject without eliciting a phase 2 or higher Graft Versus Host Disease (GVHD) response up to about 100 days after the transplantation. The method comprises (i) administering a heterogeneous population of cells comprising lymphocytes, granulocytes and monocytes, wherein at least about 30% of said lymphocytes comprise conventional T cells (Tcon); and (ii) administering a population of regulatory T cells (tregs). In this method, the heterologous cell fraction and/or Treg population comprises less than about 5EU/ml endotoxin.

In another aspect, there is provided a method of treating a human subject, the method comprising the step (a) of administering a plurality of cell populations, wherein the plurality of cell populations comprises: (i) a population of Hematopoietic Stem and Progenitor Cells (HSPCs); (ii) a population of cells comprising regulatory T cells (tregs); and (iii) a population of conventional T cells (Tcon); and administering no more than one Graft Versus Host Disease (GVHD) prophylactic agent for less than about 120 days. In this method, the HSPC population comprises less than about 2% cd3+ cells.

A further aspect provides a method of treating a human subject in need thereof, the method comprising administering to the human subject at least two pharmaceutical compositions, wherein the pharmaceutical compositions are selected from the group consisting of (a) a pharmaceutical composition comprising a population of hematopoietic stem cells and progenitor cells (HSPCs); (b) A pharmaceutical composition comprising a population of regulatory T cells (tregs); and (c) a pharmaceutical composition comprising a population of conventional T cells (Tcon). In this method, each of pharmaceutical compositions (a), (b) and (c) comprises less than about 5EU/ml endotoxin; and fewer than 15 human subjects in a group of 100 human subjects administered two or more pharmaceutical compositions develop a stage 2 or higher Graft Versus Host Disease (GVHD) response within about 30 days after administration of the pharmaceutical composition comprising the Tcon population.

An additional aspect provides a method of transplanting a population of conventional T cells (Tcon) into a human subject without eliciting a phase 2 or higher Graft Versus Host Disease (GVHD) response up to about 100 days after the transplantation. The method comprises the following steps: (i) administering a solution comprising a population of conventional T cells (Tcon); and (ii) administering a solution comprising a population of regulatory T cells (tregs). In this method, the Tcon population 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 5EU endotoxin/ml solution.

In one aspect, there is provided a method of treating hematological malignancy in a human subject in need thereof, the method comprising administering to the human subject: (a) a population of Hematopoietic Stem and Progenitor Cells (HSPCs); (b) a population of regulatory T cells (tregs); and (c) a population of conventional T cells (Tcon). In this method, the HSPC population and Treg population are administered prior to the Tcon population; and the peripheral blood of the human subject showed an elevated Treg count as compared to a healthy human subject not administered the three cell populations until about 100 days after administration of the three cell populations.

A further aspect provides a method of transplanting a population of conventional T cells (Tcon) into a human subject without eliciting a phase 2 or higher Graft Versus Host Disease (GVHD) response up to about 100 days after the transplantation. The method comprises (i) administering a population of conventional T cells (Tcon); and (ii) administering a population of regulatory T cells (tregs). In this method, the population of Tcon is administered at least about 12 hours after the population of tregs is administered; and the population of Tcon and Treg contain less than about 5EU/ml endotoxin.

It should be understood that different aspects and/or embodiments of the present invention may be used individually, jointly or in combination with one another. Any description herein of a particular composition, multicomponent drug treatment, cell population, solution, formulation, kit, and/or method applies to and may be used with any other particular composition, multicomponent drug treatment, cell population, solution, formulation, kit, and/or method. In other words, any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein.

Incorporation by reference

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.

Drawings

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

figures 1A-B show schematic diagrams of grafts (identified as high precision Orca-T or Orca T) and differences from standard of care (SOC) queues (identified as regular grafts or SOCs) according to the methods described herein.

Fig. 1C shows a schematic representation of graft generation and administration.

Figure 2A shows the body weight of patients enrolled in the study disclosed in the examples.

Figures 2B-C show HSPC and Treg cell doses administered to patients enrolled in the study disclosed in the examples.

Fig. 2D shows the purity of Treg cells administered to patients enrolled in the study disclosed in the examples.

Fig. 3A shows the time of platelet implantation in study group (identified as Orca-T) and standard of care (SOC) cohorts.

Figures 3B-3L illustrate the implantation of various cell populations in patients in the study group disclosed in the examples. The figure also shows the level of each cell type in the donor prior to sample collection. Box line graph display: the bins show the 75 th, 50 th and 25 th percentiles; the bin whisker shows the 90 th and 10 th percentiles. X-axis naming: leading digits (e.g., 01, 02, 025 … …) are used for ordering; after underlining, dsrn=healthy donor before G-CSF mobilization (pre-G-CSF mobilization), rsrn=acceptor within 1 month prior to conditioning, apher=healthy donor blood withdrawn at apheresis, d028=acceptor on day 28 post-transplant, d056-d 365=acceptor on days post-transplant. N shown represents the sample amount at each time point. The symbols represent the individual measured values. Cell number per uL blood x 10 ^-3 Equivalent to 1,000 cells per uL of blood.

Figures 3M-N show the timelines of lymphocyte and monocyte implantation in a subset of the study group (Orca-T) and standard of care cohorts.

Figure 3O shows representative flow cytometry data for the frequency of cd3+cd4+ T cells as tregs in two subjects compared to healthy controls. In healthy controls, 3.72% of circulating cd3+cd4+ T cells are tregs (cd25+cd127 dim). Of the two transplant recipients, 28.1% and 23.7% of cd3+cd4+ T cells were tregs at day +28 post-transplant, 32.3% and 17.8% of cd3+cd4+ T cells were tregs at day +56 post-transplant, and 19.2% and 20.7% of cd3+cd4+ T cells were tregs at day +100 post-transplant.

Figure 3P shows flow cytometry data for B cell markers from samples of receptors from the compositions of the present disclosure compared to healthy controls. In all cases, the Y-axis represents cd19+ staining. The left panel shows the gating of lymphocytes to identify B cells (cd19+) and T cells (cd3+). 13.4% of lymphocytes in the transplant recipients were B cells, while 9.84% of lymphocytes in the healthy controls were B cells. The second panel in the left panel shows that 98.3-100% of cells gated as CD19+ are also CD20+. The second panel on the right shows the fraction of B cells as igd+ which can be used to identify mature B cells. 92.1% of B cells in the transplant recipients were IgD+, while 89.5% of B cells in the healthy controls were IgD+. The right-most panel shows staining of CD27, which can be used, for example, to identify memory B cells, late plasmablasts, and plasma cells. 43.6% of B cells in the transplant recipients were CD27+, while 67.1% of B cells in the healthy controls were CD27+.

FIG. 4A shows the onset of ≡2 grade aGVHD in the study group (Orca-T) and standard of care cohorts by day +120 post-implantation. The Orca-T data was lower than the standard of care data at almost all time points.

FIG. 4B shows the onset of ≡3 grade aGVHD in the study group (Orca T) and standard of care cohorts by day +120 post-implantation. The Orca-T data was lower than the standard of care data at almost all time points.

Fig. 4C shows the onset of moderate to severe cGVHD in study group (Orca-T) and standard of care (SOC) cohorts by day +365 post-implantation. The Orca-T data was lower than the standard of care data at almost all time points.

Figure 4D shows non-recurrent related mortality in study group (Orca-T) and standard of care (SOC) cohorts by day +365 post-implantation. The Orca-T data was lower than the standard of care data at almost all time points.

Fig. 4E shows the recurrence rate in study group (Orca-T) and standard of care cohorts by day +365 post-implantation. At the last time point, the rate of Orca-T recurrence was 16% and the standard of care recurrence was 19%.

Figure 4F shows GVHD and relapse free survival in study group (Orca-T) and standard of care cohorts by day +365 post-transplant. The Orca-T data was higher than the standard of care data at almost all time points.

Figure 4G shows cGVHD-free survival in study groups and standard-of-care cohorts by day +365 post-implantation. The Orca-T data was higher than the standard of care data at almost all time points.

Figure 4H shows overall survival in study groups and standard of care cohorts by day +365 post-transplant. At the last time point, the overall survival rate of Orca-T was 90%, while the overall survival rate of standard care was 78%. Fig. 4I shows the number of hospitalization days in the subset of study groups and standard of care (SOC) cohorts until +365 days post-implantation.

Figure 5 summarizes the disease status of a small subset of subjects in the study group prior to and at day +90, day +180, and day +356 post-implantation. CR indicated complete remission and MRD indicated minimal residual disease.

Figures 6A-F compare aGVHD, cGVHD, relapse-free survival, GVHD and relapse-free survival (GRFS) and overall survival for a subset of patients in study groups receiving different conditioning regimens.

Figures 7A-H compare aGVHD, cGVHD, relapse-free related mortality, relapse-free survival, GVHD and relapse-free survival (GRFS) and overall survival for a subset of patients in study groups receiving different GVHD preventative agents.

Figures 8A-C show aGVHD and cGVHD incidences in patients with different serum levels of tacks Mo Sigu.

Figures 9A-B compare aGVHD and cGVHD levels in patients with different serum tacrolimus levels.

Figures 9C-D compare aGVHD and cGVHD levels in patients with different serum tacrolimus levels but given the same conditioning regimen of busulfan and cyclophosphamide (Bu/Cy).

Figures 9E-G compare aGVHD and cGVHD levels in patients with different serum tacrolimus levels but given the same conditioning regimen of systemic irradiation (TBI)/busulfan, fludarabine, thiotepa (TBI/BFT).

Fig. 9H shows the average levels of sitaglycone up to +30 days post-transplantation plotted against the proportion of donor-derived cd3+ cells at +30 days (except that the chimerism data is from day 90, where indicated by "D90").

Detailed Description

Introduction to the invention

Various embodiments of the present invention provide compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits and/or methods related to improved allogeneic hematopoietic stem cell transplantation (alloHCT).

alloHCT is the transplantation of pluripotent Hematopoietic Stem and Progenitor Cells (HSPCs), typically derived from donor bone marrow, peripheral blood or umbilical cord blood, into a recipient. The recipient may be myeloablative conditioned, which kills hematopoietic cells, including tumor cells and host immune cells. HSPCs transplanted into the recipient then reconstitute the hematopoietic compartment. HCT is useful in the treatment of cancer, known as Graft Versus Tumor (GVT), due to the ability of donor T cells to exert an anti-tumor effect. HCT is associated with improved survival in patients with hematological malignancies refractory to chemotherapy.

Surprisingly, alloHCT receptors receiving a single agent Graft Versus Host Disease (GVHD) prophylactic consisting of tacrolimus have significantly better clinical results than existing alloHCT regimens and standard of care. These recipients experience improved clinical outcomes including, for example, increased overall survival, increased recurrence-free survival, increased GVHD-free and recurrence-free survival (GRFS), faster and/or more complete implantation of various hematopoietic components (e.g., neutrophils, platelets, T cells, B cells), improved donor chimerism (e.g., T cell chimerism), reduced recurrence, reduced primary graft failure, reduced secondary graft failure, reduced treatment-related mortality, reduced acute and/or chronic GVHD, and shorter discharge time following a single agent GVHD preventative consisting of tacrolimus.

Although alloHCT is associated with improved survival in patients with hematological malignancies refractory to chemotherapy, some subjects treated with existing alloHCT regimens experience cancer recurrence and many complications may limit the efficacy of alloHCT. The effectiveness of alloHCT may be limited by, for example, primary graft failure, secondary graft failure, limited or slow implantation of various hematopoietic components (e.g., neutrophils, platelets, T cells, or B cells), and limited donor chimerism (e.g., T cell chimerism). Furthermore, alloHCT, for example, can lead to treatment-related mortality or toxicity, however, donor T cells can also attack non-tumor host cells, leading to Graft Versus Host Disease (GVHD). GVHD is a major source of post-HCT complications and can be fatal. Management of GVHD may require immunosuppressive therapy or cytotoxic mediation, which may lead to toxicity, increased susceptibility to infection, and/or inactivation of anti-tumor immunity. Early morbidity and mortality associated with acute graft versus host disease (aGVHD; which occurs within the first 100 days after transplantation) are the major factors limiting the success of HCT, and long-term morbidity associated with chronic GVHD (cGVHD) is also the major factor limiting the success of HCT. GVHD is a risk for both HLA-matched and HLA-mismatched transplants. GVHD can occur even if the donor and recipient are HLA matched, as the immune system can still recognize other differences between donor tissues.

Both GVT and GVHD are mediated primarily by conventional T cells (Tcon) that produce an immune response upon recognition of cognate antigens by T cell receptors. Depletion of T cells from hematopoietic stem cell transplantation (HCT) grafts may reduce GVHD, but may also lead to reduced GVT and an increased likelihood of cancer recurrence. In addition to Tcon, tregs are an additional subset of T cells that down regulate inflammation and promote immune tolerance. Tregs can prevent or reduce GVHD by their down-regulation of inflammation, including for example inflammation initiated by donor Tcon when they recognize the recipient antigen.

Provided herein are methods for improving alloHCT comprising administering to a subject a population of cells comprising a first cd45+ cell population comprising at least HSPCs, a population of cells enriched in tregs, and a second cd45+ cell population comprising at least Tcon. Without wishing to be bound by theory, administration of the Treg-enriched cell population reduces the incidence and/or severity of GVHD, while administration of the second cd45+ cell population comprising Tcon enhances GVT. Thus, embodiments of the present invention provide compositions, multi-component drug therapies, cell populations, solutions, formulations, kits, and/or methods for administering at least two T cell populations to enhance GVT while minimizing GVHD. Thus, the compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods disclosed herein can maintain the Graft Versus Tumor (GVT) effect of alloHCT administered to a subject having cancer (e.g., hematological cancer) while preventing or reducing Graft Versus Host Disease (GVHD) in the subject. In some embodiments, two or more cell populations are administered at different times, e.g., a first cd45+ cell population comprising at least HSPCs and a Treg-enriched cell population can be administered before a second cd45+ cell population comprising at least Tcon.

I. Cell populations

Embodiments of the present invention provide a multi-component drug therapy to be administered to a human subject in need thereof. The multicomponent treatment includes: (a) A solution comprising a first cd45+ cell population comprising Hematopoietic Stem and Progenitor Cells (HSPCs) and granulocytes, wherein up to about 10% of the first cd45+ cell population comprises granulocytes; (b) A solution comprising a population of cells enriched for regulatory T cells (tregs); (c) A solution comprising a second cd45+ cell population, wherein the second cd45+ cell population comprises at least about 20% cd3+ normal T cells (Tcon), at least about 10% monocytes and at least about 10% granulocytes; and (d) a solution comprising one or more doses of a Graft Versus Host Disease (GVHD) prophylactic agent, such as tacrolimus. In various embodiments, the HSPCs are cd34+.

In one placeIn some embodiments, the first cd45+ cell population comprising HSPCs comprises about 5 x 10 ⁵ Up to about 2X 10 ⁷ Individual HSPCs/kg of the ideal body of the human subject. In embodiments, the first cd45+ cell population comprises at least about 0.5% granulocytes, at least about 1% granulocytes, at most about 5% 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 cd45+ cell population, the Treg-enriched cell population, and the second cd45+ cell population are obtained from a single donor. In some embodiments, the first cd45+ cell population, the Treg-enriched cell population, and/or the second cd45+ cell population is allogeneic with respect to the human subject. In embodiments, the first cd45+ cell population, the Treg-enriched cell population, and/or the second cd45+ cell population are obtained from a donor that is HLA-matched relative to the human subject. In various embodiments, the first cd45+ cell population, the Treg-enriched cell population, and/or the second cd45+ cell population are obtained from a donor that is HLA-mismatched relative to the human subject. In some embodiments, the first cd45+ cell population, the Treg-enriched cell population, and/or the second cd45+ cell population are obtained from a donor that is haploid matched relative to the human subject.

In embodiments, treg is cd4+cd25+cd127dim or cd4+foxp3+. In some cases, the population of Treg-enriched cells comprises cd45+ cells, e.g., more than about 90% of the cd45+ cells are tregs. In various embodiments, the Treg-enriched cell population comprises about 1 x 10 ⁵ Up to about 1X 10 ⁷ Individual tregs/kg of the actual or ideal body weight of the human subject, or about 5 x 10 ⁵ Up to about 4X 10 ⁶ Individual tregs/kg the actual or ideal body weight of the human subject.

In some embodiments, the second population of cd45+ cells comprises about 1×10 cells ⁵ Up to about 1X 10 ⁷ The actual or ideal body weight of the human subject, or the second CD45+ cell population, comprises about 5X 10 Tcon/kg ⁵ Up to about 5X 10 ⁶ Tcon/kg the actual or ideal body weight of the human subject. In embodiments, the second population of cd45+ cells comprises at least about 0.1% cd34+ cells or from about 0.2% to about 20% cd34+ cells and/or at least about 0.1% tregs. In various embodiments, the second population of cd45+ cells comprises a population of memory T cells (Tmem), such as Tmem for cd3+cd45ra-cd45ro+. In some embodiments, the Tmem population comprises greater than about 3 x 10 ⁵ Tmem/kg of the ideal physical reality or ideal weight of the human subject. In embodiments, the Tmem population comprises about 3 x 10 ⁵ Up to about 1X 10 ⁹ Tmem/kg of the ideal physical reality or ideal weight of the human subject. In various embodiments, the second cd45+ cell population comprises a population of constant natural killer T cells (iNKT), such as iNKT that is cd3+vα24jα18+. In some embodiments, the iNKT population comprises more than about 5 x 10 ² Individual iNKT/kg ideal physical reality or ideal body weight of the human subject. In embodiments, the iNKT population comprises about 5 x 10 ² Up to about 1X 10 ⁷ Individual iNKT/kg ideal physical reality or ideal body weight of the human subject.

Provided herein are compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods for improved hematopoietic stem cell transplantation (HCT), e.g., allogeneic hematopoietic stem cell transplantation (alloHCT). The compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods disclosed herein may include one or more cell populations that may be administered in combination with a GVHD preventing agent to achieve positive clinical results. The cell population may comprise one or more types of cells, such as Hematopoietic Stem and Progenitor Cells (HSPCs), conventional T cells (Tcon), regulatory T cells (tregs), constant natural killer T cells (inkts), memory T cells (tmems), and combinations thereof.

The present disclosure provides parameters of cell populations and methods of administering cell populations that can aid in successful clinical outcome of HCT receptor subjects. Without wishing to be bound by theory, parameters that may contribute to the successful clinical outcome of HCT receptor subjects include, for example, co-administration of GVHD prophylaxis agents (e.g., tacrolimus) as described herein, the population administered, the order and time of administration of the different populations, the purity criteria of the population, the method for obtaining the population, the method for processing or storing the population, the dose of the population administered, the method for obtaining the population, and combinations thereof.

In various embodiments, administering comprises infusing the first cd45+ cell population, the Treg-enriched cell population, and the second cd45+ cell population into the human subject.

In some embodiments, the second cd45+ cell population (as disclosed herein) is administered at least about 12 hours after the first cd45+ cell population (as disclosed herein), the second cd45+ cell population is administered about 24 to about 96 hours after the first cd45+ cell population, the second cd45+ cell population is administered about 36 to about 60 hours after the first cd45+ cell population, the second cd45+ cell population is administered at least about 12 hours after the Treg-enriched cell population (as disclosed herein), the second cd45+ cell population is administered about 24 to about 96 hours after the Treg-enriched cell population, and/or the second cd45+ cell population is administered about 36 to about 60 hours after the Treg-enriched cell population.

HSPCs can have a broad ability to self-renew, as well as to differentiate into specialized cell types, e.g., the ability to reconstitute all hematopoietic cell lineages. HSPCs can undergo asynchronous replication, in which two daughter cells with different phenotypes are produced. HSPC cells can exist in mitotically quiescent form. HSPCs may be derived from bone marrow, peripheral blood, and/or umbilical cord blood.

Subpopulations of immune cells, such as conventional T cells (Tcon), regulatory T cells (Treg), constant natural killer T cells (iNKT) and memory T cells (Tmem), can contribute to various aspects of GVHD after HCT, as well as to GVT immune responses, immune reconstitution, susceptibility to infection and patient survival, for example.

GVHD can be mediated to a large extent by donor T cells that can elicit an inflammatory response upon recognition of the recipient antigen. T Cell Depletion (TCD) of a cell population for transplantation into a subject may be performed to reduce the likelihood of acute and/or chronic GVHD. T cells can be depleted using methods that include, but are not limited to, physical adsorption of T cells to protein ligands such as lectins, immune depletion using T cell specific antibodies, and immunoaffinity techniques (e.g., using T cell or lymphocyte specific antibodies in an immunoadsorption column, magnetically Activated Cell Sorting (MACS), or Fluorescence Activated Cell Sorting (FACS)). Application of TCD techniques to donor grafts may result in, for example, 10-fold to 10-fold ⁵ Double T cell depletion, and reduced incidence of GVHD. However, TCD may also lead to an increased incidence of cancer recurrence, as the lack of T cells may reduce Graft Versus Tumor (GVT) immune responses. In addition, TCD can lead to impaired immune recovery and increased susceptibility to infection.

Both GVT and GVHD can be mediated to a large extent by conventional T cells (Tcon) that produce an immune response upon recognition of cognate antigens (tumor antigens of GVT, non-tumor receptor antigens of GVHD) by T cell receptors. For example, tcon may result in GVT, GVHD, or a combination thereof. In some embodiments, administration of Tcon after Treg administration may enhance GVT immunity, and/or reduce susceptibility to infection.

Tcon can refer broadly to all cd3+ T cells, cells expressing CD3 and CD4 or cells expressing CD3 and CD8, cells expressing moderate to high levels of CD127, cells expressing CD3 and moderate to high levels of CD127, cells expressing CD3, cells expressing moderate to high levels of CD127, and cells expressing CD4 or CD 8. In some embodiments, tcon does not express vα24jα18TCR. Tcon and regulatory T cells ("tregs") may be non-mutually exclusive populations of cells. In some embodiments, tcon and Treg are mutually exclusive cell populations.

Regulatory T cells ("tregs") are specialized sub-populations of T cells that down-regulate (e.g., suppress) activation of the immune system, thereby promoting immune tolerance. Without wishing to be bound by theory, the Treg-enriched cell populations of the present disclosure contribute to positive clinical outcome 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. Administration of a Treg-enriched cell population and a cd45+ cell population comprising at least HSPCs can, for example, promote retention of Graft Versus Tumor (GVT) and reduced incidence and/or severity of GVHD. Without wishing to be bound by theory, administration of a Treg-enriched cell population may prevent GVHD, and administration of a second cd45+ cell population comprising at least Tcon may promote GVT effects, for example, relative to an alternative hematopoietic stem cell transplantation (HCT) method, i.e., a method different from the compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods disclosed and/or claimed herein. In some embodiments, administration of the Treg-enriched cell population reduces the risk of developing GVHD, and administration of the second cd45+ cell population comprising at least Tcon promotes GVT effects relative to alternative HCT methods, i.e., methods different from the compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods disclosed and/or claimed herein.

As used herein, an alternative composition lacks one or more cell populations and/or prophylactic agents disclosed and/or claimed herein. As an example, the alternative composition lacks one or more of a population of cells comprising HSPCs, a population of cells comprising tregs, a population of cells comprising Tcon, and a prophylactic agent. In some embodiments, an alternative composition or treatment regimen comprises an additional population of cells or agent, such as an additional or different GVHD preventing agent, as compared to the composition or treatment regimen of the present disclosure.

There are a number of subsets of tregs, for example, tcrαβ+cd4+ regulatory T cells, including natural regulatory T cells (nTreg) and induced regulatory T cells (iTreg). nTreg may be T cells produced in the thymus and delivered to the periphery as a long-lived lineage of autoantigen-specific lymphocytes. The iTreg can be recruited from circulating lymphocytes and acquire regulatory properties under specific stimulation conditions in the periphery. nTreg and iTreg are cd4+cd25+; both can inhibit proliferation of CD4+CD25-T cells in a dose-dependent manner. In some embodiments, tregs are non-allergic and do not proliferate under TCR stimulation. In addition to being positive for CD4 and CD25, tregs may 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+, (5) cd3+cd4+cd25+cd127dim, (6) cd3+cd4+cd25+cd127dim foxp3+, (7) cd3+foxp3+, (8) cd3+cd4+foxp3+, (9) cd3+cd4+cd25+foxp3+, (10) cd3+cd25+foxp3+, (11) cd3+cd25+cd127dim, (12) cd4+cd25+, (13) cd4+cd25+cd127dimfoxp3+, (14) cd4+xp3+, (15) cd4+cd25+foxp3+, (16) cd25+foxp3+ (17) and (17) foxp3+.

Selection based on certain expression profiles can be achieved based on extracellular markers and does not require cell permeabilization, e.g., selection based on cd4+cd25+cd127 dim.

The population of cells comprising tregs may, 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.

The cell populations of the present disclosure may comprise constant natural killer T cells (iNKT). iNKT is a subset of CD1 d-restricted Natural Killer T (NKT) cells (subs) that express a highly conserved αβ -T cell receptor consisting of the vα24jα18tcrα chain in humans (referred to herein as "vα24jα18+). iNKT cells can be identified by binding to CD1 d-multimers, such as CD1 d-multimers loaded with alpha-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 recognizes the V.alpha.24J.alpha.18 region. Examples include the vα24 antibody, the jα18 antibody, or monoclonal antibody clone 6B11, which specifically binds to a unique region of vα24jα18TCR and can be used to identify iNKT cells. iNKT may be cd3+vα24jα18+.

In some embodiments, iNKT may promote implantation, promote GVT, reduce the incidence and/or severity of GVHD, reduce susceptibility to cancer recurrence, reduce susceptibility to infection, or a combination thereof. In some embodiments, iNKT promotes Treg activity. In some embodiments, iNKT promotes HSPC activity.

The cell populations of the present disclosure may comprise memory T cells (Tmem). Tmem may refer to T cells that an antigen undergoes, which express, for example, the phenotypic markers CD45RO, tcrα, tcrβ, CD3, CD4, CD95 and IL-2rβ or the phenotypic markers CD45RO, tcrα, tcrβ, CD3, CD8, CD95 and IL-2rβ. Tmem provides immunity and can persist in an inactive state for a long period of time. Tmem is able to rapidly acquire effector functions upon re-challenge with antigen. The Tmem population may comprise any combination of T central memory cells and T effector memory cell subsets. In some embodiments, tmem is cd3+cd45ra-cd45ro+. In various methods, tmem administered to a subject receiving HCT can, for example, promote GVT, reduce GVHD, reduce susceptibility to cancer recurrence, reduce susceptibility to infection, or a combination thereof.

A. Cell harvesting and processing

In embodiments, at least one of the mobilized peripheral blood donations is processed and sorted to enrich for cd34+ cells and tregs. In some embodiments, the one or more of the mobilized peripheral blood donations has a processing and sorting time of less than about 35 hours, the one or more of the mobilized peripheral blood donations has a processing and sorting time of less than about 30 hours, the one or more of the mobilized peripheral blood donations has a processing and sorting time of less than about 25 hours, the one or more of the mobilized peripheral blood donations has a processing and sorting time of at most about 35 hours, and/or the one or more of the mobilized peripheral blood donations has a processing and sorting time of at most about 25 hours.

In various embodiments, one or more of the mobilized peripheral blood donations are processed and sorted using one or more immunoseparation particles (ISPs), e.g., ISPs include affinity reagents, such as immunomagnetic separation particles, which may be antibodies each conjugated to an iron-containing particle. In some embodiments, the affinity agent comprises a plurality of CD34 agents (e.g., anti-CD 34 antibodies) that bind to one or more CD34 receptors on HSPCs.

In some cases, at least a portion of the plurality of ISPs are attached to cd34+ receptors on HPSCs of the HSPC cell population; optionally, the average number of ISPs per HSPC in the HSPC cell population is less than about 20,000, the average number of ISPs per HSPC in the HSPC cell population is equal to or less than about 10,000, and/or the average number of ISPs per HSPC in the HSPC cell population is from about 1000 to about 20,000.

In some embodiments, the average number of ISPs per HSPC in a population of HSPC cells can be about 1,500 to about 20,000. In some embodiments, the average number of ISPs per HSPC in a population of HSPC cells can be at least about 1,500. In some embodiments, the average number of ISPs per HSPC in a population of HSPC cells can be up to about 20,000. In some embodiments, the average number of ISPs per HSPC in a HSPC cell population can be about 1,500 to about 2,000, about 1,500 to about 5,000, about 1,500 to about 6,000, about 1,500 to about 10,000, about 1,500 to about 12,000, about 1,500 to about 15,000, about 1,500 to about 20,000, about 2,000 to about 5,000, about 2,000 to about 6,000, about 2,000 to about 10,000, about 2,000 to about 12,000, about 2,000 to about 15,000, about 2,000 to about 20,000, about 5,000 to about 6,000, about 5,000 to about 10,000, about 5,000 to about 12,000, about 5,000 to about 15,000, about 6,000 to about 10,000, about 6,000 to about 12,000, about 6,000 to about 15,000, about 20,000 to about 10,000, about 12,000, about 20,000 to about 15,000, about 10,000 or about 20,000. In some embodiments, the average number of ISPs per HSPC in a population of HSPC cells can be about 1,500, 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, the average number of ISPs per HSPC in a population of HSPC cells can be at least 1,500, 2,000, 5,000, 6,000, 10,000, 12,000, 15,000, or 20,000. In some embodiments, the average number of ISPs per HSPC in a population of HSPC cells can be up to 1,500, 2,000, 5,000, 6,000, 10,000, 12,000, 15,000, or 20,000.

In some cases, at least a portion of the plurality of ISPs attach to cd25+ receptors on cells of the population of Treg cells; optionally, the average number of ISPs per T-reg cell in the Treg population is equal to or less than about 4000, or the average number of ISPs per T-reg cell in the Treg population is from about 1500 to about 2500. In some cases, at least a portion of the plurality of ISPs are attached to cd3+ receptors on cells of the heterologous cell population; optionally, the average number of ISPs per cell in the heterologous T population is less than about 4,000.

In some embodiments, the average number of ISPs per Treg cell in the population of Treg cells may be about 500 to about 4,000. In some embodiments, the average number of ISPs per Treg cell in the population of Treg cells may be at least about 500. In some embodiments, the average number of ISPs per Treg cell in the population of Treg cells may be up to about 4,000. In some embodiments, the average number of ISPs per Treg cell in the population of Treg cells may be about 500 to about 1,000, about 500 to about 1,500, about 500 to about 2,000, about 500 to about 2,500, about 500 to about 3,000, about 500 to about 4,000, about 1,000 to about 1,500, about 1,000 to about 2,000, about 1,000 to about 2,500, about 1,000 to about 3,000, about 1,000 to about 4,000, about 1,500 to about 2,000, about 1,500 to about 2,500, about 1,500 to about 3,000, about 1,500 to about 4,000, about 2,000 to about 2,500, about 2,000 to about 3,000, about 2,000 to about 4,000, about 2,500 to about 3,000, or about 3,000 to about 4,000. In some embodiments, the average number of ISPs per Treg cell in a population of Treg cells can 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, the average number of ISPs per Treg cell in the population of Treg cells may be at least 500, 1,000, 1,500, 2,000, 2,500, 3,000, or 4,000. In some embodiments, the average number of ISPs per Treg cell in the population of Treg cells may be up to 500, 1,000, 1,500, 2,000, 2,500, 3,000, or 4,000.

In some embodiments, the average number of ISPs per Tcon cell in the population of Tcon cells may be about 100 to about 1,000. In some embodiments, the average number of ISPs per Tcon cell in the population of Tcon cells may be at least about 100. In some embodiments, the average number of ISPs per Tcon cell in the population of Tcon cells may be up to about 1,000. In some embodiments, the average number of ISPs per Tcon cell in the population of Tcon cells may be about 100 to about 200, about 100 to about 500, about 100 to about 1,000, about 200 to about 500, about 200 to about 1,000, or about 500 to about 1,000. In some embodiments, the average number of ISPs per Tcon cell in the population of Tcon cells may be about 100, about 200, about 500, or about 1,000. In some embodiments, the average number of ISPs per Tcon cell in the population of Tcon cells may be at least 100, 200, 500, or 1,000. In some embodiments, the average number of ISPs per Tcon cell in the population of Tcon cells may be up to 100, 200, 500, or 1,000.

In some embodiments, the cell populations of the present disclosure are obtained from whole blood. The cell populations of the present disclosure can be obtained from peripheral blood apheresis products, e.g., mobilized by administering GCSF, GM-CSF, pleshafu (mobibil), and combinations thereof, to a donor. The cell populations of the present 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 apheresis products. In some embodiments, the cell populations of the present disclosure are obtained from a apheresis product. In some embodiments, the cell populations of the present disclosure are obtained from two apheresis products. In some embodiments, the cell populations of the present disclosure are obtained from a apheresis product from one donor and a apheresis product from at least one second donor.

In some embodiments, the cell populations of the present disclosure are obtained from bone marrow.

In some embodiments, the cell populations of the present disclosure are obtained from umbilical cord blood.

The cell populations of the present disclosure can be refined by selecting from a population of cells (e.g., peripheral blood or peripheral blood apheresis products). Methods of selecting a cell population may include methods involving positive or negative selection of the cell population of interest. Methods of selecting a cell population may include affinity reagents including, but not limited to, antibodies, full length antibodies, fragments of antibodies, naturally occurring antibodies, synthetic antibodies, engineered antibodies, full length affibodies, fragments of full length affilin, affilin, full length anti-calins (anti-calins), fragments of anti-calins, fragments of full length avimer, avimer, fragments of full length DARPin, DARPin, fragments of full length fynomer, fynomer, full length kunitz domain peptides, fragments of full length monobody, monobody, peptides, or polyamino acids. In some embodiments, the affinity reagent is directly conjugated to the detection reagent and/or purification reagent. In some cases, the detection reagent and the purification reagent are the same. In some cases, the detection reagent and the purification reagent are different. For example, the detection reagent and/or purification reagent is fluorescent, magnetic, etc. In some cases, the detection reagent and/or purification reagent is a magnetic particle for column purification. For example, magnetic column purification can be performed using columns, antibodies, buffers, preparative materials and reagents, miltenyi systems (clinic).

In various embodiments, at least one of the cell populations has a plurality of immunoisolatory particles (ISPs) attached to receptors on cells of the cell populations. In some cases, the plurality of ISPs are immunomagnetically isolated particles. In some embodiments, the plurality of ISPs comprises antibodies conjugated to iron-containing particles. In some cases, at least a portion of the plurality of ISPs are attached to cd34+ receptors on HPSCs of the HSPC cell population; optionally, the average number of ISPs per HSPC in the HSPC cell population is less than about 6,000, the average number of ISPs per HSPC in the HSPC cell population is equal to or less than about 3,000, and/or the average number of ISPs per HSPC in the HSPC cell population is from about 1700 to about 3,000. In some cases, at least a portion of the plurality of ISPs attach to cd25+ receptors on cells of the population of Treg cells; optionally, the average number of ISPs per T-reg cell in the Treg population is equal to or less than about 1700, or the average number of ISPs per T-reg cell in the Treg population is from about 1400 to about 1700. In some cases, at least a portion of the plurality of ISPs are attached to cd3+ receptors on cells of the heterologous cell population; optionally, the average number of ISPs per cell in the heterologous T population is less than about 1,000.

Affinity reagents may include immunoaffinity reagents that utilize the binding specificity of an antibody or fragment or derivative thereof to positively or negatively select a population of cells of interest. Methods of selecting cell populations may include affinity reagents and columns, such as Magnetically Activated Cell Sorting (MACS) with specific antibodies and microbeads. The method of selecting the cell population may comprise Fluorescence Activated Cell Sorting (FACS), wherein the cell population is sorted based on a staining profile using one or more fluorescence conjugated antibodies. The method of selection of the cell population may include physical adsorption, for example, physical adsorption of protein ligands such as lectins by T cells.

HSPCs can be obtained by harvesting from bone marrow or from peripheral blood. Bone marrow may be extracted from the posterior iliac crest or anterior iliac crest when the donor is under local anesthesia or general anesthesia. HSPCs may be obtained by harvesting from peripheral blood (e.g., by peripheral blood apheresis). The number of stem cells harvested can be increased by treating the donor with a mobilizing agent (i.e., an agent that mobilizes stem cells from bone marrow into peripheral blood). Non-limiting examples of mobilizing agents include granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), pleshafu, and combinations thereof. Techniques for mobilizing stem cells into peripheral blood may include administering, for example, 10 to 40 μ/kg/day of a mobilizing agent to the donor. The mobilizing agent can be administered to the donor in, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. The apheresis product may be isolated from the 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 hours after a dose of mobilizing agent.

The cd45+ cell populations of the present disclosure may comprise HSPCs. HSPCs may be selected based on the expression of CD 34. For example, the HSPCs of the present disclosure can be selected using anti-CD 34 antibodies as part of a Magnetically Activated Cell Sorting (MACS) or Fluorescent Activated Cell Sorting (FACS) system.

The number of HPSCs in a cd45+ cell population may be determined, for example, by quantifying cd34+ cells via flow cytometry. In some embodiments, the dose calculation is adjusted based on the measurement of the cell viability measurement, e.g., viability determined via flow cytometry using propidium iodide or 7-AAD or via trypan blue exclusion.

The cell populations of the present disclosure can be enriched for tregs. Tregs may be selected based on expression of markers including CD3, CD4, CD25, CD127, FOXP3, and combinations thereof.

Tregs can be selected using Magnetically Activated Cell Sorting (MACS). Tregs can be selected using Fluorescence Activated Cell Sorting (FACS). Tregs may be selected using a variety of procedures, for example, a variety of MACS selections, a variety of FACS selections, or a combination of MACS selections and FACS selections. For example, the expression of CD25 may be selected for a first time, e.g., using MACS to isolate CD25+ cells from a sample of hematopoietic cells. The second selection can be performed by contacting cd25+ cells with antibodies specific for CD4 and CD127, wherein FACS is used to isolate cells that are cd4+ CD127 dim.

Tregs can be isolated from whole blood. Tregs can be isolated from peripheral blood apheresis products. Tregs may be isolated from a cell population pre-enriched and/or depleted of one or more other cell types, for example, from a cell population depleted of cd34+ cells. In some embodiments, tregs are isolated from a flow-through fraction selected for cd34+ MACS.

The number of tregs in a cell population can be determined, for example, by flow cytometry, wherein tregs can be identified as, for example, cd4+cd25+cd127dim or cd4+foxp3+. The dose calculation may be adjusted based on the measurement of the cell viability measurement, for example, viability determined via flow cytometry using propidium iodide or 7-AAD or via trypan blue exclusion.

The second cd45+ cell population may comprise a population of Tcon. The second cd45+ cell population comprising at least Tcon may be derived from peripheral blood. The second cd45+ cell population may be derived from a peripheral blood apheresis product.

In some embodiments, the selecting step is not performed and the second cd45+ cell population comprising at least Tcon is derived directly from an aliquot of peripheral blood or apheresis product. In some embodiments, the cell population may be enriched for Tcon, for example, by sorting based on the expression of various markers using MACS, FACS, or a combination thereof. The second cd45+ cell population may be enriched by sorting cd3+ cells. The second cd4+ cell population may be enriched by sorting cd4+ and cd8+ cells. The second cd45+ cell population may be enriched by negative selection, wherein non-Tcon cells are removed, e.g., by MACS depletion of cells expressing CD34, CD19, CD25, or a combination thereof.

The number of Tcon present in the second cd45+ cell population may be quantified, for example, by quantifying cd3+ cells via flow cytometry. The number of cd3+ cells in an aliquot and the volume containing the appropriate dose of CD3 cells administered to the recipient can be determined. The dose calculation may be adjusted based on measurements of cell viability, for example, viability determined via flow cytometry using propidium iodide or 7-AAD or via trypan blue exclusion.

The apheresis product of the present disclosure can be split into two parts, one part for providing a second cd45+ cell population comprising at least Tcon and the other part for isolating and purifying a cd45+ cell population comprising at least HSPCs and a Treg-enriched cell population. In an alternative embodiment, CD34+ cells are isolated and purified from the apheresis product to produce a CD 34-negative cell fraction, and then cell Tregs are isolated from the CD 34-negative cell fraction to help provide a Treg-enriched cell population.

The cell populations of the present disclosure can comprise a population of inkts. The iNKT population may be derived from peripheral blood. The iNKT population may be derived from peripheral blood apheresis products.

For example, the cell population may be enriched for iNKT by sorting based on the expression of various markers using MACS, FACS, or a combination thereof. For example, iNKT populations can be enriched by sorting cd3+vα24jα18+ cells.

The amount of iNKT present in the population may be quantified, for example, by quantifying cd3+vα24jα18+ cells via flow cytometry. The number of cd3+vα24jα18+ cells in an aliquot and the volume containing the appropriate dose of iNKT administered to the receptor can be determined. In some embodiments, the dose calculation is adjusted based on the measurement of the cell viability measurement, for example, viability determined via flow cytometry using propidium iodide or 7-AAD or via trypan blue exclusion.

The cell population of the present disclosure may comprise a Tmem population. The Tmem population may be derived from peripheral blood. The Tmem population may be derived from a peripheral blood apheresis product.

The cell population may be enriched for Tmem, for example, by sorting based on the expression of various markers using MACS, FACS, or a combination thereof. For example, the Tmem population can be enriched by sorting cd3+cd45ra-cd45ro+ cells.

The number of Tmem 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 and the volume containing the appropriate dose of Tmem administered to the receptor can be determined. The dose calculation may be adjusted based on the measurement of the cell viability measurement, for example, viability determined via flow cytometry using propidium iodide or 7-AAD or via trypan blue exclusion.

The cell population of the present disclosure or the cell population of the present disclosure may be administered freshly after isolation, or after cryopreservation and subsequent thawing.

Freshly isolated cells ("fresh cells") from a donor may be administered to a recipient subject. Fresh cells can be stored in a buffer, for example, cliniMACS PBS-EDTA buffer containing 0.5% human serum albumin, or Plasma-Lyte-A at pH 7.4 supplemented with 2% human serum albumin. Fresh cells can be stored at reduced temperatures (e.g., 2-8 ℃) without the need for cryopreservation/freezing.

After a fresh cell population is obtained from the donor, fresh cells may be stored 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 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.

After obtaining the fresh cell population from the donor, the fresh cells may be stored for up to about 1 hour, up to about 2 hours, up to about 3 hours, up to about 4 hours, up to about 5 hours, up to about 6 hours, up to about 7 hours, up to about 8 hours, up to about 9 hours, up to about 10 hours, up to about 12 hours, up to about 14 hours, up to about 16 hours, up to about 18 hours, up to about 20, up to 22 hours, up to about 24 hours, up to about 30 hours, up to about 36 hours, up to about 40 hours, up to about 48 hours, up to about 60 hours, up to about 70 hours, up to about 72 hours, up to about 80 hours, up to about 90 hours, up to about 96 hours, up to about 120 hours, up to about 150 hours, up to about 200 hours, or up to about 300 hours prior to administration to the subject.

The cells of the present disclosure may be cryopreserved. In some embodiments, cryopreservation may be beneficial to the methods disclosed herein. For example, cryopreservation of a second cd45+ cell population comprising at least Tcon may reduce GVHD prior to subsequent thawing and administration to a subject.

Cryopreservation may include adding a preservative (e.g., DMSO) and gradually cooling the cells in a controlled rate freezer to prevent permeabilization cell damage caused by ice crystal formation. Cryopreservation may include commercial cryopreservation reagents and materials, such as freezer bags andCS10。

cryopreserved cells may be stored at low temperatures for a period of time ranging from hours to years. The cryopreserved cells may be stored at ultra low temperatures, for example, -50 ℃, -60 ℃, -70 ℃, -80 ℃, -90 ℃, -100 ℃, -110 ℃, -120 ℃, -130 ℃, -140 ℃, -150 ℃, -160 ℃, -170 ℃, -180 ℃, -190 ℃, -196 ℃ or lower. The cryopreserved cells may be stored in a storage device comprising liquid nitrogen.

Cells may be cryopreserved before or after certain steps in the methods of the disclosure, e.g., before or after a sorting step, before or after a characterization step, e.g., to determine cell viability or concentration of a particular type of cell.

In some embodiments, whole blood may be cryopreserved. Whole blood may be cryopreserved without sorting or characterization. Whole blood may be cryopreserved after sorting but without characterization. Whole blood may be cryopreserved after characterization but without sorting. Whole blood may be cryopreserved after characterization and sorting. Whole blood may be cryopreserved after quantification of the cell types of the present disclosure. Whole blood may be cryopreserved after quantification of conventional T cells (Tcon, e.g., cd3+ cells). Whole blood may be cryopreserved after quantifying viability of all cells or cell populations of the present disclosure (e.g., conventional T cells).

The peripheral blood apheresis product of the present disclosure may be cryopreserved. Peripheral blood apheresis products can be cryopreserved without sorting or characterization. Peripheral blood apheresis products can be cryopreserved after sorting but without characterization. Peripheral blood apheresis products can be cryopreserved after characterization but without sorting. Peripheral blood apheresis products can be cryopreserved after characterization and sorting. Peripheral blood apheresis products can be cryopreserved after quantification of the cell types of the present disclosure. Peripheral blood apheresis products can be cryopreserved after quantification of conventional T cells (Tcon, e.g., cd3+ cells). Peripheral blood apheresis products can be cryopreserved after quantifying viability of all cells or cell populations of the present disclosure (e.g., conventional T cells).

A cell population selected or selected from another cell population may be cryopreserved, for example, a population of cd45+ cells, HSPC, treg, tcon, iNKT, or Tmem may be cryopreserved.

The cell populations of the present disclosure can be cryopreserved for any amount of time. The cells of the present disclosure can 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 hours, 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 administering to a subject.

In some embodiments, the cell population of the present 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 hours, 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 administering to a subject.

In some embodiments, the cell populations of the present disclosure are 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 administering to a subject.

In some embodiments, the cell populations of the present disclosure are cryopreserved for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 10 days, up to about 14 days, up to about 21 days, up to about 28 days, up to about 50 days, up to about 60 days, or up to about 96 days prior to thawing and administering to a subject.

B. Donor(s)

The cell population may include cells from one or more donors, each of which has been HLA typed, e.g., to determine the degree of matching of HLA to the subject that will receive the cell population.

Human Leukocyte Antigens (HLA), also widely known as Major Histocompatibility Complex (MHC) antigens, can be protein molecules expressed on the surface of cells that can confer antigenic properties on the cells. HLA/MHC antigens are target molecules that can be recognized by T cells and Natural Killer (NK) cells as being derived from the same hematopoietic stem cell source ("self") as immune effector cells or from another hematopoietic cell source ("non-self"). HLA class I antigens (A, B and C in humans) can be expressed by most cells, while HLA class II antigens (DR, DP and DQ in humans) can be expressed primarily on professional antigen presenting cells. Both HLA classes may be associated with GVHD.

HLA antigens are encoded by highly polymorphic genes; there is a series of alleles for each of the HLA class I and class II genes. The allele products may differ in one or more amino acids in the alpha and/or beta domains. The set of specific antibodies or nucleic acid reagents can be used to determine the individual's HLA haplotype, e.g., using leukocytes expressing class I and class II molecules to determine the individual's HLA haplotype. HLA alleles can be described at different levels of detail. Most designations begin with HLA-and locus designations, followed by an x and a few (even) numbers, designating alleles. The first two digits may designate a set of alleles. The third through fourth digits (when present) may designate synonymous alleles. The fifth through sixth digits (when present) may represent any synonymous mutation within the coding framework of a gene. The seventh and eighth digits (when present) may distinguish mutations outside the coding region. Letters such as L, N, Q or S may follow the name of the allele to specify the expression level or other known non-genomic data about it. Thus, a fully described allele may be up to 9 digits long, excluding HLA prefix and locus symbol.

Haplotypes are formed from a set of HLA alleles inherited from one parent. HLA haploid match may refer to a donor-acceptor pair in which one chromosome is matched between the donor and acceptor over at least HLA-A, HLA-B and HLA-DR. Haploid pairings may or may not match on other alleles, such as other HLA genes on other chromosomes, or additional histocompatibility loci on either chromosome. Such donors may often occur in the home, e.g., parents may be in line with the child haploids; and siblings may be haploid-matched.

The cell population may be from a donor that has been HLA-typed on any number of HLA alleles. The donor and subject may be HLA matched, e.g., matched across all typed HLA alleles. The donor and the subject may be HLA mismatched, e.g., at least one HLA antigen may be mismatched between the donor and the recipient.

In some embodiments, the donor and subject may be HLA-typed at six alleles consisting of HLA-A, HLA-B, and HLA-DR alleles. The donor and the subject may be matched at, for example, 3/6, 4/6, 5/6 or 6/6 of the allele. In some embodiments, the donor and the subject match at least 5/6 alleles. In some embodiments, the donor and the subject match at the 6/6 allele.

In some embodiments, the donor and subject may be HLA-typed at eight alleles consisting of HLA-A, HLA-B, HLA-C, and HLA-DR alleles (e.g., HLA-DRB1 alleles). The donor and the subject may be matched at, for example, 4/8, 5/8, 6/8, 7/8 or 8/8 of the allele. In some embodiments, the donor and the subject match at least 6/8 alleles. In some embodiments, the donor and the subject match at least 7/8 alleles. In some embodiments, the donor and the subject match at 8/8 alleles.

In some embodiments, the donor and subject may be HLA-typed at ten alleles consisting of HLA-A, HLA-B, HLA-C, and HLA-DR alleles (e.g., HLA-DRB1 alleles). The donor and the subject may be matched at, for example, 5/10, 6/10, 7/10, 8/10, 9/10 or 10/10 of the allele. In some embodiments, the donor and the subject match at least 7/10 alleles. In some embodiments, the donor and the subject match at least 8/10 alleles. In some embodiments, the donor and the subject match at least 9/10 alleles. In some embodiments, the donor and the subject match at 10/10 alleles.

Cell populations can be generated from matched sibling donors that are 8/8 matched for HLA-A, -B, -C, and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched sibling donors that match 7/8 for HLA-A, -B, -C, and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched sibling donors that are 6/8 matched for HLA-A, -B, -C, and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched sibling donors that are 10/10 matched for HLA-A, -B, -C, -DQB1, and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched sibling donors that are 9/10 matched for HLA-A, -B, -C, -DQB1, and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched sibling donors that are 8/10 matched for HLA-A, -B, -C, -DQB1, and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched sibling donors that match 7/10 for HLA-A, -B, -C, -DQB1, and-DRB 1 (all typed using DNA-based high resolution methods).

Cell populations can be generated from matched unrelated donors that are 8/8 matched for HLA-A, -B, -C and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched unrelated donors that match 7/8 for HLA-A, -B, -C and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched unrelated donors that are 6/8 matched for HLA-A, -B, -C and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched unrelated donors that are 10/10 matched for HLA-A, -B, -C, -DQB1, and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched unrelated donors that are 9/10 matched for HLA-A, -B, -C, -DQB1, and-DRB 1 (all typed using DNA-based high resolution methods). Cell populations can be generated from matched unrelated donors that match 8/10 for HLA-A, -B, -C, -DQB1, and-DRB 1 (all typed using DNA-based high resolution methods).

The cell population may be derived from a heterologous donor. The cell population may be produced by a donor of the first blood relative as the subject. The cell population may be produced by a donor of the second blood relative as the subject. The cell population may be generated from a donor that is independent of the subject. The cell population may be generated from a donor that is HLA matched to the recipient subject. The cell population may be generated from a donor that is HLA mismatched to the recipient subject. The cell population may be produced by a donor that haploids with the recipient subject. The cell population may be generated by a donor associated with the recipient subject, e.g., parent, child, sibling, grandparent, grandchild, palliative, tertiary, or coumarone. The population of cells may be produced by a donor at least 16 years old. The population of cells may be produced by a donor at least 18 years old.

The cell population may be generated from a donor that meets the donor qualification criteria for living, leukocyte-enriched cells or tissues as defined by 21CFR ≡ 1271 2018 and related FDA industry guidelines. For example, the population of cells may be generated from donors meeting qualification criteria outlined in one or more of the following: eligibility Determination for Donors of Human Cells, tissue, 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, tissue, 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, tissue, 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). The cell population may be generated from a donor (NMDP donor) that meets any donation standard as specified by the standard NMDP guidelines.

The cell population may be produced by a donor that does not show signs of active infection. The cell population may be generated from a donor that is not seropositive for HIV-1 or-2, HTLV-1 or-2. The cell population may be generated from a donor that is not positive for anti-Hepatitis C (HCV) antibodies or HCV NAT. The cell population may be generated from a donor tested negative for chronic HBV infection. The cell population may be generated by a donor that does not have a high likelihood of a zika virus infection as defined by any one of the following: (i) Medical diagnosis of Zika virus infection in the past 6 months; (ii) Living or traveling in the past 6 months in the region where active Zika virus was transmitted; (iii) Unprotected behavior occurred with persons known to have any one of risk factors (i) or (ii) over the past 6 months. The cell population may be generated by a donor that does not have an indication or symptom consistent with an active zika virus infection.

One or more cell populations of the present disclosure can be obtained from a single donor, e.g., from mobilized peripheral blood apheresis of a single donor. HSPC, treg, tcon, iNKT, tmem or any combination thereof may be obtained from a single donor.

One or more cell populations of the present disclosure can be obtained from one donor, and one or more additional cell populations of the present disclosure can be obtained from a second donor. One cell population of the present disclosure may be obtained from a single donor, and a second cell population of the present disclosure may be obtained from multiple donors. The populations of the present disclosure can be obtained from multiple donors, e.g., from mobilized peripheral blood apheresis of multiple donors. HSPCs can be obtained from multiple donors. Tregs can be obtained from multiple donors. Tcon can be obtained from multiple donors. iNKT can be obtained from multiple donors. Tmem can be obtained from multiple donors.

C. Dose of cell populations

The dose administered to the cell population of the subject may be based on the body weight of the subject. In some cases, the body weight of the subject may be used to determine the dose of one or more cell populations to be administered to the subject. In some cases, the cell dose may be based on the ideal body weight of the subject, rather than their actual weight, e.g., the "actual body weight". Ideal body weight may be the preferred method of dose calculation to avoid incorrect cell doses due to excessive body fat and/or muscle mass. Ideal volume of objectWeight can be calculated using their height and sex. Other methods of calculating the ideal body weight of the subject may be used. For example, other methods of determining the body fat percentage of a subject.

HSPC

The first population of cd45+ cells comprising at least HSPCs may comprise at least about 1 x 10 ⁴ At least about 1X 10 ⁵ At least about 5X 10 ⁵ At least about 6X 10 ⁵ At least about 7X 10 ⁵ At least about 8X 10 ⁵ At least about 9X 10 ⁵ At least about 1X 10 ⁶ At least about 1.1X10 ⁶ At least about 1.2 x 10 ⁶ At least about 1.3X10 ⁶ At least about 1.4X10 ⁶ At least about 1.5X10 ⁶ At least about 1.6X10 ⁶ At least about 1.7X10 ⁶ At least about 1.8X10 ⁶ At least about 1.9X10 ⁶ At least about 2X 10 ⁶ At least about 2.1X10 ⁶ At least about 2.2 x 10 ⁶ At least about 2.3X10 ⁶ At least about 2.4X10 ⁶ At least about 2.5X10 ⁶ At least about 2.6X10 ⁶ At least about 2.7X10 ⁶ At least about 2.8X10 ⁶ At least about 2.9X10 ⁶ At least about 3X 10 ⁶ At least about 3.1X10 ⁶ At least about 3.2 x 10 ⁶ At least about 3.3X10 ⁶ At least about 3.4X10 ⁶ At least about 3.5X10 ⁶ At least about 3.6X10 ⁶ At least about 3.7X10 ⁶ At least about 3.8X10 ⁶ At least about 3.9X10 ⁶ At least about 4X 10 ⁶ At least about 4.1X10 ⁶ At least about 4.2 x 10 ⁶ At least about 4.3X10 ⁶ At least about 4.4X10 ⁶ At least about 4.5X10 ⁶ At least about 4.6X10 ⁶ At least about 4.7X10 ⁶ At least about 4.8X10 ⁶ At least about 4.9X10 ⁶ At least about 5X 10 ⁶ At least about 5.1X10 ⁶ At least about 5.2 x 10 ⁶ At least about 5.3X10 ⁶ At least about 5.4X10 ⁶ At least about 5.5X10 ⁶ At least about 5.6X10 ⁶ At least about 5.7X10 ⁶ At least about 5.8X10 ⁶ At least about 5.9X10 ⁶ At least about 6X 10 ⁶ At least about 6.5X10 ⁶ At least about 7X 10 ⁶ At least about 7.5X10 ⁶ At least about 8X 10 ⁶ At least about 8.5X10 ⁶ At least about 9X 10 ⁶ At least about 9.5X10 ⁶ At least about 1X 10 ⁷ At least about 1.5X10 ⁷ At least about 2X 10 ⁷ At least about 2.5X10 ⁷ At least about 3X 10 ⁷ At least about 3.5X10 ⁷ At least about 4X 10 ⁷ At least about 4.5X10 ⁷ At least about 5X 10 ⁷ At least about 5.5X10 ⁷ At least about 6X 10 ⁷ At least about 6.5X10 ⁷ At least about 7X 10 ⁷ At least about 7.5X10 ⁷ At least about 8X 10 ⁷ At least about 8.5X10 ⁷ At least about 9X 10 ⁷ At least about 9.5X10 ⁷ At least about 1X 10 ⁸ At least about 1X 10 ⁸ At least about 1.5X10 ⁸ At least about 2X 10 ⁸ At least about 2.5X10 ⁸ At least about 3X 10 ⁸ At least about 3.5X10 ⁸ At least about 4X 10 ⁷ At least about 4.5X10 ⁸ At least about 5X 10 ⁸ At least about 5.5X10 ⁸ At least about 6X 10 ⁸ At least about 6.5X10 ⁸ At least about 7X 10 ⁸ At least about 7.5X10 ⁸ At least about 8X 10 ⁸ At least about 8.5X10 ⁸ At least about 9X 10 ⁸ At least about 9.5X10 ⁸ At least about 1X 10 ⁹ Or more cells of the first cd45+ cell population and/or HSPCs (e.g., cd34+ cells)/kg of actual or desired body weight of the recipient subject.

The first population of cd45+ cells may comprise up to about 1 x 10 cells ⁴ Up to about 1X 10 ⁵ Up to about 5X 10 ⁵ Up to about 6X 10 ⁵ Up to about 7X 10 ⁵ Up to about 8X 10 ⁵ Up to about 9X 10 ⁵ Up to about 1X 10 ⁶ Up to about 1.1X10 ⁶ Up to about 1.2X10 ⁶ Up to about 1.3X10 ⁶ Up to about 1.4X10 ⁶ Up to about 1.5X10 ⁶ Up to about 1.6X10 ⁶ Up to about 1.7X10 ⁶ Up to about 1.8X10 ⁶ Up to about 1.9X10 ⁶ Up to about 2X 10 ⁶ Up to about 2.1X10 ⁶ Up to about 2.2X10 ⁶ Up to about 2.3X10 ⁶ Up to about 2.4X10 ⁶ Up to about 2.5X10 ⁶ Up to about 2.6X10 ⁶ Up to about 2.7X10 ⁶ Up to about 2.8X10 ⁶ Up to about 2.9X10 ⁶ Up to about 3X 10 ⁶ Up to about 3.1X10 ⁶ Up to about 3.2 x 10 ⁶ Up to about 3.3X10 ⁶ Up to about 3.4X10 ⁶ Up to about 3.5X10 ⁶ Up to about 3.6X10 ⁶ Up to about 3.7X10 ⁶ Up to about 3.8X10 ⁶ Up to about 3.9X10 ⁶ Up to about 4X 10 ⁶ Up to about 4.1X10 ⁶ Up to about 4.2 x 10 ⁶ Up to about 4.3X10 ⁶ Up to about 4.4X10 ⁶ Up to about 4.5X10 ⁶ Up to about 4.6X10 ⁶ Up to about 4.7X10 ⁶ Up to about 4.8X10 ⁶ Up to about 4.9X10 ⁶ Up to about 5X 10 ⁶ Up to about 5.1X10 ⁶ Up to about 5.2 x 10 ⁶ Up to about 5.3X10 ⁶ Up to about 5.4X10 ⁶ Up to about 5.5X10 ⁶ Up to about 5.6X10 ⁶ Up to about 5.7X10 ⁶ Up to about 5.8X10 ⁶ Up to about 5.9X10 ⁶ Up to about 6X 10 ⁶ Up to about 6.5X10 ⁶ Up to about 7X 10 ⁶ Up to about 7.5X10 ⁶ Up to about 8X 10 ⁶ Up to about 8.5X10 ⁶ Up to about 9X 10 ⁶ Up to about 9.5X10 ⁶ Up to about 1X 10 ⁷ Up to about 1.5X10 ⁷ Up to about 2X 10 ⁷ Up to about 2.5X10 ⁷ Up to about 3X 10 ⁷ Up to about 3.5X10 ⁷ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁷ Up to about 5X 10 ⁷ Up to about 5.5X10 ⁷ Up to about 6X 10 ⁷ Up to about 6.5X10 ⁷ Up to about 7X 10 ⁷ Up to about 7.5X10 ⁷ Up to about 8X 10 ⁷ Up to about 8.5X10 ⁷ Up to about 9X 10 ⁷ Up to about 9.5X10 ⁷ Up to about 1X 10 ⁸ Up to about 1X 10 ⁸ Up to about 1.5X10 ⁸ Up to about 2X 10 ⁸ Up to about 2.5X10 ⁸ Up to about 3X 10 ⁸ Up to about 3.5X10 ⁸ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁸ Up to about 5X 10 ⁸ Up to about 5.5X10 ⁸ Up to about 6X 10 ⁸ Up to about 6.5X10 ⁸ Up to about 7X 10 ⁸ Up to about 7.5X10 ⁸ Up to about 8X 10 ⁸ Up to about 8.5X10 ⁸ Up to about 9X 10 ⁸ Up to about 9.5X10 ⁸ Or up to about 1X 10 ⁹ The actual or ideal body weight of the cells and/or HSPCs (e.g. cd34+ cells) of the first cd45+ cell population per kg recipient subject.

For example, the first cd45+ cell population may comprise 1×10 cells ⁴ Up to 1X 10 ⁹ 、1×10 ⁵ Up to 1X 10 ⁸ 、1×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 1.5X10 ⁷ 、5×10 ⁵ Up to 1X 10 ⁷ 、5×10 ⁵ To 9X 10 ⁶ 、5×10 ⁵ Up to 8X 10 ⁶ 、5×10 ⁵ To 7X 10 ⁶ 、5×10 ⁵ Up to 6X 10 ⁶ 、5×10 ⁵ Up to 5X 10 ⁶ 、5×10 ⁵ Up to 4X 10 ⁶ 、5×10 ⁵ Up to 3X 10 ⁶ 、5×10 ⁵ Up to 2X 10 ⁶ 、5×10 ⁵ Up to 1X 10 ⁶ 、1×10 ⁶ Up to 1.5X10 ⁷ 、1×10 ⁶ Up to 1X 10 ⁷ 、1×10 ⁶ To 9X 10 ⁶ 、1×10 ⁶ Up to 8X 10 ⁶ 、1×10 ⁶ To 7X 10 ⁶ 、1×10 ⁶ Up to 6X 10 ⁶ 、1×10 ⁶ Up to 5X 10 ⁶ 、1×10 ⁶ Up to 4X 10 ⁶ 、1×10 ⁶ Up to 3X 10 ⁶ 、1×10 ⁶ Up to 2X 10 ⁶ 、1.5×10 ⁶ Up to 1.5X10 ⁷ 、1.5×10 ⁶ Up to 1X 10 ⁷ 、1.5×10 ⁶ To 9X 10 ⁶ 、1.5×10 ⁶ Up to 8X 10 ⁶ 、1.5×10 ⁶ To 7X 10 ⁶ 、1.5×10 ⁶ Up to 6X 10 ⁶ 、1.5×10 ⁶ Up to 5X 10 ⁶ 、1.5×10 ⁶ Up to 4X 10 ⁶ 、1.5×10 ⁶ Up to 3X 10 ⁶ 、1.5×10 ⁶ Up to 2X 10 ⁶ 、2×10 ⁶ Up to 1.5X10 ⁷ 、2×10 ⁶ Up to 1X 10 ⁷ 、2×10 ⁶ To 9X 10 ⁶ 、2×10 ⁶ Up to 8X 10 ⁶ 、2×10 ⁶ To 7X 10 ⁶ 、2×10 ⁶ Up to 6X 10 ⁶ 、2×10 ⁶ Up to 5X 10 ⁶ 、2×10 ⁶ Up to 4X 10 ⁶ 、2×10 ⁶ Up to 3X 10 ⁶ 、2.5×10 ⁶ Up to 1.5X10 ⁷ 、2.5×10 ⁶ Up to 1X 10 ⁷ 、2.5×10 ⁶ To 9X 10 ⁶ 、2.5×10 ⁶ Up to 8X 10 ⁶ 、2.5×10 ⁶ To 7X 10 ⁶ 、2.5×10 ⁶ Up to 6X 10 ⁶ 、2.5×10 ⁶ Up to 5X 10 ⁶ 、2.5×10 ⁶ Up to 4X 10 ⁶ Or 2.5X10 ⁶ Up to 3X 10 ⁶ The actual or ideal body weight of the cells and/or HSPCs (e.g. cd34+ cells) of the first cd45+ cell population per kg recipient subject.

The first population of cd45+ cells may have a determined level of purity for cd34+ cells. For example, the first population of cd45+ cells may comprise at least about 5%, at least about 10%, 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 88%, at least about 92%, at least about 90%, at least about 91%, at least about 94%, or at least about 94% more than about 95% of the total cells.

The first population of cd45+ cells may have a defined level of contaminating cd3+ cells. In some embodiments, up to about 1X 10 ² Up to about 2X 10 ² Up to about 3X 10 ² Up to about 4X 10 ² Up to about 5X 10 ² Up to about 6X 10 ² Up to about 7X 10 ² Up to about 8X 10 ² Up to about 9X 10 ² Up to about 1X 10 ³ Up to about 2X 10 ³ Up to about 3X 10 ³ Up to about 4X 10 ³ Up to about 5X 10 ³ Up to about 6X 10 ³ Up to about 7X 10 ³ Up to about 8X 10 ³ Up to about 9X 10 ³ Up to about 1X 10 ⁴ Up to about 2X 10 ⁴ Up to about 3X 10 ⁴ Up to about 4X 10 ⁴ Up to about 5X 10 ⁴ Up to about 6X 10 ⁴ Up to about 7X 10 ⁴ Up to about 8X 10 ⁴ Up to about 9X 10 ⁴ Or up to about 1X 10 ⁵ The body weight of the individual cd3+ cells/kg recipient subjects is present in the first cd45+ cell population or in the desired body weight.

In some embodiments of the present invention, in some embodiments, the 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 up to about 1.1%, up to about 1.2%, up to about 1.3%, up to about 1.4%, up to about 1.5%, up to about 1.6%, up to about 1.7%, up to about 1.8%, up to about 1.9%, up to about 2%, up to about 2.1%, up to about 2.2%, up to about 2.3%, up to about 2.4%, up to about 2.5%, up to about 2.6%, up to about 2.7%, up to about 2.8%, up to about 2.9%, up to about 3%, up to about 3.1%, up to about 3.2%, up to about 3.3%, up to about 3.4%, up to about 3.5%, up to about 3.6%, up to about 3.7%, up to about 3.8%, up to about 3.9%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, or up to about 10% cd3+ cells.

In some embodiments, the first cd45+ cell population comprises less than about 5EU endotoxin/ml solution, less than about 1EU endotoxin/ml solution, and/or less than about 0.5EU endotoxin/ml solution.

The first cd45+ cell population may comprise from 0.5EU/ml endotoxin to 10EU/ml endotoxin. The first cd45+ cell population may comprise at least 0.5EU/ml endotoxin. The first cd45+ cell population may comprise up to 10EU/ml of endotoxin. The first CD45+ cell population may comprise 10EU/ml of endotoxin to 8EU/ml of endotoxin, 10EU/ml of endotoxin to 6EU/ml of endotoxin, 10EU/ml of endotoxin to 5EU/ml of endotoxin, 10EU/ml of endotoxin to 4EU/ml of endotoxin, 10EU/ml of endotoxin to 2EU/ml of endotoxin, 10EU/ml of endotoxin to 1EU/ml of endotoxin, 10EU/ml of endotoxin to 0.5EU/ml of endotoxin, 8EU/ml of endotoxin to 6EU/ml of endotoxin, 8EU/ml of endotoxin to 5EU/ml of endotoxin, 8EU/ml of endotoxin to 4EU/ml of endotoxin, 8EU/ml of endotoxin to 2EU/ml of endotoxin, 8EU/ml of endotoxin to 1EU/ml of endotoxin 8EU/ml endotoxin to 0.5EU/ml endotoxin, 6EU/ml endotoxin to 5EU/ml endotoxin, 6EU/ml endotoxin to 4EU/ml endotoxin, 6EU/ml endotoxin to 2EU/ml endotoxin, 6EU/ml endotoxin to 1EU/ml endotoxin, 6EU/ml endotoxin to 0.5EU/ml endotoxin, 5EU/ml endotoxin to 4EU/ml endotoxin, 5EU/ml endotoxin to 2EU/ml endotoxin, 5EU/ml endotoxin to 1EU/ml endotoxin, 5EU/ml endotoxin to 0.5EU/ml endotoxin, 4EU/ml endotoxin to 2EU/ml endotoxin, 4EU/ml endotoxin to 1EU/ml endotoxin, 4EU/ml endotoxin to 0.5EU/ml endotoxin, 2EU/ml endotoxin to 1EU/ml endotoxin, 2EU/ml endotoxin to 0.5EU/ml endotoxin or 1EU/ml endotoxin to 0.5EU/ml endotoxin. The first cd45+ cell population may comprise 10EU/ml of endotoxin, 8EU/ml of endotoxin, 6EU/ml of endotoxin, 5EU/ml of endotoxin, 4EU/ml of endotoxin, 2EU/ml of endotoxin, 1EU/ml of endotoxin, or 0.5EU/ml of endotoxin. The first cd45+ cell population may comprise at least 8EU/ml endotoxin, 6EU/ml endotoxin, 5EU/ml endotoxin, 4EU/ml endotoxin, 2EU/ml endotoxin, 1EU/ml endotoxin, or 0.5EU/ml endotoxin. The first cd45+ cell population may comprise up to 10EU/ml of endotoxin, 8EU/ml of endotoxin, 6EU/ml of endotoxin, 5EU/ml of endotoxin, 4EU/ml of endotoxin, 2EU/ml of endotoxin, or 1EU/ml of endotoxin.

The first population of cd45+ cells may comprise 0.5% w/w to 10% w/w unbound reagent. These unbound reagents may include any affinity reagent used to sort HSPCs, such as antibodies or purified particles or magnetic particles. The first population of cd45+ cells may comprise at least 0.5% w/w unbound reagent. The first cd45+ cell population may comprise up to 10% w/w unbound reagent. The first population of CD45+ cells may 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 of unbound reagent. The first cd45+ cell population may 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 reagent. The first cd45+ cell population may 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 reagent. The first cd45+ cell population may comprise up to 10%, 8%, 6%, 5%, 4%, 2% or 1% of unbound reagent.

The first population of CD45+ cells may comprise 5X 10 cells ³ Up to 90X 10 ³ Individual microbeads/cells. Such microbeads may include microbeads for purifying HSPC populations, e.g., anti-CD 34 antibodies comprising microbeads for sorting HSPC populations. The first population of cd45+ cells may comprise at least 5 x 10 ³ Individual microbeads/cells. The first population of cd45+ cells may comprise up to 90 x 10 ³ Individual microbeads/cells. The first population of CD45+ cells may comprise 90X 10 cells ³ Up to 70X 10 ³ 、90×10 ³ Up to 50X 10 ³ 、90×10 ³ Up to 40X 10 ³ 、90×10 ³ Up to 30X 10 ³ 、90×10 ³ Up to 20X 10 ³ 、90×10 ³ Up to 10X 10 ³ 、90×10 ³ Up to 5X 10 ³ 、70×10 ³ Up to 50X 10 ³ 、70×10 ³ Up to 40X 10 ³ 、70×10 ³ Up to 30X 10 ³ 、70×10 ³ Up to 20X 10 ³ 、70×10 ³ Up to 10X 10 ³ 、70×10 ³ Up to 5X 10 ³ 、50×10 ³ Up to 40X 10 ³ 、50×10 ³ Up to 30X 10 ³ 、50×10 ³ Up to 20X 10 ³ 、50×10 ³ Up to 10X 10 ³ 、50×10 ³ Up to 5X 10 ³ 、40×10 ³ Up to 30X 10 ³ 、40×10 ³ Up to 20X 10 ³ 、40×10 ³ Up to 10X 10 ³ 、40×10 ³ Up to 5X 10 ³ 、30×10 ³ Up to 20X 10 ³ 、30×10 ³ Up to 10X 10 ³ 、30×10 ³ Up to 5X 10 ³ 、20×10 ³ Up to 10X 10 ³ 、20×10 ³ Up to 5X 10 ³ Or 10X 10 ³ To 5×10 ³ Individual microbeads/cells. The first population of CD45+ cells may comprise 90X 10 cells ³ 、70×10 ³ 、50×10 ³ 、40×10 ³ 、30×10 ³ 、20×10 ³ 、10×10 ³ Or 5X 10 ³ Individual microbeads/cells. The first population of cd45+ cells may comprise at least 70 x 10 ³ 、50×10 ³ 、40×10 ³ 、30×10 ³ 、20×10 ³ 、10×10 ³ Or 5X 10 ³ Individual microbeads/cells. The first population of cd45+ cells may comprise up to 90 x 10 ³ 、70×10 ³ 、50×10 ³ 、40×10 ³ 、30×10 ³ 、20×10 ³ Or 10X 10 ³ Individual microbeads/cells.

Treg

The Treg-enriched cell population may comprise at least about 1 x 10 ⁴ At least about 1X 10 ⁵ At least about 5X 10 ⁵ At least about 6X 10 ⁵ At least about 7X 10 ⁵ At least about 8X 10 ⁵ At least about 9X 10 ⁵ At least about 1X 10 ⁶ At least about 1.1X10 ⁶ At least about 1.2 x 10 ⁶ At least about 1.3X10 ⁶ At least about 1.4X10 ⁶ At least about 1.5X10 ⁶ At least about 1.6X10 ⁶ At least about 1.7X10 ⁶ At least about 1.8X10 ⁶ At least about 1.9X10 ⁶ At least about 2X 10 ⁶ At least about 2.1X10 ⁶ At least about 2.2 x 10 ⁶ At least about 2.3X10 ⁶ At least about 2.4X10 ⁶ At least about 2.5X10 ⁶ At least about 2.6X10 ⁶ At least about 2.7X10 ⁶ At least about 2.8X10 ⁶ At least about 2.9X10 ⁶ At least about 3X 10 ⁶ At least about 3.1X10 ⁶ At least about 3.2 x 10 ⁶ At least about 3.3X10 ⁶ At least about 3.4X10 ⁶ At least about 3.5X10 ⁶ At least about 3.6X10 ⁶ At least about 3.7X10 ⁶ At least about 3.8X10 ⁶ At least about 3.9X10 ⁶ At least about 4X 10 ⁶ At least about 4.1X10 ⁶ At least about 4.2 x 10 ⁶ At least about 4.3X10 ⁶ At least about 4.4X10 ⁶ At least about 4.5X10 ⁶ At least about 4.6X10 ⁶ At least about 4.7X10 ⁶ At least about 4.8X10 ⁶ At least about 4.9X10 ⁶ At least about 5X 10 ⁶ At least about 5.1X10 ⁶ At least about 5.2 x 10 ⁶ At least about 5.3X10 ⁶ At least about 5.4X10 ⁶ At least about 5.5X10 ⁶ At least about 5.6X10 ⁶ At least about 5.7X10 ⁶ At least about 5.8X10 ⁶ At least about 5.9X10 ⁶ At least about 6X 10 ⁶ At least about 6.5X10 ⁶ At least about 7X 10 ⁶ At least about 7.5X10 ⁶ At least about 8X 10 ⁶ At least about 8.5X10 ⁶ At least about 9X 10 ⁶ At least about 9.5X10 ⁶ At least about 1X 10 ⁷ At least about 1.5X10 ⁷ At least about 2X 10 ⁷ At least about 2.5X10 ⁷ At least about 3X 10 ⁷ At least about 3.5X10 ⁷ At least about 4X 10 ⁷ At least about 4.5X10 ⁷ At least about 5X 10 ⁷ At least about 5.5X10 ⁷ At least about 6X 10 ⁷ At least about 6.5X10 ⁷ At least about 7X 10 ⁷ At least about 7.5X10 ⁷ At least about 8X 10 ⁷ At least about 8.5X10 ⁷ At least about 9X 10 ⁷ At least about 9.5X10 ⁷ At least about 1X 10 ⁸ At least about 1X 10 ⁸ At least about 1.5X10 ⁸ At least about 2X 10 ⁸ At least about 2.5X10 ⁸ At least about 3X 10 ⁸ At least about 3.5X10 ⁸ At least about 4X 10 ⁷ At least about 4.5X10 ⁸ At least about 5X 10 ⁸ At least about 5.5X10 ⁸ At least about 6X 10 ⁸ At least about 6.5X10 ⁸ At least about 7X 10 ⁸ At least about 7.5X10 ⁸ At least about 8X 10 ⁸ At least about 8.5X10 ⁸ At least about 9X 10 ⁸ At least about 9.5X10 ⁸ At least about 1X 10 ⁹ Or the actual or ideal body weight of the cells of the population of more Treg-enriched cells and/or the Treg/kg recipient subject (e.g., where Treg is cd4+cd25+cd127dim, cd3+cd4+cd25+, cd3+cd4+cd25+cd127 dim) Foxp3+, 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+cd127 dim.

The Treg-enriched cell population may comprise up to about 1 x 10 ⁴ Up to about 1X 10 ⁵ Up to about 5X 10 ⁵ Up to about 6X 10 ⁵ Up to about 7X 10 ⁵ Up to about 8X 10 ⁵ Up to about 9X 10 ⁵ Up to about 1X 10 ⁶ Up to about 1.1X10 ⁶ Up to about 1.2X10 ⁶ Up to about 1.3X10 ⁶ Up to about 1.4X10 ⁶ Up to about 1.5X10 ⁶ Up to about 1.6X10 ⁶ Up to about 1.7X10 ⁶ Up to about 1.8X10 ⁶ Up to about 1.9X10 ⁶ Up to about 2X 10 ⁶ Up to about 2.1X10 ⁶ Up to about 2.2X10 ⁶ Up to about 2.3X10 ⁶ Up to about 2.4X10 ⁶ Up to about 2.5X10 ⁶ Up to about 2.6X10 ⁶ Up to about 2.7X10 ⁶ Up to about 2.8X10 ⁶ Up to about 2.9X10 ⁶ Up to about 3X 10 ⁶ Up to about 3.1X10 ⁶ Up to about 3.2 x 10 ⁶ Up to about 3.3X10 ⁶ Up to about 3.4X10 ⁶ Up to about 3.5X10 ⁶ Up to about 3.6X10 ⁶ Up to about 3.7X10 ⁶ Up to about 3.8X10 ⁶ Up to about 3.9X10 ⁶ Up to about 4X 10 ⁶ Up to about 4.1X10 ⁶ Up to about 4.2 x 10 ⁶ Up to about 4.3X10 ⁶ Up to about 4.4X10 ⁶ Up to about 4.5X10 ⁶ Up to about 4.6X10 ⁶ Up to about 4.7X10 ⁶ Up to about 4.8X10 ⁶ Up to about 4.9X10 ⁶ Up to about 5X 10 ⁶ Up to about 5.1X10 ⁶ Up to about 5.2 x 10 ⁶ Up to about 5.3X10 ⁶ Up to about 5.4X10 ⁶ Up to about 5.5X10 ⁶ Up to about 5.6X10 ⁶ Up to about 5.7X10 ⁶ Up to about 5.8X10 ⁶ Up to about 5.9X10 ⁶ Up to about 6X 10 ⁶ Up to about 6.5X10 ⁶ Up to about 7X 10 ⁶ Up to about 7.5X10 ⁶ Up to about 8X 10 ⁶ Up to about 8.5X10 ⁶ Up to about 9X 10 ⁶ Up to about 9.5X10 ⁶ Up to about 1X 10 ⁷ Up to about 1.5X10 ⁷ Up to about 2X 10 ⁷ Up to about 2.5X10 ⁷ Up to about 3X 10 ⁷ Up to about 3.5X10 ⁷ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁷ Up to about 5X 10 ⁷ Up to about 5.5X10 ⁷ Up to about 6X 10 ⁷ Up to about 6.5X10 ⁷ Up to about 7X 10 ⁷ Up to about 7.5X10 ⁷ Up to about 8X 10 ⁷ Up to about 8.5X10 ⁷ Up to about 9X 10 ⁷ Up to about 9.5X10 ⁷ Up to about 1X 10 ⁸ Up to about 1X 10 ⁸ Up to about 1.5X10 ⁸ Up to about 2X 10 ⁸ Up to about 2.5X10 ⁸ Up to about 3X 10 ⁸ Up to about 3.5X10 ⁸ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁸ Up to about 5X 10 ⁸ Up to about 5.5X10 ⁸ Up to about 6X 10 ⁸ Up to about 6.5X10 ⁸ Up to about 7X 10 ⁸ Up to about 7.5X10 ⁸ Up to about 8X 10 ⁸ Up to about 8.5X10 ⁸ Up to about 9X 10 ⁸ Up to about 9.5X10 ⁸ Or up to about 1X 10 ⁹ The actual or ideal body weight of the cells of the individual Treg-enriched cell populations and/or Treg/kg recipient subjects (e.g., wherein Treg is cd4+cd25+cd127dim, cd3+cd4+cd25+, cd3+cd4+cd25+cd127dim foxp3+, cd3+foxp3+, cd3+cd4+cd25+foxp3+, cd3+cd25+foxp3+, cd3+cd25+cd127dim, cd4+cd25+, cd4+cd25+cd25+cd127 dimfoxp3+, foxp3+, cd4+foxp3+, foxp3+, cd25+foxp3+ or cd25+cd127 dim.

For example, the Treg-enriched cell population may comprise 1×10 ⁴ Up to 1X 10 ⁹ 、1×10 ⁵ Up to 1X 10 ⁸ 、1×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 1.5X10 ⁷ 、5×10 ⁵ Up to 1X 10 ⁷ 、5×10 ⁵ To 9X 10 ⁶ 、5×10 ⁵ Up to 8X 10 ⁶ 、5×10 ⁵ To 7X 10 ⁶ 、5×10 ⁵ Up to 6X 10 ⁶ 、5×10 ⁵ Up to 5X 10 ⁶ 、5×10 ⁵ Up to 4X 10 ⁶ 、5×10 ⁵ Up to 3X 10 ⁶ 、5×10 ⁵ Up to 2X 10 ⁶ 、5×10 ⁵ Up to 1X 10 ⁶ 、1×10 ⁶ Up to 1.5X10 ⁷ 、1×10 ⁶ Up to 1X 10 ⁷ 、1×10 ⁶ To 9X 10 ⁶ 、1×10 ⁶ Up to 8X 10 ⁶ 、1×10 ⁶ To 7X 10 ⁶ 、1×10 ⁶ Up to 6X 10 ⁶ 、1×10 ⁶ Up to 5X 10 ⁶ 、1×10 ⁶ Up to 4X 10 ⁶ 、1×10 ⁶ Up to 3X 10 ⁶ 、1×10 ⁶ Up to 2X 10 ⁶ 、1.5×10 ⁶ Up to 1.5X10 ⁷ 、1.5×10 ⁶ Up to 1X 10 ⁷ 、1.5×10 ⁶ To 9X 10 ⁶ 、1.5×10 ⁶ Up to 8X 10 ⁶ 、1.5×10 ⁶ To 7X 10 ⁶ 、1.5×10 ⁶ Up to 6X 10 ⁶ 、1.5×10 ⁶ Up to 5X 10 ⁶ 、1.5×10 ⁶ Up to 4X 10 ⁶ 、1.5×10 ⁶ Up to 3X 10 ⁶ 、1.5×10 ⁶ Up to 2X 10 ⁶ 、2×10 ⁶ Up to 1.5X10 ⁷ 、2×10 ⁶ Up to 1X 10 ⁷ 、2×10 ⁶ To 9X 10 ⁶ 、2×10 ⁶ Up to 8X 10 ⁶ 、2×10 ⁶ To 7X 10 ⁶ 、2×10 ⁶ Up to 6X 10 ⁶ 、2×10 ⁶ Up to 5X 10 ⁶ 、2×10 ⁶ Up to 4X 10 ⁶ 、2×10 ⁶ Up to 3X 10 ⁶ 、2.5×10 ⁶ Up to 1.5X10 ⁷ 、2.5×10 ⁶ Up to 1X 10 ⁷ 、2.5×10 ⁶ To 9X 10 ⁶ 、2.5×10 ⁶ Up to 8X 10 ⁶ 、2.5×10 ⁶ To 7X 10 ⁶ 、2.5×10 ⁶ Up to 6X 10 ⁶ 、2.5×10 ⁶ Up to 5X 10 ⁶ 、2.5×10 ⁶ Up to 4X 10 ⁶ Or 2.5X10 ⁶ Up to 3X 10 ⁶ Actual body weight or idealized body of cells of individual Treg-enriched cell populations and/or Treg/kg recipient subjectsThe weight of the material (e.g., wherein Treg is cd4+cd25+cd127dim, cd3+cd4+cd25+, cd3+cd4+cd25+cd127dim foxp3+, cd3+foxp3+, cd3+cd4+cd25+foxp3+, cd3+cd25+foxp3+, cd3+cd25+cd127dim, cd4+cd25+, cd4+cd25+cd25+cd127 dimfoxp3+, foxp3+, cd4+foxp3+, foxp3+, cd25+foxp3+ or cd25+cd127 dim.

The population of Treg-enriched cells may have a determined level of purity for the Treg cells. For example, the number of the cells to be processed, the Treg-enriched cell population of the present disclosure may comprise at least about 5%, at least about 10%, 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 59%, by percentage of total cells, by percentage of nucleated cells, or by percentage of cd45+ cells. 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 of Treg cells (e.g., wherein Treg is cd4+cd25+cd127dim, cd3+cd4+cd25+, cd3+cd4+cd25+cd127dim foxp3+, cd3+foxp3+, cd3+cd4+foxp3+, cd3+cd4+cd25+foxp3+, cd3+cd25+foxp3+, cd3+foxp3+, cd3+cd25+cd127dim, cd4+cd25+, cd4+cd25+cd25+cd127 dimfoxp3+, foxp3+, cd4+foxp3+, cd4+cd25+foxp3+, cd25+foxp3+ or cd25+cd127 dim).

The Treg-enriched cell population of the present disclosure may comprise, as a percentage of total cells, nucleated cells, or cd45+ cells, 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%, 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%, based on the percentage of total cells, nucleated cells, or cd45+ cells. 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%, 86% to 97%, and 97% 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 (e.g., wherein Treg is cd4+cd25+cd127dim, cd3+cd4+cd25+, cd3+cd4+cd25+cd127dim foxp3+, cd3+foxp3+, cd3+cd4+cd25+foxp3+, cd3+cd25+foxp3+, cd3+cd25+cd127dim, cd4+cd25+, cd4+cd25+cd25+cd127 dimfoxp3+, foxp3+, cd4+foxp3+, foxp3+, cd25+foxp3+ or cd25+cd127 dim.

The Treg-enriched cell populations of the present disclosure can have a defined level of contaminating non-Treg cells. In some embodiments, up to about 1X 10 ² Up to about 2X 10 ² Up to about 3X 10 ² Up to about 4X 10 ² Up to about 5X 10 ² Up to about 6X 10 ² Up to about 7X 10 ² Up to about 8X 10 ² Up to about 9X 10 ² Up to about 1X 10 ³ Up to about 2X 10 ³ Up to about 3X 10 ³ Up to about 4X 10 ³ Up to about 5X 10 ³ Up to about 6X 10 ³ Up to about 7X 10 ³ Up to about 8X 10 ³ Up to about 9X 10 ³ Up to about 1X 10 ⁴ Up to about 2X 10 ⁴ Up to about 3X 10 ⁴ Up to about 4X 10 ⁴ Up to about 5X 10 ⁴ Up to about 6X 10 ⁴ Up to about 7X 10 ⁴ Up to about 8X 10 ⁴ Up to about 9X 10 ⁴ Or up to about 1X 10 ⁵ The individual non-Treg cells/kg body weight or ideal body weight are present in the Treg-enriched cell population of the present disclosure, wherein the non-Treg cells are FOXP 3-or cd127+/bright.

In some embodiments of the present invention, in some embodiments, treg-enriched cell populations of the present disclosure comprise 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 1.1.2%, at most about 1.3% >; up to about 1.4%, up to about 1.5%, up to about 1.6%, up to about 1.7%, up to about 1.8%, up to about 1.9%, up to about 2%, up to about 2.1%, up to about 2.2%, up to about 2.3%, up to about 2.4%, up to about 2.5%, up to about 2.6%, up to about 2.7%, up to about 2.8%, up to about 2.9%, up to about 3%, up to about 3.1%, up to about 3.2%, up to about 3.3%, up to about 3.4%, up to about 3.5%, up to about 3.6%, up to about 3.7%, up to about 3.8%, up to about 3.9%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9% or up to about 10% of non-Treg cells, wherein the non-Treg cells are FOXP 3-or CD127+/bright.

In various embodiments, the Treg-enriched cell population comprises less than about 5EU endotoxin/ml solution, less than about 1EU endotoxin/ml solution, and/or less than about 0.5EU endotoxin/ml solution.

In some embodiments, at least one of the cell populations comprises less than about 5EU endotoxin/ml of the corresponding suspension.

The Treg-enriched cell population may comprise from 0.5EU/ml endotoxin to 10EU/ml endotoxin. The Treg-enriched cell population may comprise at least 0.5EU/ml endotoxin. The Treg-enriched cell population may comprise up to 10EU/ml endotoxin. The Treg-enriched cell population may comprise 10EU/ml of endotoxin to 8EU/ml of endotoxin, 10EU/ml of endotoxin to 6EU/ml of endotoxin, 10EU/ml of endotoxin to 5EU/ml of endotoxin, 10EU/ml of endotoxin to 4EU/ml of endotoxin, 10EU/ml of endotoxin to 2EU/ml of endotoxin, 10EU/ml of endotoxin to 1EU/ml of endotoxin, 10EU/ml of endotoxin to 0.5EU/ml of endotoxin, 8EU/ml of endotoxin to 6EU/ml of endotoxin, 8EU/ml of endotoxin to 5EU/ml of endotoxin, 8EU/ml of endotoxin to 4EU/ml of endotoxin, 8EU/ml of endotoxin to 2EU/ml of endotoxin, 8EU/ml of endotoxin to 1EU/ml of endotoxin 8EU/ml endotoxin to 0.5EU/ml endotoxin, 6EU/ml endotoxin to 5EU/ml endotoxin, 6EU/ml endotoxin to 4EU/ml endotoxin, 6EU/ml endotoxin to 2EU/ml endotoxin, 6EU/ml endotoxin to 1EU/ml endotoxin, 6EU/ml endotoxin to 0.5EU/ml endotoxin, 5EU/ml endotoxin to 4EU/ml endotoxin, 5EU/ml endotoxin to 2EU/ml endotoxin, 5EU/ml endotoxin to 1EU/ml endotoxin, 5EU/ml endotoxin to 0.5EU/ml endotoxin, 4EU/ml endotoxin to 2EU/ml endotoxin, 4EU/ml endotoxin to 1EU/ml endotoxin, 4EU/ml endotoxin to 0.5EU/ml endotoxin, 2EU/ml endotoxin to 1EU/ml endotoxin, 2EU/ml endotoxin to 0.5EU/ml endotoxin or 1EU/ml endotoxin to 0.5EU/ml endotoxin. The Treg-enriched cell population may comprise 10EU/ml endotoxin, 8EU/ml endotoxin, 6EU/ml endotoxin, 5EU/ml endotoxin, 4EU/ml endotoxin, 2EU/ml endotoxin, 1EU/ml endotoxin or 0.5EU/ml endotoxin. The Treg-enriched cell population can comprise at least 8EU/ml endotoxin, 6EU/ml endotoxin, 5EU/ml endotoxin, 4EU/ml endotoxin, 2EU/ml endotoxin, 1EU/ml endotoxin, or 0.5EU/ml endotoxin. The Treg-enriched cell population may comprise up to 10EU/ml endotoxin, 8EU/ml endotoxin, 6EU/ml endotoxin, 5EU/ml endotoxin, 4EU/ml endotoxin, 2EU/ml endotoxin or 1EU/ml endotoxin.

The Treg-enriched cell population may comprise from 0.5% w/w to 10% w/w unbound reagent. These unbound reagents may include any affinity reagent for sorting tregs, such as antibodies or purified particles or magnetic particles. The Treg-enriched cell population may comprise at least 0.5% w/w unbound reagent. The Treg-enriched cell population may comprise up to 10% w/w unbound reagent. The Treg-enriched cell population may 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 of unbound reagent. The Treg-enriched cell population may 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 reagent. The Treg-enriched cell population may 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 reagent. The Treg-enriched cell population may comprise up to 10% w/w, 8% w/w, 6% w/w, 5% w/w, 4% w/w, 2% w/w or 1% w/w unbound reagent.

The Treg-enriched cell population may comprise 1 x 10 ³ Up to 50X 10 ³ Individual microbeads/cells. Such microbeads may include microbeads for purifying the population of tregs, e.g., anti-CD 25 antibodies comprising microbeads for sorting the population of tregs, or anti-CD 4 antibodies comprising microbeads for sorting the population of tregs, and/or anti-CD 127 antibodies comprising microbeads for sorting the population of tregs. The Treg-enriched cell population may comprise at least 1 x 10 ³ Individual microbeads/cells. The Treg-enriched cell population may comprise up to 50 x 10 ³ Individual microbeads/cells. The Treg-enriched cell population may comprise 50 x 10 ³ Up to 40X 10 ³ 、50×10 ³ Up to 30X 10 ³ 、50×10 ³ Up to 20X 10 ³ 、50×10 ³ Up to 10X 10 ³ 、50×10 ³ Up to 5X 10 ³ 、50×10 ³ Up to 4X 10 ³ 、50×10 ³ Up to 2X 10 ³ 、50×10 ³ Up to 1X 10 ³ 、40×10 ³ Up to 30X 10 ³ 、40×10 ³ Up to 20X 10 ³ 、40×10 ³ Up to 10X 10 ³ 、40×10 ³ Up to 5X 10 ³ 、40×10 ³ Up to 4X 10 ³ 、40×10 ³ Up to 2X 10 ³ 、40×10 ³ Up to 1X 10 ³ 、30×10 ³ Up to 20X 10 ³ 、30×10 ³ Up to 10X 10 ³ 、30×10 ³ Up to 5X 10 ³ 、30×10 ³ Up to 4X 10 ³ 、30×10 ³ Up to 2X 10 ³ 、30×10 ³ Up to 1X 10 ³ 、20×10 ³ Up to 10X 10 ³ 、20×10 ³ Up to 5X 10 ³ 、20×10 ³ Up to 4X 10 ³ 、20×10 ³ Up to 2X 10 ³ 、20×10 ³ Up to 1X 10 ³ 、10×10 ³ Up to 5X 10 ³ 、10×10 ³ Up to 4X 10 ³ 、10×10 ³ Up to 2X 10 ³ 、10×10 ³ Up to 1X 10 ³ 、5×10 ³ Up to 4X 10 ³ 、5×10 ³ Up to 2X 10 ³ 、5×10 ³ Up to 1X 10 ³ 、4×10 ³ Up to 2X 10 ³ 、4×10 ³ Up to 1X 10 ³ Or 2X 10 ³ Up to 1X 10 ³ Individual microbeads/cells. The Treg-enriched cell population may comprise 50 x 10 ³ 、40×10 ³ 、30×10 ³ 、20×10 ³ 、10×10 ³ 、5×10 ³ 、4×10 ³ 、2×10 ³ Or 1X 10 ³ Individual microbeads/cells.

Tcon

The second population of CD45+ cells comprising at least Tcon may comprise at least about 1X 10 ⁴ At least about 1X 10 ⁵ At least about 5X 10 ⁵ At least about 6X 10 ⁵ At least about 7X 10 ⁵ At least about 8X 10 ⁵ At least about 9X 10 ⁵ At least about 1X 10 ⁶ At least about 1.1X10 ⁶ At least about 1.2 x 10 ⁶ At least about 1.3X10 ⁶ At least about 1.4X10 ⁶ At least about 1.5X10 ⁶ At least about 1.6X10 ⁶ At least about 1.7X10 ⁶ At least about 1.8X10 ⁶ At least about 1.9X10 ⁶ At least about 2X 10 ⁶ At least about 2.1X10 ⁶ At least about 2.2 x 10 ⁶ At least about 2.3X10 ⁶ At least about 2.4X10 ⁶ At least about 2.5X10 ⁶ At least about 2.6X10 ⁶ At least about 2.7X10 ⁶ At least about 2.8X10 ⁶ At least about 2.9X10 ⁶ At least about 3X 10 ⁶ At least about 3.1X10 ⁶ At least about 3.2 x 10 ⁶ At least about 3.3X10 ⁶ At least about 3.4X10 ⁶ At least about 3.5X10 ⁶ At least about 3.6X10 ⁶ At least about 3.7X10 ⁶ At least about 3.8X10 ⁶ At least about 3.9X10 ⁶ At least about 4X 10 ⁶ At least about 4.1X10 ⁶ At least about 4.2 x 10 ⁶ At least about 4.3X10 ⁶ At least about 4.4X10 ⁶ At least about 4.5X10 ⁶ To (1)Less than about 4.6X10 ⁶ At least about 4.7X10 ⁶ At least about 4.8X10 ⁶ At least about 4.9X10 ⁶ At least about 5X 10 ⁶ At least about 5.1X10 ⁶ At least about 5.2 x 10 ⁶ At least about 5.3X10 ⁶ At least about 5.4X10 ⁶ At least about 5.5X10 ⁶ At least about 5.6X10 ⁶ At least about 5.7X10 ⁶ At least about 5.8X10 ⁶ At least about 5.9X10 ⁶ At least about 6X 10 ⁶ At least about 6.5X10 ⁶ At least about 7X 10 ⁶ At least about 7.5X10 ⁶ At least about 8X 10 ⁶ At least about 8.5X10 ⁶ At least about 9X 10 ⁶ At least about 9.5X10 ⁶ At least about 1X 10 ⁷ At least about 1.5X10 ⁷ At least about 2X 10 ⁷ At least about 2.5X10 ⁷ At least about 3X 10 ⁷ At least about 3.5X10 ⁷ At least about 4X 10 ⁷ At least about 4.5X10 ⁷ At least about 5X 10 ⁷ At least about 5.5X10 ⁷ At least about 6X 10 ⁷ At least about 6.5X10 ⁷ At least about 7X 10 ⁷ At least about 7.5X10 ⁷ At least about 8X 10 ⁷ At least about 8.5X10 ⁷ At least about 9X 10 ⁷ At least about 9.5X10 ⁷ At least about 1X 10 ⁸ At least about 1X 10 ⁸ At least about 1.5X10 ⁸ At least about 2X 10 ⁸ At least about 2.5X10 ⁸ At least about 3X 10 ⁸ At least about 3.5X10 ⁸ At least about 4X 10 ⁷ At least about 4.5X10 ⁸ At least about 5X 10 ⁸ At least about 5.5X10 ⁸ At least about 6X 10 ⁸ At least about 6.5X10 ⁸ At least about 7X 10 ⁸ At least about 7.5X10 ⁸ At least about 8X 10 ⁸ At least about 8.5X10 ⁸ At least about 9X 10 ⁸ At least about 9.5X10 ⁸ At least about 1X 10 ⁹ Or a cell of a second, or more, cd45+ cell population and/or an actual or ideal body weight of a Tcon/kg recipient subject (e.g., where Tcon is cd3+ or cd3+cd127+/bright).

The second CD45+ cell population comprising at least Tcon may compriseAbout 1×10 more ⁴ Up to about 1X 10 ⁵ Up to about 5X 10 ⁵ Up to about 6X 10 ⁵ Up to about 7X 10 ⁵ Up to about 8X 10 ⁵ Up to about 9X 10 ⁵ Up to about 1X 10 ⁶ Up to about 1.1X10 ⁶ Up to about 1.2X10 ⁶ Up to about 1.3X10 ⁶ Up to about 1.4X10 ⁶ Up to about 1.5X10 ⁶ Up to about 1.6X10 ⁶ Up to about 1.7X10 ⁶ Up to about 1.8X10 ⁶ Up to about 1.9X10 ⁶ Up to about 2X 10 ⁶ Up to about 2.1X10 ⁶ Up to about 2.2X10 ⁶ Up to about 2.3X10 ⁶ Up to about 2.4X10 ⁶ Up to about 2.5X10 ⁶ Up to about 2.6X10 ⁶ Up to about 2.7X10 ⁶ Up to about 2.8X10 ⁶ Up to about 2.9X10 ⁶ Up to about 3X 10 ⁶ Up to about 3.1X10 ⁶ Up to about 3.2 x 10 ⁶ Up to about 3.3X10 ⁶ Up to about 3.4X10 ⁶ Up to about 3.5X10 ⁶ Up to about 3.6X10 ⁶ Up to about 3.7X10 ⁶ Up to about 3.8X10 ⁶ Up to about 3.9X10 ⁶ Up to about 4X 10 ⁶ Up to about 4.1X10 ⁶ Up to about 4.2 x 10 ⁶ Up to about 4.3X10 ⁶ Up to about 4.4X10 ⁶ Up to about 4.5X10 ⁶ Up to about 4.6X10 ⁶ Up to about 4.7X10 ⁶ Up to about 4.8X10 ⁶ Up to about 4.9X10 ⁶ Up to about 5X 10 ⁶ Up to about 5.1X10 ⁶ Up to about 5.2 x 10 ⁶ Up to about 5.3X10 ⁶ Up to about 5.4X10 ⁶ Up to about 5.5X10 ⁶ Up to about 5.6X10 ⁶ Up to about 5.7X10 ⁶ Up to about 5.8X10 ⁶ Up to about 5.9X10 ⁶ Up to about 6X 10 ⁶ Up to about 6.5X10 ⁶ Up to about 7X 10 ⁶ Up to about 7.5X10 ⁶ Up to about 8X 10 ⁶ Up to about 8.5X10 ⁶ Up to about 9X 10 ⁶ Up to about 9.5X10 ⁶ Up to about 1X 10 ⁷ Up to about 1.5X10 ⁷ At most about2×10 ⁷ Up to about 2.5X10 ⁷ Up to about 3X 10 ⁷ Up to about 3.5X10 ⁷ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁷ Up to about 5X 10 ⁷ Up to about 5.5X10 ⁷ Up to about 6X 10 ⁷ Up to about 6.5X10 ⁷ Up to about 7X 10 ⁷ Up to about 7.5X10 ⁷ Up to about 8X 10 ⁷ Up to about 8.5X10 ⁷ Up to about 9X 10 ⁷ Up to about 9.5X10 ⁷ Up to about 1X 10 ⁸ Up to about 1X 10 ⁸ Up to about 1.5X10 ⁸ Up to about 2X 10 ⁸ Up to about 2.5X10 ⁸ Up to about 3X 10 ⁸ Up to about 3.5X10 ⁸ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁸ Up to about 5X 10 ⁸ Up to about 5.5X10 ⁸ Up to about 6X 10 ⁸ Up to about 6.5X10 ⁸ Up to about 7X 10 ⁸ Up to about 7.5X10 ⁸ Up to about 8X 10 ⁸ Up to about 8.5X10 ⁸ Up to about 9X 10 ⁸ Up to about 9.5X10 ⁸ Or up to about 1X 10 ⁹ The actual or ideal body weight of the cells of the second CD45+ cell population and/or the Tcon/kg recipient subject (e.g., where Tcon is CD3+ or CD3+CD127+/bright).

For example, the second CD45+ cell population comprising at least Tcon may comprise 1X 10 cells ⁴ Up to 1X 10 ⁹ 、1×10 ⁵ Up to 1X 10 ⁸ 、1×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 1.5X10 ⁷ 、5×10 ⁵ Up to 1X 10 ⁷ 、5×10 ⁵ To 9X 10 ⁶ 、5×10 ⁵ Up to 8X 10 ⁶ 、5×10 ⁵ To 7X 10 ⁶ 、5×10 ⁵ Up to 6X 10 ⁶ 、5×10 ⁵ Up to 5X 10 ⁶ 、5×10 ⁵ Up to 4X 10 ⁶ 、5×10 ⁵ Up to 3X 10 ⁶ 、5×10 ⁵ Up to 2X 10 ⁶ 、5×10 ⁵ Up to 1X 10 ⁶ 、1×10 ⁶ Up to 1.5X10 ⁷ 、1×10 ⁶ Up to 1X 10 ⁷ 、1×10 ⁶ To 9X 10 ⁶ 、1×10 ⁶ Up to 8X 10 ⁶ 、1×10 ⁶ To 7X 10 ⁶ 、1×10 ⁶ Up to 6X 10 ⁶ 、1×10 ⁶ Up to 5X 10 ⁶ 、1×10 ⁶ Up to 4X 10 ⁶ 、1×10 ⁶ Up to 3X 10 ⁶ 、1×10 ⁶ Up to 2X 10 ⁶ 、1.5×10 ⁶ Up to 1.5X10 ⁷ 、1.5×10 ⁶ Up to 1X 10 ⁷ 、1.5×10 ⁶ To 9X 10 ⁶ 、1.5×10 ⁶ Up to 8X 10 ⁶ 、1.5×10 ⁶ To 7X 10 ⁶ 、1.5×10 ⁶ Up to 6X 10 ⁶ 、1.5×10 ⁶ Up to 5X 10 ⁶ 、1.5×10 ⁶ Up to 4X 10 ⁶ 、1.5×10 ⁶ Up to 3X 10 ⁶ 、1.5×10 ⁶ Up to 2X 10 ⁶ 、2×10 ⁶ Up to 1.5X10 ⁷ 、2×10 ⁶ Up to 1X 10 ⁷ 、2×10 ⁶ To 9X 10 ⁶ 、2×10 ⁶ Up to 8X 10 ⁶ 、2×10 ⁶ To 7X 10 ⁶ 、2×10 ⁶ Up to 6X 10 ⁶ 、2×10 ⁶ Up to 5X 10 ⁶ 、2×10 ⁶ Up to 4X 10 ⁶ 、2×10 ⁶ Up to 3X 10 ⁶ 、2.5×10 ⁶ Up to 1.5X10 ⁷ 、2.5×10 ⁶ Up to 1X 10 ⁷ 、2.5×10 ⁶ To 9X 10 ⁶ 、2.5×10 ⁶ Up to 8X 10 ⁶ 、2.5×10 ⁶ To 7X 10 ⁶ 、2.5×10 ⁶ Up to 6X 10 ⁶ 、2.5×10 ⁶ Up to 5X 10 ⁶ 、2.5×10 ⁶ Up to 4X 10 ⁶ Or 2.5X10 ⁶ Up to 3X 10 ⁶ The actual or ideal body weight of the cells of the second CD45+ cell population and/or the Tcon/kg recipient subject (e.g., where Tcon is CD3+ or CD3+CD127+/bright).

The second cd45+ cell population of the present disclosure may have a determined purity level for Tcon cells. For example, the number of the cells to be processed, the second cd45+ cell population of the present disclosure may comprise at least about 5%, at least about 10%, 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% by percentage of total cells 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 of Tcon cells (e.g., wherein Tcon is CD3+ or CD3+CD127+/bright).

The second cd45+ cell population of the present disclosure may 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%, 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% based on the percentage of total cells, nucleated cells, or cd45+ cells. 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%, 86% to 97%, and 97% 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% Tcon (e.g., where Tcon is cd3+ or cd3+cd127+/bright).

The second cd45+ cell population of the present disclosure may have a defined level of contaminating non-Tcon cells. In some embodiments, up to about 1X 10 ² Up to about 2X 10 ² Up to about 3X 10 ² Up to about 4X 10 ² Up to about 5X 10 ² Up to about 6X 10 ² Up to about 7X 10 ² Up to about 8X 10 ² Up to about 9X 10 ² Up to about 1X 10 ³ Up to about 2X 10 ³ Up to about 3X 10 ³ Up to about 4X 10 ³ Up to about 5X 10 ³ Up to about 6X 10 ³ Up to about 7X 10 ³ Up to about 8X 10 ³ Up to about 9X 10 ³ Up to about 1X 10 ⁴ Up to about 2X 10 ⁴ Up to about 3X 10 ⁴ Up to about 4X 10 ⁴ Up to about 5X 10 ⁴ Up to about 6X 10 ⁴ Up to about 7X 10 ⁴ Up to about 8X 10 ⁴ Up to about 9X 10 ⁴ Or up to about 1X 10 ⁵ The actual or ideal body weight of the individual non-Tcon cells/kg recipient subject is present in the second CD45+ cell population of the disclosure (e.g., wherein the non-Tcon cells are notTcon cells are CD 3-or CD3+CD127 dim).

In some embodiments of the present invention, in some embodiments, the second cd45+ cell population of the present disclosure comprises up to about 0.001%, up to about 0.002%, up to about 0.003%, up to about 0.004%, up to about 0.005%, up to about 0.006%, up to about 0.007%, up to about 0.008%0.009%, up to about 0.01%, up to about 0.02%, up to about 0.03%, up to about 0.04%, up to about 0.05%, up to about 0.06%, up to about 0.07%, up to about 0.08%, up to about 0.09%, up to about 0.1%, up to about 0.2%, up to about 0.3%, up to about 0.4%, up to about 0.5%, up to about 0.6%, up to about 0.7%, up to about 0.8%, up to about 0.9%, up to about 1%, up to about 1.1.2%, up to about 1.3% >, up to about 1.4%, up to about 1.5%, up to about 1.6%, up to about 1.7%, up to about 1.8%, up to about 1.9%, up to about 2%, up to about 2.1%, up to about 2.2%, up to about 2.3%, up to about 2.4%, up to about 2.5%, up to about 2.6%, up to about 2.7%, up to about 2.8%, up to about 2.9%, up to about 3%, up to about 3.1%, up to about 3.2%, up to about 3.3%, up to about 3.4%, up to about 3.5%, up to about 3.6%, up to about 3.7%, up to about 3.8%, up to about 3.9%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9% or up to about 10% of non-Tcon cells, (e.g., wherein the non-Tcon cells are CD 3-or CD3+CD127 dim).

In some cases, the Treg-enriched cell population comprises less than about 5EU endotoxin/ml solution, less than about 1EU endotoxin/ml solution, and/or less than about 0.5EU endotoxin/ml solution.

The second CD45+ cell population comprising at least Tcon may comprise from 0.5EU/ml endotoxin to 10EU/ml endotoxin. The second cd45+ cell population may comprise at least 0.5EU/ml endotoxin. The second cd45+ cell population may comprise up to 10EU/ml of endotoxin. The second CD45+ cell population may comprise 10EU/ml of endotoxin to 8EU/ml of endotoxin, 10EU/ml of endotoxin to 6EU/ml of endotoxin, 10EU/ml of endotoxin to 5EU/ml of endotoxin, 10EU/ml of endotoxin to 4EU/ml of endotoxin, 10EU/ml of endotoxin to 2EU/ml of endotoxin, 10EU/ml of endotoxin to 1EU/ml of endotoxin, 10EU/ml of endotoxin to 0.5EU/ml of endotoxin, 8EU/ml of endotoxin to 6EU/ml of endotoxin, 8EU/ml of endotoxin to 5EU/ml of endotoxin, 8EU/ml of endotoxin to 4EU/ml of endotoxin, 8EU/ml of endotoxin to 2EU/ml of endotoxin, 8EU/ml of endotoxin to 1EU/ml of endotoxin 8EU/ml endotoxin to 0.5EU/ml endotoxin, 6EU/ml endotoxin to 5EU/ml endotoxin, 6EU/ml endotoxin to 4EU/ml endotoxin, 6EU/ml endotoxin to 2EU/ml endotoxin, 6EU/ml endotoxin to 1EU/ml endotoxin, 6EU/ml endotoxin to 0.5EU/ml endotoxin, 5EU/ml endotoxin to 4EU/ml endotoxin, 5EU/ml endotoxin to 2EU/ml endotoxin, 5EU/ml endotoxin to 1EU/ml endotoxin, 5EU/ml endotoxin to 0.5EU/ml endotoxin, 4EU/ml endotoxin to 2EU/ml endotoxin, 4EU/ml endotoxin to 1EU/ml endotoxin, 4EU/ml endotoxin to 0.5EU/ml endotoxin, 2EU/ml endotoxin to 1EU/ml endotoxin, 2EU/ml endotoxin to 0.5EU/ml endotoxin or 1EU/ml endotoxin to 0.5EU/ml endotoxin. The second cd45+ cell population may comprise about 10EU/ml of endotoxin, 8EU/ml of endotoxin, 6EU/ml of endotoxin, 5EU/ml of endotoxin, 4EU/ml of endotoxin, 2EU/ml of endotoxin, 1EU/ml of endotoxin, or 0.5EU/ml of endotoxin. The second cd45+ cell population may comprise at least 8EU/ml endotoxin, 6EU/ml endotoxin, 5EU/ml endotoxin, 4EU/ml endotoxin, 2EU/ml endotoxin, 1EU/ml endotoxin, or 0.5EU/ml endotoxin. The second cd45+ cell population may comprise up to 10EU/ml of endotoxin, 8EU/ml of endotoxin, 6EU/ml of endotoxin, 5EU/ml of endotoxin, 4EU/ml of endotoxin, 2EU/ml of endotoxin, or 1EU/ml of endotoxin.

The second population of cd45+ cells may comprise less than 0.1% w/w to 3% w/w unbound reagent. These unbound reagents may include any affinity reagent for sorting Tcon or other cell populations, such as antibodies or purified particles or magnetic particles. The second population of cd45+ cells may comprise less than about 0.1% w/w unbound reagent. The second cd45+ cell population may comprise less than 3% w/w to 2% w/w, 3% w/w to 1% w/w, 3% w/w to 0.5% w/w, 3% w/w to 0.25% w/w, 3% w/w to 0.1% w/w, 2% w/w to 1% w/w, 2% w/w to 0.5% w/w, 2% w/w to 0.25% w/w, 2% w/w to 0.1% w/w, 1% w/w to 0.5% w/w, 1% w/w to 0.25% w/w, 1% w/w to 0.1% w/w, 0.5% w/w to 0.25% w/w, 0.5% w/w to 0.1% w/w, or 0.25% w/w to 0.1% w/w of unbound reagent. The second population of cd45+ cells may comprise less than about 3% w/w, 2% w/w, 1% w/w, 0.5% w/w, 0.25% w/w, or 0.1% w/w unbound reagent.

The second cd45+ cell population may comprise less than 50 to 2,000 microbeads per cell. Such microbeads may include microbeads for purifying a Tcon population or other cell populations, such as CD25 microbeads, or CD4 microbeads, or CD127 microbeads, or CD34 microbeads for sorting cell populations. The second cd45+ cell population may comprise less than 2,000 microbeads per cell. The second population of cd45+ cells may comprise less than 2,000 to 1,000, 2,000 to 700, 2,000 to 500, 2,000 to 300, 2,000 to 100, 2,000 to 50, 1,000 to 700, 1,000 to 500, 1,000 to 300, 1,000 to 100, 1,000 to 50, 700 to 500, 700 to 300, 700 to 100, 700 to 50, 500 to 300, 500 to 100, 500 to 50, 300 to 100, 300 to 50, or 100 to 50 microbeads/cell. The second cd45+ cell population may comprise about 2,000, 1,000, 700, 500, 300, 100, or 50 microbeads/cell. The second cd45+ cell population may not comprise microbeads per cell.

In the second cd45+ cell population, the ratio of Tcon to Treg administered to the subject may be, for example, about 1:100, 1:50, 1:25, 1:20, 1:15, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1.5:2, 1:1.5, 1:1, 1.5:1, 2:1, 2:1.5, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 50:1, or 100:1.

Cells of the second cd45+ cell population comprising at least Tcon may be cryopreserved for any amount of time. The cells of the second cd45+ cell population 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 longer prior to thawing and administering to the subject.

In some embodiments, cells of the second cd45+ cell population comprising at least Tcon are 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 administering to the subject.

In some embodiments, the cells of the second cd45+ cell population are 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 administering to the subject.

In some embodiments, the cells of the second cd45+ cell population are cryopreserved for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 10 days, up to about 14 days, up to about 21 days, up to about 28 days, up to about 50 days, up to about 60 days, or up to about 96 days prior to thawing and administering to the subject.

iNKT

The population of cells comprising the iNKT population may comprise at least about 1 x 10 ⁴ At least about 1X 10 ⁵ At least about 5X 10 ⁵ At least about 6X 10 ⁵ At least about 7X 10 ⁵ At least about 8X 10 ⁵ At least about 9X 10 ⁵ At least about 1X 10 ⁶ At least about 1.1X10 ⁶ At least about 1.2 x 10 ⁶ At least about 1.3X10 ⁶ At least about 1.4X10 ⁶ At least about 1.5X10 ⁶ At least about 1.6X10 ⁶ At least about 1.7X10 ⁶ At least about 1.8X10 ⁶ At least about 1.9X10 ⁶ At least about 2X 10 ⁶ At least about 2.1X10 ⁶ At least about 2.2 x 10 ⁶ At least about 2.3X10 ⁶ At least about 2.4X10 ⁶ At least about 2.5X10 ⁶ At least about 2.6X10 ⁶ At least about 2.7X10 ⁶ At least about 2.8X10 ⁶ At least about 2.9X10 ⁶ At least about 3X 10 ⁶ At least about 3.1X10 ⁶ At least about 3.2 x 10 ⁶ At least about 3.3X10 ⁶ At least about 3.4X10 ⁶ At least about 3.5X10 ⁶ At least about 3.6X10 ⁶ At least about 3.7X10 ⁶ At least about 3.8X10 ⁶ At least about 3.9X10 ⁶ At least about 4X 10 ⁶ At least about 4.1X10 ⁶ At least about 4.2 x 10 ⁶ At least about 4.3X10 ⁶ At least about 4.4X10 ⁶ At least about 4.5X10 ⁶ At least about 4.6X10 ⁶ At least about 4.7X10 ⁶ At least about 4.8X10 ⁶ At least about 4.9X10 ⁶ At least about 5X 10 ⁶ At least about 5.1X10 ⁶ At least about 5.2 x 10 ⁶ At least about 5.3X10 ⁶ At least about 5.4X10 ⁶ At least about 5.5X10 ⁶ At least about 5.6X10 ⁶ At least about 5.7X10 ⁶ At least about 5.8X10 ⁶ At least about 5.9X10 ⁶ To (1)Less than about 6X 10 ⁶ At least about 6.5X10 ⁶ At least about 7X 10 ⁶ At least about 7.5X10 ⁶ At least about 8X 10 ⁶ At least about 8.5X10 ⁶ At least about 9X 10 ⁶ At least about 9.5X10 ⁶ At least about 1X 10 ⁷ At least about 1.5X10 ⁷ At least about 2X 10 ⁷ At least about 2.5X10 ⁷ At least about 3X 10 ⁷ At least about 3.5X10 ⁷ At least about 4X 10 ⁷ At least about 4.5X10 ⁷ At least about 5X 10 ⁷ At least about 5.5X10 ⁷ At least about 6X 10 ⁷ At least about 6.5X10 ⁷ At least about 7X 10 ⁷ At least about 7.5X10 ⁷ At least about 8X 10 ⁷ At least about 8.5X10 ⁷ At least about 9X 10 ⁷ At least about 9.5X10 ⁷ At least about 1X 10 ⁸ At least about 1X 10 ⁸ At least about 1.5X10 ⁸ At least about 2X 10 ⁸ At least about 2.5X10 ⁸ At least about 3X 10 ⁸ At least about 3.5X10 ⁸ At least about 4X 10 ⁷ At least about 4.5X10 ⁸ At least about 5X 10 ⁸ At least about 5.5X10 ⁸ At least about 6X 10 ⁸ At least about 6.5X10 ⁸ At least about 7X 10 ⁸ At least about 7.5X10 ⁸ At least about 8X 10 ⁸ At least about 8.5X10 ⁸ At least about 9X 10 ⁸ At least about 9.5X10 ⁸ At least about 1X 10 ⁹ Or an actual body weight or an ideal body weight of a subject with more iNKT/kg receptors (e.g., where iNKT is cd3+vα24jα18+).

The population of cells comprising the iNKT population may comprise up to about 1 x 10 ⁴ Up to about 1X 10 ⁵ Up to about 5X 10 ⁵ Up to about 6X 10 ⁵ Up to about 7X 10 ⁵ Up to about 8X 10 ⁵ Up to about 9X 10 ⁵ Up to about 1X 10 ⁶ Up to about 1.1X10 ⁶ Up to about 1.2X10 ⁶ Up to about 1.3X10 ⁶ Up to about 1.4X10 ⁶ Up to about 1.5X10 ⁶ Up to about 1.6X10 ⁶ Up to about 1.7X10 ⁶ Up to about 1.8X10 ⁶ At most about 1.9×10 ⁶ Up to about 2X 10 ⁶ Up to about 2.1X10 ⁶ Up to about 2.2X10 ⁶ Up to about 2.3X10 ⁶ Up to about 2.4X10 ⁶ Up to about 2.5X10 ⁶ Up to about 2.6X10 ⁶ Up to about 2.7X10 ⁶ Up to about 2.8X10 ⁶ Up to about 2.9X10 ⁶ Up to about 3X 10 ⁶ Up to about 3.1X10 ⁶ Up to about 3.2 x 10 ⁶ Up to about 3.3X10 ⁶ Up to about 3.4X10 ⁶ Up to about 3.5X10 ⁶ Up to about 3.6X10 ⁶ Up to about 3.7X10 ⁶ Up to about 3.8X10 ⁶ Up to about 3.9X10 ⁶ Up to about 4X 10 ⁶ Up to about 4.1X10 ⁶ Up to about 4.2 x 10 ⁶ Up to about 4.3X10 ⁶ Up to about 4.4X10 ⁶ Up to about 4.5X10 ⁶ Up to about 4.6X10 ⁶ Up to about 4.7X10 ⁶ Up to about 4.8X10 ⁶ Up to about 4.9X10 ⁶ Up to about 5X 10 ⁶ Up to about 5.1X10 ⁶ Up to about 5.2 x 10 ⁶ Up to about 5.3X10 ⁶ Up to about 5.4X10 ⁶ Up to about 5.5X10 ⁶ Up to about 5.6X10 ⁶ Up to about 5.7X10 ⁶ Up to about 5.8X10 ⁶ Up to about 5.9X10 ⁶ Up to about 6X 10 ⁶ Up to about 6.5X10 ⁶ Up to about 7X 10 ⁶ Up to about 7.5X10 ⁶ Up to about 8X 10 ⁶ Up to about 8.5X10 ⁶ Up to about 9X 10 ⁶ Up to about 9.5X10 ⁶ Up to about 1X 10 ⁷ Up to about 1.5X10 ⁷ Up to about 2X 10 ⁷ Up to about 2.5X10 ⁷ Up to about 3X 10 ⁷ Up to about 3.5X10 ⁷ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁷ Up to about 5X 10 ⁷ Up to about 5.5X10 ⁷ Up to about 6X 10 ⁷ Up to about 6.5X10 ⁷ Up to about 7X 10 ⁷ Up to about 7.5X10 ⁷ Up to about 8X 10 ⁷ Up to about 8.5X10 ⁷ Up to about 9X 10 ⁷ Up to about 9.5X10 ⁷ Up to about 1X 10 ⁸ Up to about 1X 10 ⁸ Up to about 1.5X10 ⁸ Up to about 2X 10 ⁸ Up to about 2.5X10 ⁸ Up to about 3X 10 ⁸ Up to about 3.5X10 ⁸ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁸ Up to about 5X 10 ⁸ Up to about 5.5X10 ⁸ Up to about 6X 10 ⁸ Up to about 6.5X10 ⁸ Up to about 7X 10 ⁸ Up to about 7.5X10 ⁸ Up to about 8X 10 ⁸ Up to about 8.5X10 ⁸ Up to about 9X 10 ⁸ Up to about 9.5X10 ⁸ Or up to about 1X 10 ⁹ Actual or ideal body weight of individual iNKT/kg recipient subjects (e.g., where iNKT is cd3+vα24jα18+).

For example, a cell population comprising a population of inkts may comprise 1 x 10 ⁴ Up to 1X 10 ⁹ 、1×10 ⁵ Up to 1X 10 ⁸ 、1×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 1.5X10 ⁷ 、5×10 ⁵ Up to 1X 10 ⁷ 、5×10 ⁵ To 9X 10 ⁶ 、5×10 ⁵ Up to 8X 10 ⁶ 、5×10 ⁵ To 7X 10 ⁶ 、5×10 ⁵ Up to 6X 10 ⁶ 、5×10 ⁵ Up to 5X 10 ⁶ 、5×10 ⁵ Up to 4X 10 ⁶ 、5×10 ⁵ Up to 3X 10 ⁶ 、5×10 ⁵ Up to 2X 10 ⁶ 、5×10 ⁵ Up to 1X 10 ⁶ 、1×10 ⁶ Up to 1.5X10 ⁷ 、1×10 ⁶ Up to 1X 10 ⁷ 、1×10 ⁶ To 9X 10 ⁶ 、1×10 ⁶ Up to 8X 10 ⁶ 、1×10 ⁶ To 7X 10 ⁶ 、1×10 ⁶ Up to 6X 10 ⁶ 、1×10 ⁶ Up to 5X 10 ⁶ 、1×10 ⁶ Up to 4X 10 ⁶ 、1×10 ⁶ Up to 3X 10 ⁶ 、1×10 ⁶ Up to 2X 10 ⁶ 、1.5×10 ⁶ Up to 1.5X10 ⁷ 、1.5×10 ⁶ Up to 1X 10 ⁷ 、1.5×10 ⁶ To 9X 10 ⁶ 、1.5×10 ⁶ Up to 8X 10 ⁶ 、1.5×10 ⁶ To 7X 10 ⁶ 、1.5×10 ⁶ Up to 6X 10 ⁶ 、1.5×10 ⁶ Up to 5X 10 ⁶ 、1.5×10 ⁶ Up to 4X 10 ⁶ 、1.5×10 ⁶ Up to 3X 10 ⁶ 、1.5×10 ⁶ Up to 2X 10 ⁶ 、2×10 ⁶ Up to 1.5X10 ⁷ 、2×10 ⁶ Up to 1X 10 ⁷ 、2×10 ⁶ To 9X 10 ⁶ 、2×10 ⁶ Up to 8X 10 ⁶ 、2×10 ⁶ To 7X 10 ⁶ 、2×10 ⁶ Up to 6X 10 ⁶ 、2×10 ⁶ Up to 5X 10 ⁶ 、2×10 ⁶ Up to 4X 10 ⁶ 、2×10 ⁶ Up to 3X 10 ⁶ 、2.5×10 ⁶ Up to 1.5X10 ⁷ 、2.5×10 ⁶ Up to 1X 10 ⁷ 、2.5×10 ⁶ To 9X 10 ⁶ 、2.5×10 ⁶ Up to 8X 10 ⁶ 、2.5×10 ⁶ To 7X 10 ⁶ 、2.5×10 ⁶ Up to 6X 10 ⁶ 、2.5×10 ⁶ Up to 5X 10 ⁶ 、2.5×10 ⁶ Up to 4X 10 ⁶ Or 2.5X10 ⁶ Up to 3X 10 ⁶ Actual or ideal body weight of individual iNKT/kg recipient subjects (e.g., where iNKT is cd3+vα24jα18+).

The iNKT populations of the present disclosure can have a determined purity level for iNKT cells. For example, the number of the cells to be processed, the iNKT population of the present disclosure may comprise at least about 5%, at least about 10%, 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 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 iNKT cells (e.g., wherein iNKT is cd3+vα24jα18+).

The iNKT population of the present disclosure may 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%, 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%, 96% to 100%, 97% to 99% based on the percentage of total cells, nucleated cells, or cd45+ cells. 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%, 97% to 97%, and 97% to 97% of the composition 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% iNKT (e.g., where iNKT is cd3+vα24jα18+).

The iNKT populations of the present disclosure may have a defined level of contaminating non-iNKT cells. In some embodiments, up to about 1X 10 ² Up to about 2X 10 ² Up to about 3X 10 ² Up to about 4X 10 ² Up to about 5X 10 ² Up to about 6X 10 ² Up to about 7X 10 ² Up to about 8X 10 ² Up to about 9X 10 ² Up to about 1X 10 ³ Up to about 2X 10 ³ Up to about 3X 10 ³ Up to about 4X 10 ³ Up to about 5X 10 ³ Up to about 6X 10 ³ Up to about 7X 10 ³ Up to about 8X 10 ³ Up to about 9X 10 ³ Up to about 1X 10 ⁴ Up to about 2X 10 ⁴ Up to about 3X 10 ⁴ Up to about 4X 10 ⁴ Up to about 5X 10 ⁴ Up to about 6X 10 ⁴ Up to about 7X 10 ⁴ Up to about 8X 10 ⁴ Up to about 9X 10 ⁴ Or up to about 1X 10 ⁵ The actual or ideal body weight of a non-iNKT cell/kg recipient subject is present in the iNKT population of the present disclosure (e.g., wherein the non-iNKT cell is vα24jα18-).

In some embodiments of the present invention, in some embodiments, the iNKT population of the present 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 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% >; up to about 1.4%, up to about 1.5%, up to about 1.6%, up to about 1.7%, up to about 1.8%, up to about 1.9%, up to about 2%, up to about 2.1%, up to about 2.2%, up to about 2.3%, up to about 2.4%, up to about 2.5%, up to about 2.6%, up to about 2.7%, up to about 2.8%, up to about 2.9%, up to about 3%, up to about 3.1%, up to about 3.2%, up to about 3.3%, up to about 3.4%, up to about 3.5%, up to about 3.6%, up to about 3.7%, up to about 3.8%, up to about 3.9%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, or up to about 10% of non iNKT cells, (e.g., wherein the non-iNKT cells are vα24jα18-).

Tmem

The cell population comprising the Tmem population may comprise at least about 1 x 10 ⁴ At least about 1X 10 ⁵ At least about 5X 10 ⁵ At least about 6X 10 ⁵ At least about 7X 10 ⁵ At least about 8X 10 ⁵ At least about 9X 10 ⁵ At least about 1X 10 ⁶ At least about 1.1X10 ⁶ At least about 1.2 x 10 ⁶ At least about 1.3X10 ⁶ At least about 1.4X10 ⁶ At least about 1.5X10 ⁶ At least about 1.6X10 ⁶ At least about 1.7X10 ⁶ At least about 1.8X10 ⁶ At least about 1.9X10 ⁶ At least about 2X 10 ⁶ At least about 2.1X10 ⁶ At least about 2.2 x 10 ⁶ At least about 2.3X10 ⁶ At least about 2.4X10 ⁶ At least about 2.5X10 ⁶ At least about 2.6X10 ⁶ At least about 2.7X10 ⁶ At least about 2.8X10 ⁶ At least about 2.9X10 ⁶ At least about 3X 10 ⁶ At least about 3.1X10 ⁶ At least about 3.2 x 10 ⁶ At least about 3.3X10 ⁶ At least about 3.4X10 ⁶ At least about 3.5X10 ⁶ At least about 3.6X10 ⁶ At least about 3.7X10 ⁶ At least about 3.8X10 ⁶ At least about 3.9X10 ⁶ At least about 4X 10 ⁶ At least about 4.1X10 ⁶ At least about 4.2 x 10 ⁶ At least about 4.3X10 ⁶ At least about 4.4X10 ⁶ At least about 4.5X10 ⁶ At least about 4.6X10 ⁶ At least about 4.7X10 ⁶ At least about 4.8X10 ⁶ At least about 4.9X10 ⁶ At least about 5X 10 ⁶ At least about 5.1X10 ⁶ At least about 5.2 x 10 ⁶ At least about 5.3X10 ⁶ At least about 5.4X10 ⁶ At least about 5.5X10 ⁶ At least about 5.6X10 ⁶ At least about 5.7X10 ⁶ At least about 5.8X10 ⁶ At least about 5.9X10 ⁶ At least about 6X 10 ⁶ At least about 6.5X10 ⁶ At least about 7X 10 ⁶ At least about 7.5X10 ⁶ At least about 8X 10 ⁶ At least about 8.5X10 ⁶ At least about 9X 10 ⁶ At least about 9.5X10 ⁶ At least about 1X 10 ⁷ At least about 1.5X10 ⁷ At least about 2X 10 ⁷ At least about 2.5X10 ⁷ At least about 3X 10 ⁷ At least about 3.5X10 ⁷ At least about 4X 10 ⁷ At least about 4.5X10 ⁷ At least about 5X 10 ⁷ At least about 5.5X10 ⁷ At least about 6X 10 ⁷ At least about 6.5X10 ⁷ At least about 7X 10 ⁷ At least about 7.5X10 ⁷ At least about 8X 10 ⁷ At least about 8.5X10 ⁷ At least about 9X 10 ⁷ At least about 9.5X10 ⁷ At least about 1X 10 ⁸ At least about 1X 10 ⁸ At least about 1.5X10 ⁸ At least about 2X 10 ⁸ At least about 2.5X10 ⁸ At least about 3X 10 ⁸ At least about 3.5X10 ⁸ At least about 4X 10 ⁷ At least about 4.5X10 ⁸ At least about 5X 10 ⁸ At least about 5.5X10 ⁸ At least about 6X 10 ⁸ At least about 6.5X10 ⁸ At least about 7X 10 ⁸ At least about 7.5X10 ⁸ At least about 8X 10 ⁸ At least about 8.5X10 ⁸ At least about 9X 10 ⁸ At least about 9.5X10 ⁸ At least about 1X 10 ⁹ Or more Tmem/kg of the actual body weight or ideal body weight of the subject (e.g., wherein Tmem is cd3+cd45ra-cd45ro+).

The cell population comprising the Tmem population may comprise up to about 1 x 10 ⁴ Up to about 1X 10 ⁵ Up to about 5X 10 ⁵ Up to about 6X 10 ⁵ Up to about 7X 10 ⁵ Up to about 8X 10 ⁵ Up to about 9X 10 ⁵ Up to about 1X 10 ⁶ Up to about 1.1X10 ⁶ Up to about 1.2X10 ⁶ Up to about 1.3X10 ⁶ Up to about 1.4X10 ⁶ Up to about 1.5X10 ⁶ Up to about 1.6X10 ⁶ Up to about 1.7X10 ⁶ Up to about 1.8X10 ⁶ Up to about 1.9X10 ⁶ Up to about 2X 10 ⁶ Up to about 2.1X10 ⁶ Up to about 2.2X10 ⁶ Up to about 2.3X10 ⁶ Up to about 2.4X10 ⁶ Up to about 2.5X10 ⁶ Up to about 2.6X10 ⁶ Up to about 2.7X10 ⁶ Up to about 2.8X10 ⁶ Up to about 2.9X10 ⁶ Up to about 3X 10 ⁶ Up to about 3.1X10 ⁶ Up to about 3.2 x 10 ⁶ Up to about 3.3X10 ⁶ Up to about 3.4X10 ⁶ Up to about 3.5X10 ⁶ Up to about 3.6X10 ⁶ Up to about 3.7X10 ⁶ Up to about 3.8X10 ⁶ Up to about 3.9X10 ⁶ Up to about 4X 10 ⁶ Up to about 4.1X10 ⁶ Up to about 4.2 x 10 ⁶ Up to about 4.3X10 ⁶ Up to about 4.4X10 ⁶ Up to about 4.5X10 ⁶ Up to about 4.6X10 ⁶ Up to about 4.7X10 ⁶ Up to about 4.8X10 ⁶ Up to about 4.9X10 ⁶ Up to about 5X 10 ⁶ Up to about 5.1X10 ⁶ Up to about 5.2 x 10 ⁶ Up to about 5.3X10 ⁶ Up to about 5.4X10 ⁶ Up to about 5.5X10 ⁶ Up to about 5.6X10 ⁶ Up to about 5.7X10 ⁶ Up to about 5.8X10 ⁶ Up to about 5.9X10 ⁶ Up to about 6X 10 ⁶ Up to about 6.5X10 ⁶ Up to about 7X 10 ⁶ Up to about 7.5X10 ⁶ Up to about 8X 10 ⁶ Up to about 8.5X10 ⁶ Up to about 9X 10 ⁶ Up to about 9.5X10 ⁶ Up to about 1X 10 ⁷ Up to about 1.5X10 ⁷ Up to about 2X 10 ⁷ Up to about 2.5X10 ⁷ Up to about 3X 10 ⁷ Up to about 3.5X10 ⁷ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁷ Up to about 5X 10 ⁷ Up to about 5.5X10 ⁷ Up to about 6X 10 ⁷ Up to about 6.5X10 ⁷ Up to about 7X 10 ⁷ Up to about 7.5X10 ⁷ Up to about 8X 10 ⁷ Up to about 8.5X10 ⁷ Up to about 9X 10 ⁷ Up to about 9.5X10 ⁷ Up to about 1X 10 ⁸ Up to about 1X 10 ⁸ Up to about 1.5X10 ⁸ Up to about 2X 10 ⁸ Up to about 2.5X10 ⁸ Up to about 3X 10 ⁸ Up to about 3.5X10 ⁸ Up to about 4X 10 ⁷ Up to about 4.5X10 ⁸ Up to about 5X 10 ⁸ Up to about 5.5X10 ⁸ Up to about 6X 10 ⁸ Up to about 6.5X10 ⁸ Up to about 7X 10 ⁸ Up to about 7.5X10 ⁸ Up to about 8X 10 ⁸ Up to about 8.5X10 ⁸ Up to about 9X 10 ⁸ Up to about 9.5X10 ⁸ Or up to about 1X 10 ⁹ The actual or ideal body weight of the individual Tmem/kg recipient subject (e.g., where Tmem is cd3+cd45ra-cd45ro+).

For example, a cell population comprising a Tmem population may comprise 1 x 10 ⁴ Up to 1X 10 ⁹ 、1×10 ⁵ Up to 1X 10 ⁸ 、1×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 2X 10 ⁷ 、5×10 ⁵ Up to 1.5X10 ⁷ 、5×10 ⁵ Up to 1X 10 ⁷ 、5×10 ⁵ To 9X 10 ⁶ 、5×10 ⁵ Up to 8X 10 ⁶ 、5×10 ⁵ To 7X 10 ⁶ 、5×10 ⁵ Up to 6X 10 ⁶ 、5×10 ⁵ Up to 5X 10 ⁶ 、5×10 ⁵ Up to 4X 10 ⁶ 、5×10 ⁵ Up to 3X 10 ⁶ 、5×10 ⁵ Up to 2X 10 ⁶ 、5×10 ⁵ Up to 1X 10 ⁶ 、1×10 ⁶ Up to 1.5X10 ⁷ 、1×10 ⁶ Up to 1X 10 ⁷ 、1×10 ⁶ To 9X 10 ⁶ 、1×10 ⁶ Up to 8X 10 ⁶ 、1×10 ⁶ To 7X 10 ⁶ 、1×10 ⁶ Up to 6X 10 ⁶ 、1×10 ⁶ Up to 5X 10 ⁶ 、1×10 ⁶ Up to 4X 10 ⁶ 、1×10 ⁶ Up to 3X 10 ⁶ 、1×10 ⁶ Up to 2X 10 ⁶ 、1.5×10 ⁶ Up to 1.5X10 ⁷ 、1.5×10 ⁶ Up to 1X 10 ⁷ 、1.5×10 ⁶ To 9X 10 ⁶ 、1.5×10 ⁶ Up to 8X 10 ⁶ 、1.5×10 ⁶ To 7X 10 ⁶ 、1.5×10 ⁶ Up to 6X 10 ⁶ 、1.5×10 ⁶ Up to 5X 10 ⁶ 、1.5×10 ⁶ Up to 4X 10 ⁶ 、1.5×10 ⁶ Up to 3X 10 ⁶ 、1.5×10 ⁶ Up to 2X 10 ⁶ 、2×10 ⁶ Up to 1.5X10 ⁷ 、2×10 ⁶ Up to 1X 10 ⁷ 、2×10 ⁶ To 9X 10 ⁶ 、2×10 ⁶ Up to 8X 10 ⁶ 、2×10 ⁶ To 7X 10 ⁶ 、2×10 ⁶ Up to 6X 10 ⁶ 、2×10 ⁶ Up to 5X 10 ⁶ 、2×10 ⁶ Up to 4X 10 ⁶ 、2×10 ⁶ Up to 3X 10 ⁶ 、2.5×10 ⁶ Up to 1.5X10 ⁷ 、2.5×10 ⁶ Up to 1X 10 ⁷ 、2.5×10 ⁶ To 9X 10 ⁶ 、2.5×10 ⁶ Up to 8X 10 ⁶ 、2.5×10 ⁶ To 7X 10 ⁶ 、2.5×10 ⁶ Up to 6X 10 ⁶ 、2.5×10 ⁶ Up to 5X 10 ⁶ 、2.5×10 ⁶ Up to 4X 10 ⁶ Or 2.5X10 ⁶ Up to 3X 10 ⁶ The actual or ideal body weight of the individual Tmem/kg recipient subject (e.g., where Tmem is cd3+cd45ra-cd45ro+).

The Tmem populations of the present disclosure can have a determined purity level for Tmem cells. For example, tmem populations of the present disclosure can comprise at least about 5%, at least about 10%, 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 92%, at least about 90%, at least about 91%, at least about 94%, at least about 95%, or more (e.g., at least about 94% more than about 95% of cells, at least about 96%, or at least about 95% of total cells. Wherein Tmem is CD3+CD45RA-CD45RO+).

The Tmem population of the present disclosure may 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%, 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%, 95% to 99%, 96% to 100%, 97% to 100%, based on the percentage of total cells, nucleated cells, or cd45+ cells. 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%, 97% to 97%, and 97% to 97% of the composition 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% Tmem (for example, where Tmem is cd3+cd45RA-CD45 RO).

The Tmem populations of the present disclosure can have a defined level of contaminating non-Tmem cells. In some embodiments, up to about 1X 10 ² Up to about 2X 10 ² Up to about 3X 10 ² Up to about 4X 10 ² Up to about 5X 10 ² Up to about 6X 10 ² Up to about 7X 10 ² Up to about 8X 10 ² Up to about 9X 10 ² Up to about 1X 10 ³ Up to about 2X 10 ³ Up to about 3X 10 ³ Up to about 4X 10 ³ Up to about 5X 10 ³ Up to about 6X 10 ³ Up to about 7X 10 ³ Up to about 8X 10 ³ Up to about 9X 10 ³ Up to about 1X 10 ⁴ Up to about 2X 10 ⁴ Up to about 3X 10 ⁴ Up to about 4X 10 ⁴ Up to about 5X 10 ⁴ Up to about 6X 10 ⁴ Up to about 7X 10 ⁴ Up to about 8X 10 ⁴ Up to about 9X 10 ⁴ Or up to about 1X 10 ⁵ The actual body weight or ideal body weight of the individual non-Tmem cells/kg recipient subject is present in the population of Tmem of the present disclosure (e.g., wherein the non-Tmem cells are CD45 RO-).

In some embodiments of the present invention, in some embodiments, the Tmem population of the present 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 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 1.3% >, up to about 1.4%, up to about 1.5%, up to about 1.6%, up to about 1.7%, up to about 1.8%, up to about 1.9%, up to about 2%, up to about 2.1%, up to about 2.2%, up to about 2.3%, up to about 2.4%, up to about 2.5%, up to about 2.6%, up to about 2.7%, up to about 2.8%, up to about 2.9%, up to about 3%, up to about 3.1%, up to about 3.2%, up to about 3.3%, up to about 3.4%, up to about 3.5%, up to about 3.6%, up to about 3.7%, up to about 3.8%, up to about 3.9%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9% or up to about 10% of non-Tmem cells (e.g., wherein the non-Tmem cells are CD45 RO-).

D. Sequence and timing of administration of cell populations

In one aspect, a multi-component drug therapy is provided to be administered to a human subject in need thereof. The multicomponent treatment includes: (a) A solution comprising a first cd45+ cell population comprising Hematopoietic Stem and Progenitor Cells (HSPCs) and granulocytes, wherein up to about 10% of the first cd45+ cell population comprises granulocytes; (b) A solution comprising a population of cells enriched for regulatory T cells (tregs); (c) A solution comprising a second cd45+ cell population, wherein the second cd45+ cell population comprises at least about 20% cd3+ normal T cells (Tcon), at least about 10% monocytes and at least about 10% granulocytes; and (d) a solution comprising one or more doses of a Graft Versus Host Disease (GVHD) prophylaxis agent.

In another aspect, a method of treating a human subject diagnosed with a hematological malignancy is provided. The method comprises administering to a human subject a solution comprising a first cd45+ cell population, a solution comprising a cell population enriched for regulatory T cells (tregs), a solution comprising a second cd45+ cell population, and a solution comprising one or more doses of a GVHD preventing agent. In this aspect, the solution comprising the first cd45+ cell population, the solution comprising the cell population enriched for regulatory tregs, the solution comprising the second cd45+ cell population, and the solution comprising one or more doses of a GVHD prophylaxis agent are as defined according to any of the multicomponent drug therapies disclosed herein.

Disclosed herein are methods for enhanced allogeneic hematopoietic stem cell transplantation comprising administering to a subject a solution comprising a population of cells.

In some embodiments, a cell population comprising a first cd45+ cell population comprising at least HSPCs, a regulatory T cell (Treg) -enriched cell population, and a second cd45+ cell population comprising at least Tcon is administered to a subject.

The first cd45+ cell population and the Treg-enriched cell population may be administered at the same or similar times or at different times. In some embodiments, the first cd45+ cell population and the Treg-enriched cell population are administered on the same day.

In various embodiments, the first cd45+ cell population and the Treg-enriched cell population are administered before the second cd45+ cell population.

The first cd45+ cell population and the Treg-enriched cell population may be administered up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours apart.

The second cd45+ cell population may be administered to the subject after the first cd45+ cell population.

The second cd45+ cell population may be administered to the subject at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the first cd45+ cell population.

In some embodiments, the second cd45+ cell population is administered to the subject up to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 120 hours after the first cd45+ cell population.

The second CD45+ cell population may be administered to the subject after the first CD45+ cell population, for example, about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-60, 60-66, or 66.

The second cd45+ cell population may be administered to the subject after the Treg-enriched cell population.

The population Tcon can be administered to the subject greater than at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the Treg-enriched cell population.

In some embodiments, the second cd45+ cell population is administered to the subject up to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the Treg-enriched cell population.

The second population of CD45+ cells may be administered after the population of Treg-enriched cells, for example, about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-60, 60-66, 66-60 or 66.

The second cd45+ cell population may be administered to the subject after the first cd45+ cell population and the Treg-enriched cell population.

The second cd45+ cell population may be administered to the subject after the first cd45+ cell population and the Treg-enriched cell population, for example, for at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

In some embodiments, the second cd45+ cell population is administered to the subject up to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the first cd45+ cell population and the Treg-enriched cell population.

The second CD45+ cell population may be administered to the subject, for example, after the first CD45+ cell population and the Treg-enriched cell population, about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-48, 36-42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-60, 54-66, 54-60, 72-72, 72-60, or 72-60.

In some embodiments, a population of Hematopoietic Stem and Progenitor Cells (HSPCs), a population of cells enriched for regulatory T cells (tregs), a population of conventional T cells (Tcon), and a population of constant natural killer T cells (inkts) are administered to a subject.

The iNKT population may be administered to the subject at the same time as the first cd45+ cell population or at a similar time. In some embodiments, the iNKT population is administered to the subject after the first cd45+ cell population.

The iNKT population may be administered to the subject greater than at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the first cd45+ cell population.

In some embodiments, the iNKT population is administered to the subject up to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the first cd45+ cell population.

The iNKT population may be administered after the first cd45+ cell population, for example, about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-66, or 72-72.

The iNKT population may be administered to the subject at the same time or at a similar time as the Treg-enriched cell population. In some embodiments, the iNKT population is administered to the subject after the Treg-enriched cell population.

The iNKT population may be administered to the subject greater than at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the Treg-enriched cell population.

In some embodiments, the iNKT population is administered to the subject up to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the Treg-enriched cell population.

The iNKT population may be administered to the subject after the Treg-enriched cell population, for example, about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-60, 60-66, or 72.

In some embodiments, a population of Hematopoietic Stem and Progenitor Cells (HSPCs), a population of cells enriched for regulatory T cells (tregs), a population of conventional T cells (Tcon), and a population of memory T cells (Tmem) are administered to a subject.

The Tmem population can be administered to the subject at the same time or at a similar time as the first cd45+ cell population. In some embodiments, the Tmem population is administered to the subject after the first cd45+ cell population.

The Tmem population can be administered to the subject greater than at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the first cd45+ cell population.

In some embodiments, the Tmem population is administered to the subject up to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the first cd45+ cell population.

The Tmem population may be administered after the first cd45+ cell population, for example, about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-66, 30-60, 66-72, or 72.

The Tmem population can be administered to the subject at the same time or at a similar time as the Treg-enriched cell population. In some embodiments, the Tmem population is administered to the subject after the Treg-enriched cell population.

The Tmem population can be administered to the subject greater than at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the Treg-enriched cell population.

In some embodiments, the Tmem population is administered to the subject up to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the Treg-enriched cell population.

The Tmem population may be administered to the subject after the Treg-enriched cell population, for example, about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-66, or 72.

In embodiments, the population of Tcon is administered at least about 12 hours after the population of cells comprising tregs, e.g., the population of Tcon is administered about 24 to about 96 hours after the population of cells comprising tregs, or the population of Tcon is administered about 36 to about 60 hours after the population of cells comprising tregs.

Heterogeneous cell populations

In various embodiments, the heterologous cell population comprises from about 0.2% to about 2.0% hematopoietic stem cells and progenitor cells.

In some embodiments, the hematological malignancy is acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, multiple myeloma, lymphoma, hodgkin's lymphoma, and non-hodgkin's lymphoma.

In embodiments, the gene expression level of the T-reg cells is correlated with cells harvested from the donor within about 60 hours prior to administration to the patient.

In various embodiments, the number of T-reg cells in the T-reg population is about equal to the number of T-con cells in the heterologous cell population. In some cases, the T-reg cells in the T-reg population inhibit activation of conventional T cells in the heterologous cell population by an amount up to about 20% of the healthy tissue of the patient.

In some embodiments, the peripheral blood of the patient exhibits an elevated ratio of Treg to cd4+ T cells up to about 100 days after administration of the cell population as compared to a healthy human subject not administered the cell population.

In embodiments, at least about 50% of the cells in the HSPC cell population are colony forming units.

In various embodiments, at least one of the cell populations has an elevated amount of granulocyte colony stimulating factor as compared to the non-mobilized blood. In some cases, the at least one cell population is a heterologous cell population.

In one aspect, a method of transplanting a population of cells into a human patient as part of a treatment regimen for a hematological malignancy is provided. The method comprises administering to a patient a population of Hematopoietic Stem and Progenitor Cells (HSPCs) comprising HSPCs and a liquid suspending the HSPCs; administering to a patient a population of regulatory T cells (tregs) to be administered to the patient, the population of tregs comprising tregs and a liquid suspending the tregs; and administering to the patient a heterologous cell population to be administered to the patient, the heterologous cell population comprising lymphocytes, granulocytes, monocytes and a liquid suspending said cells, wherein at least about 30% of said lymphocytes comprise conventional T cells (Tcon); and administering to the patient a single Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) comprising tacrolimus (tacrolimus GHVDPA) over a period of up to about 180 days, wherein tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the patient's blood above a threshold level during said period of time; and wherein the risk and/or severity of GHVD associated with a treatment regimen for hematological malignancy is significantly reduced.

In some embodiments, the heterologous cell population may be administered to a subject. The heterogeneous cell population may include many different cell types found in the peripheral blood of human donors. The heterologous cell population may comprise granulocytes, monocytes and lymphocytes. The heterologous cell population can comprise T cells (e.g., tcon, treg, tmem, naive T cells, cd4+ T cells, NK-T cells), B cells, NK cells, HSPCs, dendritic cells (e.g., plasmacytoid dendritic cells and myeloid dendritic cells), and other cell populations found in peripheral blood. In addition to other populations described herein, a heterologous cell population can be administered to a subject. For example, a heterologous cell population can be administered with HSPCs as described herein. In some cases, the heterologous cell population can be administered with HSPCs and tregs as described herein. In some cases, the heterologous cell population can be administered with tregs as described herein. In some cases, a heterologous cell population may be administered with a Tcon as described herein. In some cases, a heterologous cell population may be administered in place of the Tcon population as described herein.

In some embodiments, the heterologous cell population administered to the subject may comprise a combination of granulocytes and monocytes. The combination of granulocytes and monocytes may comprise 30% to 80% of the heterogeneous cell population. At least 30% of the heterogeneous cell population may comprise a combination of granulocytes and monocytes. Up to 80% of the heterogeneous cell population may comprise a combination of granulocytes and monocytes. In some cases, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80%, 50% to 60%, 50% to 70%, 50% to 80%, 60% to 70%, 60% to 80%, or 70% to 80% of the heterologous cell population may comprise a combination of granulocytes and monocytes. In some cases, 30%, 40%, 50%, 60%, 70%, or 80% of the heterologous cell population may comprise a combination of granulocytes and monocytes. In some cases, at least 30%, 40%, 50%, 60%, or 70% of the heterologous cell population may comprise a combination of granulocytes and monocytes. In some cases, up to 40%, 50%, 60%, 70% or 80% of the heterologous cell population may comprise a combination of granulocytes and monocytes.

In some embodiments, the heterologous cell population administered to the subject may comprise lymphocytes. Lymphocytes include cd45+ cells. Lymphocytes may comprise 8% to 50% of the heterogeneous cell population. In some cases, at least 8% of the heterogeneous cell population may comprise lymphocytes. In some cases, up to 50% of the heterogeneous cell population may comprise lymphocytes. In some cases, 8% to 10%, 8% to 20%, 8% to 25%, 8% to 30%, 8% to 40%, 8% to 45%, 8% to 50%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 40%, 10% to 45%, 10% to 50%, 20% to 25%, 20% to 30%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 40%, 30% to 45%, 30% to 50%, 40% to 45%, 40% to 50%, or 45% to 50% of the heterogeneous cell population may comprise lymphocytes. In some cases, 8%, 10%, 20%, 25%, 30%, 40%, 45%, or 50% of the heterologous cell population may comprise lymphocytes. In some cases, at least 8%, 10%, 20%, 25%, 30%, 40% or 45% of the heterologous cell population may comprise lymphocytes. In some cases, up to 10%, 20%, 25%, 30%, 40%, 45% or 50% of the heterologous cell population may comprise lymphocytes.

In some embodiments, lymphocytes in the heterogeneous cell population may comprise Tcon. Tcon may account for 40% to 85% of lymphocyte subpopulations of the heterologous cell population. In some cases, at least 40% of the lymphocyte subpopulations may comprise Tcon. In some cases, up to 85% of the lymphocyte subpopulations may contain Tcon. In some cases, 40% to 50%, 40% to 60%, 40% to 65%, 40% to 70%, 40% to 75%, 40% to 80%, 40% to 85%, 50% to 60%, 50% to 65%, 50% to 70%, 50% to 75%, 50% to 80%, 50% to 85%, 60% to 65%, 60% to 70%, 60% to 75%, 60% to 80%, 60% to 85%, 65% to 70%, 65% to 75%, 65% to 80%, 65% to 85%, 70% to 75%, 70% to 80%, 70% to 85%, 75% to 80%, 75% to 85%, or 80% to 85% of the lymphocyte subpopulation may comprise Tcon. In some cases, 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the lymphocyte subpopulations may comprise Tcon. In some cases, at least 40%, 50%, 60%, 65%, 70%, 75% or 80% of the lymphocyte subpopulations may comprise Tcon. In some cases, up to 50%, 60%, 65%, 70%, 75%, 80% or 85% of the lymphocyte subpopulations may comprise Tcon.

In some embodiments, the cd3+ lymphocytes in the heterologous cell population may comprise cd4+ T cells. In some cases, 30% to 70% of the cd3+ lymphocyte subpopulations may comprise cd4+ T cells. In some cases, at least 30% of the cd3+ lymphocyte subpopulations may comprise cd4+ T cells. In some cases, up to 70% of the cd3+ lymphocyte subpopulations may comprise cd4+ T cells. In some cases, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 40% to 50%, 40% to 60%, 40% to 70%, 50% to 60%, 50% to 70%, or 60% to 70% of the cd3+ lymphocyte subpopulations may comprise cd4+ T cells. In some cases, 30%, 40%, 50%, 60% or 70% of the cd3+ lymphocyte subpopulations may comprise cd4+ T cells. In some cases, at least 30%, 40%, 50%, or 60% of the cd3+ lymphocyte subpopulations may comprise cd4+ T cells. In some cases, up to 40%, 50%, 60%, or 70% of the cd3+ lymphocyte subpopulations may comprise cd4+ T cells.

In some embodiments, the cd3+ lymphocytes in the heterologous cell population may comprise cd8+ T cells. In some cases, 20% to 65% of the cd3+ lymphocyte subpopulations may comprise cd8+ T cells. In some cases, at least 20% of the cd3+ lymphocyte subpopulations may comprise cd8+ T cells. In some cases, up to 65% of the cd3+ lymphocyte subpopulations may comprise cd8+ T cells. In some cases, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 20% to 65%, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 65%, 40% to 50%, 40% to 60%, 40% to 65%, 50% to 60%, 50% to 65%, or 60% to 65% of the cd3+ lymphocyte subpopulations may comprise cd8+ T cells. In some cases, 20%, 30%, 40%, 50%, 60%, or 65% of the cd3+ lymphocyte subpopulations may comprise cd8+ T cells. In some cases, at least 20%, 30%, 40%, 50% or 60% of the cd3+ lymphocyte subpopulations may comprise cd8+ T cells. In some cases, up to 30%, 40%, 50%, 60% or 65% of the cd3+ lymphocyte subpopulations may comprise cd8+ T cells.

In some embodiments, the lymphocytes in the heterogeneous cell population may comprise B cells. In some cases, 4% to 35% of the lymphocyte subpopulations may comprise B cells. In some cases, at least 4% of the lymphocyte subpopulations may comprise B cells. In some cases, up to 35% of the lymphocyte subpopulations may contain B cells. In some cases, 4% to 5%, 4% to 10%, 4% to 20%, 4% to 30%, 4% to 35%, 5% to 10%, 5% to 20%, 5% to 30%, 5% to 35%, 10% to 20%, 10% to 30%, 10% to 35%, 20% to 30%, 20% to 35%, or 30% to 35% of the lymphocyte subpopulation may comprise B cells. In some cases, 4%, 5%, 10%, 20%, 30%, or 35% of the lymphocyte subpopulations may comprise B cells. In some cases, at least 4%, 5%, 10%, 20% or 30% of the lymphocyte subpopulations may comprise B cells. In some cases, up to 5%, 10%, 20%, 30% or 35% of the lymphocyte subpopulations may comprise B cells. The B cells may be CD45+CD19+ cells or CD45+CD19+CD3-cells.

In some embodiments, the lymphocytes in the heterogeneous cell population may comprise NK cells. In some cases, 4% to 35% of the lymphocyte subpopulations may comprise NK cells. In some cases, at least 4% of the lymphocyte subpopulations may comprise NK cells. In some cases, up to 35% of the lymphocyte subpopulations may comprise NK cells. In some cases, 4% to 5%, 4% to 10%, 4% to 20%, 4% to 30%, 4% to 35%, 5% to 10%, 5% to 20%, 5% to 30%, 5% to 35%, 10% to 20%, 10% to 30%, 10% to 35%, 20% to 30%, 20% to 35%, or 30% to 35% of the lymphocyte subpopulations may comprise NK cells. In some cases, 4%, 5%, 10%, 20%, 30% or 35% of the lymphocyte subpopulations may comprise NK cells. In some cases, at least 4%, 5%, 10%, 20% or 30% of the lymphocyte subpopulations may comprise NK cells. In some cases, up to 5%, 10%, 20%, 30% or 35% of the lymphocyte subpopulations may comprise NK cells. NK cells may be CD45+CD56+ cells or CD45+CD56+CD3-cells.

In some embodiments, the cd3+ lymphocytes in the heterologous cell population may comprise NK-T cells. In some cases, 3% to 30% of the cd3+ lymphocyte subpopulations may comprise NK-T cells. In some cases, at least 4% of the cd3+ lymphocyte subpopulations may comprise NK-T cells. In some cases, up to 35% of the cd3+ lymphocyte subpopulations may comprise NK-T cells. In some cases, 3% to 5%, 3% to 10%, 3% to 20%, 3% to 30%, 5% to 10%, 5% to 20%, 5% to 30%, 10% to 20%, 10% to 30%, 20% to 30% of the cd3+ lymphocyte subpopulations may comprise NK-T cells. In some cases, 3%, 5%, 10%, 20% or 30% of the cd3+ lymphocyte subpopulations may comprise NK-T cells. In some cases, at least 3%, 5%, 10%, or 20% of the cd3+ lymphocyte subpopulations may comprise NK-T cells. In some cases, up to 10%, 20% or 30% of the cd3+ lymphocyte subpopulations may comprise NK-T cells. NK-T cells can be CD45+CD56+ cells or CD45+CD56+CD3+ cells.

In some embodiments, the lymphocytes in the heterogeneous cell population may comprise cd34+ cells. In some cases, 0.1% to 2% of the lymphocyte subpopulations may comprise cd34+ cells. In some cases, at least 0.1% of the lymphocyte subpopulations may comprise cd34+ cells. In some cases, up to 2% of the lymphocyte subpopulations may comprise cd34+ cells. In some cases, 0.1% to 0.5%, 0.1% to 1%, 0.1% to 1.5%, 0.1% to 2%, 0.5% to 1%, 0.5% to 1.5%, 0.5% to 2%, 1% to 1.5%, 1% to 2%, or 1.5% to 2% of the lymphocyte subpopulation may comprise cd34+ cells. In some cases, 0.1%, 0.5%, 1%, 1.5%, or 2% of the lymphocyte subpopulations may comprise cd34+ cells. In some cases, at least 0.1%, 0.5%, 1% or 1.5% of the lymphocyte subpopulations may comprise cd34+ cells. In some cases, up to 0.5%, 1%, 1.5% or 2% of the lymphocyte subpopulations may comprise cd34+ cells.

GVHD preventive agent

Subjects administered the compositions of the present disclosure (e.g., cell components comprising a cell population described herein) and GVHD prophylaxis agents (e.g., single GVHD prophylaxis agents) exhibit low incidence of ≡1 grade aGVHD, e.g., lower incidence of ≡1 grade aGVHD than subjects administered alternative compositions. The single GVHD prophylaxis agent may be a calcineurin inhibitor such as tacrolimus or another agent acting on the same target as tacrolimus or comprising an active fragment of tacrolimus. In some embodiments, the low dose GVHD prophylaxis agent is tacrolimus at a target trough level of about 5ng/mL to about 10 ng/mL. In some embodiments, the low dose GVHD prophylaxis agent is tacrolimus at a target trough level of about 4ng/mL to about 6 ng/mL.

The single GVHD prophylaxis agent may be sirolimus (sirolimus). In some embodiments, the low dose GVHD prophylaxis agent is sirolimus having a target trough level of about 3ng/mL to about 8 ng/mL. In some embodiments, the low dose GVHD prophylaxis agent is sirolimus having a target trough level of about 4ng/mL to about 8 ng/mL.

The methods for alloHCT of the present disclosure utilize tacrolimus. In the alloHCT regimen as disclosed herein, combining tacrolimus with one or more cell populations has been shown to result in a dramatic improvement in clinical outcome.

In one aspect, a multi-component drug therapy is provided to be administered to a human subject in need thereof. The multicomponent treatment includes: (a) A solution comprising a first cd45+ cell population comprising Hematopoietic Stem and Progenitor Cells (HSPCs) and granulocytes, wherein up to about 10% of the first cd45+ cell population comprises granulocytes; (b) A solution comprising a population of cells enriched for regulatory T cells (tregs); (c) A solution comprising a second cd45+ cell population, wherein the second cd45+ cell population comprises at least about 20% cd3+ normal T cells (Tcon), at least about 10% monocytes and at least about 10% granulocytes; and (d) a solution comprising one or more doses of a Graft Versus Host Disease (GVHD) prophylactic agent, wherein the GVHD prophylactic agent is tacrolimus.

In various embodiments, tacrolimus is administered in an amount to maintain a target blood level of at least about 3ng/ml for at least about 20 days after administration of the second cd45+ cell population, in an amount to maintain a target blood level of about 4ng/ml or higher for at least about 40 days after administration of the second cd45+ cell population, and/or in an amount to maintain a target blood level of about 4ng/ml or higher for at least about 40 days after administration of the second cd45+ cell population. In some cases, tacrolimus is administered in an amount to maintain a target blood level of up to about 10ng/ml for at least 30 days after administration of the second cd45+ cell population.

In some embodiments, tacrolimus is formulated for oral administration or for intravenous administration.

In embodiments, the methods disclosed herein further comprise administering to the patient a single Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) comprising tacrolimus (tacrolimus GHVDPA) over a period of up to about 180 days; wherein tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the blood of the patient above a threshold level during said period of time; and wherein the risk and/or severity of GHVD is significantly reduced. In various embodiments, the threshold level is above about 4ng tacrolimus/ml patient blood, or the threshold level is above about 5ng tacrolimus/ml patient blood. In some embodiments, tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the blood of the patient below an upper threshold level during said period of time. In some cases, the upper threshold level is below about 10ng tacrolimus/ml patient blood.

In embodiments, the tacrolimus Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) is administered intravenously or orally. In various embodiments, the administration of the tacrolimus Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) begins about 12 to about 24 hours after administration of T-con. In some cases, tacrolimus GHVDPA is administered for a period of up to about 90 days, for a period of up to about 60 days. In some embodiments, tacrolimus GHVDPA is initially administered to a patient at about 0.03mg/kg of the patient's actual or ideal body weight per day. In some cases, the dose of tacrolimus gvhpa administered to the patient begins to taper about 90 days after the first dose is administered to the patient, or about 45 days after the first dose is administered to the patient.

Tacrolimus is a macrolide that can exhibit immunosuppressive activity in vivo and can prevent or reduce the response of T-lymphocyte activation to antigenic or mitogenic stimuli. Thus, tacrolimus may be used to reduce the risk of GVHD in alloHCT receptor subjects. However, the methods disclosed herein, which utilize tacrolimus as a single agent prophylactic, obtain clinical results that are superior to those observed in other alloHCT methods utilizing tacrolimus GVHD prophylactic. For example, in some embodiments, the alloHCT methods of the present disclosure that utilize tacrolimus achieve superior relapse-free survival compared to standard care regimens that include more effective GVHD prevention with methotrexate plus tacrolimus, and even compared to alloHCT methods that utilize drugs with similar targets and mechanisms of action (sirolimus). Thus, although tacrolimus may be referred to herein as a GVHD prophylactic, it may also contribute to additional therapeutic effects beyond or not directly related to GVHD prevention. Thus, as used herein, the term "tacrolimus as a single agent prophylactic agent for GVHD" also includes tacrolimus as a single agent prophylactic agent for additional therapeutic effects. In other words, the terms "tacrolimus as a single agent prophylactic agent" and "tacrolimus as a single agent GVHD prophylactic agent" are synonymous.

Treatment with tacrolimus as a single agent prophylactic may result in, for example, reduced cytokine production and reduced T cell signaling. Tacrolimus can bind to FKBP-12 protein and form a complex with calcium-dependent protein, thereby inhibiting calcineurin activity. This prevents or reduces dephosphorylation and translocation of Nuclear Factor (NFAT), a nuclear component thought to initiate transcription of genes that drive expression of lymphokines, of activated T cells. Tacrolimus also inhibits transcription of genes encoding IL-3, IL-4, IL-5, GM-CSF and TNF, all of which are involved in the early stages of T cell activation.

In some embodiments, tacrolimus is administered orally to a subject as a single agent prophylactic. Absorption of tacrolimus from the gastrointestinal tract after oral administration may be incomplete and variable. The absolute bioavailability of tacrolimus in healthy subjects after oral administration can be 18±5%. The rate and extent of absorption may vary based on whether tacrolimus is administered with food. In some embodiments, tacrolimus is administered parenterally, e.g., intravenously or subcutaneously. In some embodiments, tacrolimus is administered by the topical, intramuscular, intradermal, intraperitoneal, intrathecal or epidural route. Tacrolimus can be administered as a slow release formulation. In some embodiments, tacrolimus is used as the free base. In some embodiments, tacrolimus is used as a pharmaceutically acceptable salt.

In some embodiments, the methods of the present disclosure may allow for the use of low doses of tacrolimus. In some embodiments, a low dose of tacrolimus may improve donor T cell chimerism in a subject. In some embodiments, low doses of tacrolimus may improve alloHCT implantation as disclosed herein. In some embodiments, low doses of tacrolimus may reduce the incidence or relative risk of adverse effects such as vision confusion, liver and kidney toxicity, seizures, tremors, hypertension, hypomagnesemia, diabetes, hyperkalemia, itching, insomnia, and confusion that may be associated with high doses of tacrolimus.

In some embodiments, tacrolimus is initially administered to a human subject at about 0.03mg/kg of the actual or ideal body weight of the human subject per day, or tacrolimus is initially administered about 12 hours to about 24 hours after the administration of the second cd45+ cell population, as disclosed herein.

The circulating level of tacrolimus may be monitored and the dosage adjusted accordingly to achieve the target concentration. For example, the whole blood concentration of tacrolimus may be monitored and the dosage adjusted and administered to achieve the target trough level. The target trough level of tacrolimus may be, for example, less than about 25ng/mL, less than about 20ng/mL, less than about 15ng/mL, less than about 14ng/mL, less than about 13ng/mL, less than about 12ng/mL, less than about 11ng/mL, less than about 10ng/mL, less than about 9ng/mL, less than about 8ng/mL, less than about 7ng/mL, less than about 6ng/mL, less than about 5ng/mL, less than about 4ng/mL, less than about 3ng/mL, less than about 2ng/mL, or less than about 1ng/mL.

In some embodiments of the present invention, in some embodiments, tacrolimus has a target trough level of about 1-25ng/mL, about 1-20ng/mL, about 1-15ng/mL, about 1-12ng/mL, about 1-11ng/mL, about 1-10ng/mL, about 1-9ng/mL, about 1-8ng/mL, about 1-7ng/mL, about 1-6ng/mL, about 1-5ng/mL, about 1-4ng/mL, about 1-3ng/mL, about 1-2ng/mL, about 2-25ng/mL, about 2-20ng/mL, about 2-15ng/mL, about 2-12ng/mL, about 2-11ng/mL, about 2-10ng/mL, about 2-9ng/mL, about 2-8ng/mL, about 2-7ng/mL, about 2-6ng/mL, about 2-5ng/mL, about 2-4ng/mL, about 2-3ng/mL, about 3-25ng/mL, about 3-20ng/mL, about 3-15ng/mL, about 3-12ng/mL, about 3-11ng/mL, about 3-10ng/mL, about 3-9ng/mL, about 3-8ng/mL, about 3-7ng/mL, about 3-6ng/mL, about 3-5ng/mL, about 3-4ng/mL, about 4-25ng/mL, about 4-20ng/mL, about 4-15ng/mL, about 4-12ng/mL, about 4-11ng/mL, about 4-10ng/mL, about, about 4-9ng/mL, about 4-8ng/mL, about 4-7ng/mL, about 4-6ng/mL, about 4-5ng/mL, about 5-25ng/mL, about 5-20ng/mL, about 5-15ng/mL, about 5-12ng/mL, about 5-11ng/mL, about 5-10ng/mL, about 5-9ng/mL, about 5-8ng/mL, about 5-7ng/mL, about 5-6ng/mL, about 6-25g/mL, about 6-20ng/mL, about 6-15ng/mL, about 6-12ng/mL, about 6-11ng/mL, about 6-10ng/mL, about 6-9ng/mL, about 6-8 mL, about 6-7ng/mL, about 8-25ng/mL, about 8-20ng/mL, about 8-15ng/mL, about 8-12ng/mL, about 8-11ng/mL, about 8-10ng/mL, about 10 ng-10 ng/mL.

In some embodiments, the target trough level of tacrolimus is from about 6ng/mL to about 10ng/mL. In some embodiments, the target trough level of tacrolimus is from about 6ng/mL to about 9ng/mL. In some embodiments, the target trough level of tacrolimus is from about 6ng/mL to about 8ng/mL. In some embodiments, the target trough level of tacrolimus is from about 5ng/mL to about 10ng/mL. In some embodiments, the target trough level of tacrolimus is from about 5ng/mL to about 9ng/mL. In some embodiments, the target trough level of tacrolimus is from about 5ng/mL to about 8ng/mL. In some embodiments, the target trough level of tacrolimus is from about 4ng/mL to about 10ng/mL. In some embodiments, the target trough level of tacrolimus is from about 4ng/mL to about 9ng/mL. In some embodiments, the target trough level of tacrolimus is from about 4ng/mL to about 8ng/mL.

In some embodiments, the dosage of tacrolimus or the target trough level of tacrolimus as a single agent prophylactic may be adjusted based on the clinical parameters disclosed herein. For example, in some cases, if a subject exhibits less donor T cell chimerism than desired, e.g., when assessed after a suitable time following administration of a cell population of the disclosure, e.g., about 14 days, 15 days, 20 days, 21 days, 25 days, 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100 days, 110 days, 120 days, 130 days, 140 days, 150 days, 160 days, 170 days, 180 days, or 1 year after administration of a cell population of the disclosure (e.g., a first CD45+ cell population), the dosage or target trough level of tacrolimus may be reduced by less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50% or less than 45% of the percentage of peripheral blood donor-derived cd3+ cells. In some embodiments, the target valley level of tacrolimus may be increased if the subject exhibits an indication of GVHD as disclosed herein.

In some embodiments, the subject achieves a chimerism of at least about 80% on about day 30. In some embodiments, the subject achieves at least about 80% chimerism on about day 30 and has a target trough level of tacrolimus between about 6.5ng/mL and about 9 ng/mL.

Administration of tacrolimus to a subject can begin before or after administration of the cell population disclosed herein (e.g., the first cd45+ cell population). In some embodiments, the administration of tacrolimus to a subject begins prior to the administration of the cell population disclosed herein (e.g., the first cd45+ cell population). In some embodiments, the administration of tacrolimus to a subject begins after administration of the cell population disclosed herein (e.g., the first cd45+ cell population). In some embodiments, the administration of tacrolimus to a subject begins at about the same time as the administration of the cell population disclosed herein (e.g., the first cd45+ cell population).

In some embodiments, the administration of tacrolimus to a subject begins at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 14 days, or at least 20 days prior to the administration of the cell population disclosed herein (e.g., cd45+ cell population). In some embodiments, the administration of tacrolimus to a subject begins at most 1 day, at most 2 days, at most 3 days, at most 4 days, at most 5 days, at most 6 days, at most 7 days, at most 10 days, at most 14 days, or at most 20 days prior to the administration of the cell population disclosed herein (e.g., cd45+ cell population). In some embodiments, the administration of tacrolimus to a subject begins 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, or 20 days prior to the administration of the cell population disclosed herein (e.g., cd45+ cell population). In some embodiments, the administration of tacrolimus to the subject begins 1 day prior to the administration of the first cd45+ cell population. In some embodiments, the administration of tacrolimus to the subject begins 1 day prior to the administration of the second cd45+ cell population.

In embodiments, tacrolimus is initially administered to a human subject at about 0.03mg/kg of the actual or ideal body weight of the human subject per day, or tacrolimus is initially administered from about 12 hours to about 24 hours after the administration of the second cd45+ cell population, as disclosed herein.

In some embodiments, the administration of tacrolimus to a subject begins at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 14 days, or at least 20 days after administration of the cell population disclosed herein (e.g., cd45+ cell population). In some embodiments, the administration of tacrolimus to a subject begins at most 1 day, at most 2 days, at most 3 days, at most 4 days, at most 5 days, at most 6 days, at most 7 days, at most 10 days, at most 14 days, or at most 20 days after administration of the cell population disclosed herein (e.g., cd45+ cell population). In some embodiments, the administration of tacrolimus to a subject begins 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, or 20 days after administration of the cell population disclosed herein (e.g., cd45+ cell population). In some embodiments, the administration of tacrolimus to the subject begins 1 day after the administration of the cd45+ cell population. In some embodiments, the administration of tacrolimus to the subject begins 1 day after the administration of the second cd45+ cell population. In some embodiments, the administration of tacrolimus to a subject begins on the same day as the administration of the cell population of the present disclosure.

Tacrolimus may be administered to a subject for any amount of time after administration of the cell population of the present disclosure (e.g., cd45+ cell population). In some embodiments, after administration of a cell population of the present disclosure (e.g., a cd45+ cell population), tacrolimus is administered to a subject for the first 7 days, 14 days, first 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 200 days, 365 days, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years.

In some embodiments, following administration of a cell population of the present disclosure (e.g., a cd45+ cell population), tacrolimus is administered to a subject for less than about 20 days, less than about 30 days, less than about 40 days, less than about 50 days, less than about 60 days, less than about 70 days, less than about 80 days, less than about 90 days, less than about 100 days, less than about 110 days, less than about 120 days, less than about 150 days, less than about 200 days, less than about 365 days, less than about 13 months, less than about 14 months, less than about 15 months, less than about 16 months, less than about 17 months, less than about 18 months, less than about 19 months, less than about 20 months, less than about 21 months, less than about 22 months, less than about 23 months, less than about 2 years, less than about 2.5 years, less than about 3 years, less than about 3.5 years, less than about 4 years, less than about 4.5 years, or less than about 5 years.

In various embodiments, the dose of tacrolimus begins to taper about 90 days after administration of the first dose to the human subject, or the dose of tacrolimus begins to taper about 45 days after administration of the first dose to the human subject.

In some embodiments, the subject achieves a chimerism of at least about 80% on about day 30. In some embodiments, the subject achieves at least about 80% chimerism on about day 30 and has a target trough level of about 6.5ng/mL to about 9 ng/mL.

III. objects

Provided herein are compositions for administration to a subject having cancer and methods of administration thereof. The compositions and methods are useful for treating or alleviating cancer in a subject. In some embodiments, a second cd45+ cell population comprising at least Tcon is administered to the subject in order to elicit a Graft Versus Tumor (GVT) immune response and reduce Graft Versus Host Disease (GVHD).

In some embodiments, the subject is at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 years old. In some embodiments, the subject is at least 18 years old. In some embodiments, the subject is at least 16 years old. In some embodiments, the subject is at least 13 years old.

In some embodiments, the subject is at most 50, at most 55, at most 60, at most 65, at most 70, at most 75, or at most 80 years old. In some embodiments, the subject is at most 65 years old. In some embodiments, the subject is up to 70 years old.

A. Pathology of diseases

In another aspect, a method of treating a human subject diagnosed with a hematological malignancy is provided. The method comprises administering to a human subject a solution comprising a first cd45+ cell population, a solution comprising a cell population enriched for regulatory T cells (tregs), a solution comprising a second cd45+ cell population, and a solution comprising one or more doses of a GVHD preventing agent (e.g., tacrolimus). In this aspect, the solution comprising the first cd45+ cell population, the solution comprising the cell population enriched for regulatory tregs, the solution comprising the second cd45+ cell population, and the solution comprising one or more doses of a GVHD prophylaxis agent are as defined according to any of the multicomponent drug therapies disclosed herein.

The methods of the present disclosure can be used to treat a subject (e.g., a human subject) having cancer. In some embodiments, the subject has been treated for cancer, for example, by treatment with a chemotherapeutic agent or with radiation. The methods of the present disclosure are useful for treating hematological malignancies, such as leukemia or lymphoma. Examples of hematological malignancies that can be treated by the methods of the present disclosure include, but are not limited to, acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), multiple myeloma, and lymphomas such as hodgkin's lymphoma and non-hodgkin's lymphoma. The cancer may be a solid tumor. In some embodiments, the cancer is a primary or metastatic tumor.

Types of cancers that may be treated using the methods of the invention include, but are not limited to, leukemia, lymphoma, adrenocortical carcinoma, anal carcinoma, aplastic anemia, cholangiocarcinoma, bladder carcinoma, bone cancer, bone metastasis, brain cancer, central Nervous System (CNS) cancer, peripheral Nervous System (PNS) cancer, breast cancer, cervical cancer, childhood non-hodgkin's lymphoma, colorectal cancer, endometrial cancer, esophageal cancer, ewing's tumor family (e.g., ewing's sarcoma), eye cancer, gall bladder cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, gestational trophoblastic disease, hairy cell leukemia, hodgkin's lymphoma, kaposi's sarcoma, kidney cancer, laryngeal pharynx cancer, acute lymphoblastic leukemia, acute myelogenous leukemia, childhood leukemia, chronic lymphoblastic leukemia, liver cancer, lung carcinoid carcinoma, breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal and paranasal sinus cancer, neuroblastoma, oral and oral cavity cancer, oral cavity and oropharynx cancer, pancreatic cancer, ovarian cancer, melanoma, skin carcinoma; thymus cancer, thyroid cancer, uterine cancer (e.g., uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell carcinoma or epidermoid carcinoma, bronchial adenoma, choriocarcinoma, head and neck cancer, teratocarcinoma, and Fahrenheit macroglobulinemia.

Patients with high risk hematological malignancies are rarely cured by standard chemotherapy. High-risk malignancies include, for example, leukemia or lymphoma that progress beyond first remission, or leukemia or lymphoma that relapse refractory.

In some embodiments, the human subject or patient has been previously or is being treated for hematological malignancy.

Subjects receiving the compositions of the present disclosure may have, for example, acute myeloid leukemia, acute lymphoid leukemia, mixed phenotype leukemia, myelofibrosis, high risk myelodysplastic syndrome, very high risk myelodysplastic syndrome, myelofibrosis (MF) that meets the transplantation qualification of national integrated cancer network guidelines (National Comprehensive Cancer Network Guidelines), moderate-2-or high risk MF according to IPSS, DIPSS, or DIPSS-plus scoring systems, moderate-1-risk MF associated with high risk features such as high symptomatic burden, low platelet count, or complex cytogenetics, primary myelofibrosis, myelofibrosis evolving from another myeloproliferative tumor, myelodysplastic syndrome, non-hodgkin lymphoma, non-malignant indication of allogeneic hematopoietic stem cell transplantation (alloHCT).

In some embodiments, the subject has acute myeloid leukemia. In some embodiments, the subject has acute lymphoid leukemia. In some embodiments, the subject has mixed phenotype leukemia. In some embodiments, the subject has a high risk of myelodysplastic syndrome. In some embodiments, the subject has an extremely high risk of myelodysplastic syndrome. In some embodiments, the subject has Myelofibrosis (MF) that meets the transplantation qualification of the national integrated cancer network guideline. In some embodiments, the subject suffers from moderate-2-or high risk myelofibrosis according to the IPSS, DIPSS, or DIPSS-plus scoring system. In some embodiments, the subject suffers from moderate-1-risk myelofibrosis associated with high risk features such as high symptomatic burden, low platelet count, or complex cytogenetics. In some embodiments, the subject has primary myelofibrosis. In some embodiments, the subject has myelofibrosis. In some embodiments, the subject has myelofibrosis evolving from another myeloproliferative neoplasm. In some embodiments, the subject has myelodysplastic syndrome. In some embodiments, the subject has non-hodgkin's lymphoma. In some embodiments, the subject has a non-malignant indication of alloHCT.

The subject may be in Complete Remission (CR). The subject may be in Complete Remission (CRi) with incomplete hematological recovery, e.g., there is no known minimal residual disease. The subject may have minimal residual disease. The subject may have no evidence of minimal residual disease. The subject may have active disease. The subject may have leukemia that is not in morphological CR (e.g., acute myeloid leukemia, acute lymphoid leukemia, or mixed phenotype leukemia), wherein the bone marrow infiltration by leukemia blast cells is less than or equal to 10%. The subject may have leukemia at morphological CR (e.g., acute myeloid leukemia, acute lymphoid leukemia, or mixed phenotype leukemia), with minimal residual positives as evidenced by multiparameter flow cytometry analysis or by nucleic acid-based techniques.

Complete Remission (CR) of acute myeloid leukemia, acute lymphoid leukemia, or mixed phenotype leukemia can be expressed by meeting all of the following criteria: (i) Bone marrow blast<5%; (ii) no circulating blast and blast using Auer rods; (ii) No extramedullary disease 4.ANC is not less than 1.0X10 ⁹ L (1,000/. Mu.L); (iii) Platelet count is greater than or equal to 100X 10 ⁹ L (100,000/. Mu.L); and (iv) independence of erythrocyte infusion. Except residual neutropenia<1.0×10 ⁹ L) or thrombocytopenia<100×10 ⁹ In addition to/L), complete Remission (CRi) with incomplete hematological recovery can be demonstrated by compliance with all CR criteria.

B. Sensitivity to

In some embodiments, the subject is not known to be allergic or hypersensitivity to tacrolimus or intolerant. In some embodiments, the subject is not known to be allergic or hypersensitivity to sirolimus or intolerant.

In some embodiments, the subject is insensitive to iron dextran (e.g., a subject sensitive to iron dextran is not eligible to receive the composition of the present disclosure; in some cases, this may be because magnetic beads are used in some embodiments to isolate, deplete and/or purify cell types).

In some embodiments, the subject is not sensitive to products derived from cyanine dyes (e.g., a subject sensitive to products derived from cyanine dyes is not eligible to receive the compositions of the present disclosure).

In some embodiments, the subject is not susceptible to a protein product derived from a murine source (e.g., a subject susceptible to a protein product derived from a murine source is not eligible to receive the composition of the present disclosure).

In some embodiments, the subject is not susceptible to protein products derived from bovine sources (e.g., a subject susceptible to protein products derived from bovine sources is not eligible to receive the compositions of the present disclosure).

In some embodiments, the subject is not susceptible to protein products derived from an algae source (e.g., a subject susceptible to protein products derived from an algae source is not eligible to receive the composition of the present disclosure).

In some embodiments, the subject is not sensitive to a protein product derived from streptomyces avidin (Streptomyces avidinii) (e.g., a subject sensitive to a protein product derived from streptomyces avidin is not eligible to receive the composition of the present disclosure).

C. Organ function and biomarkers

The subject may have an estimated glomerular filtration rate (egffr) of >30 mL/min. The subject may have an estimated glomerular filtration rate (egffr) of >40 mL/min. The object may have an eGCF >50 mL/min. The subject may have an estimated glomerular filtration rate (egffr) of >60 mL/min.

The subject may have a resting cardiac ejection fraction of ≡45% or a foreshortening fraction of ≡27% as measured by echocardiography or radionuclide scanning (MUGA).

The subject may have a lung carbon monoxide Dispersion (DLCO) (adjusted for hemoglobin) of 50% or more.

The subject may have a negative serum or urine β -HCG test, for example, in females with fertility within 3 weeks after enrollment.

The subject may have total bilirubin of < 2 times the upper limit of normal value (ULN).

The subject may have gilbert syndrome, wherein hemolysis has been excluded.

The subject may have an ALT reading that is within 3 times the Upper Limit of Normal (ULN). The subject may have an AST reading within 3 times the upper normal value limit (ULN).

D. Additional therapies and other subject characteristics

In some embodiments, the subject does not receive past alloHCT. In some embodiments, the subject is not a candidate for autograft. In some embodiments, the subject does not receive a corticosteroid or other immunosuppressive therapy. In some embodiments, the subject is receiving a local corticosteroid or oral systemic corticosteroid dose of less than or equal to 10 mg/day. In some embodiments, the subject does not receive Donor Lymphocyte Infusion (DLI). In some embodiments, the subject does not receive a T cell depleting drug, e.g., cyclophosphamide (Cy) after transplantation, pre-transplantation anti-thymocyte globulin (ATG), or alemtuzumab (alemtuzumab). In some embodiments, the subject that has been previously exposed to the T cell depleting agent undergoes 5 half-life elution of the agent prior to the planned day 0 of transplantation (day of infusion of Treg and HSPC components of the graft). In some embodiments, the subject is not positive for anti-donor HLA antibodies against mismatched alleles in the selected donor, as determined by any of: (a) a positive cross-match test of any titer; or (b) the presence of anti-donor HLA antibodies directed against any HLA locus. In some embodiments, the subject has a Karnofsky performance score of ≡70%. In some embodiments, the subject does not have a hematopoietic cell transplantation specific co-disease index (HCT-CI) of > 4. In some embodiments, the subject does not have an uncontrolled bacterial, viral, or fungal infection. In some embodiments, the subject does not receive antimicrobial therapy and the infection is progressed or is not clinically ameliorated. In some embodiments, the subject is not seropositive for HIV-1 or-2, HTLV-1 or-2, hepatitis B surface antigen or hepatitis C antibody. In some embodiments, the subject does not have an uncontrolled autoimmune disease requiring active immunosuppressive therapy. In some embodiments, the subject does not have a concurrent malignancy or active disease within 1 year, e.g., excluding non-melanoma skin cancers that have been curative resections. In some embodiments, the subject does not exhibit a psychosocial environment that prevents the patient from being able to undergo transplantation or to participate responsible for follow-up care. In some embodiments, the subject is not pregnant or is in lactation. In some embodiments, the subject does not have a serious medical condition or abnormality in clinical laboratory testing that the medical professional judges to interfere with the safety of the subject upon receipt of the compositions of the present disclosure. In some embodiments, the subject is eligible to receive myeloablative alloHCT.

In some embodiments, the subject receives a prophylactic agent to reduce the risk of bacterial, fungal, and/or viral infection, e.g., during peri-transplantation (peri-transfer) phase.

In some embodiments, the subject receives supportive therapy for HCT-associated toxicity. In some embodiments, the subject does not receive supportive therapy for HCT-associated toxicity. In some embodiments, the subject receives a growth factor. In some embodiments, the subject does not receive a growth factor. In some embodiments, the subject receives intravenous immunoglobulin. In some embodiments, the subject does not receive intravenous immunoglobulin. In some embodiments, the subject receives an analgesic. In some embodiments, the subject does not receive an analgesic. In some embodiments, the subject receives an antiemetic. In some embodiments, the subject does not receive an antiemetic. In some embodiments, the subject receives electrolyte substitution. In some embodiments, the subject does not receive electrolyte substitution. In some embodiments, the subject receives a tyrosine kinase inhibitor (e.g., FLT3 inhibitor). In some embodiments, the subject does not receive a tyrosine kinase inhibitor (e.g., FLT3 inhibitor). In some embodiments, the subject receives prednisone or an equivalent thereof, e.g., at a dose of ∈10 mg/day. In some embodiments, the subject does not receive prednisone or an equivalent thereof. In some embodiments, the subject is treated with a corticosteroid to manage GVHD. In some embodiments, the subject is not receiving corticosteroid therapy to manage GVHD. In some embodiments, the subject is treated with a high dose corticosteroid to manage GVHD. In some embodiments, the subject is not receiving high dose corticosteroid therapy to manage GVHD. In some embodiments, the subject is treated with a corticosteroid to manage, for example, adrenal insufficiency, hypersensitivity or other non-cancer related symptoms, including pre-operative administration of known hypersensitivity to scanning contrast agents. In some embodiments, the subject is not receiving corticosteroid therapy. In some embodiments, the subject receives an immunosuppressive drug. In some embodiments, the subject does not receive an immunosuppressive drug. In some embodiments, the subject receives donor lymphocyte infusion. In some embodiments, the subject does not receive donor lymphocyte infusion.

E. Conditioning regimen

Conditioning regimens may be used as part of the alloHCT regimens of the present disclosure. Chemotherapy and/or radiation administered shortly before implantation is referred to as a conditioning regimen. The conditioning regimen can help eradicate the patient's disease prior to infusion of HSPCs, suppress immune responses, and allow the donor HSPCs to reconstitute empty hematopoietic compartments created by the conditioning regimen. In some embodiments of the methods of the present disclosure, the subject may be treated with myeloablative conditioning prior to infusion of the cell populations described herein. In some embodiments of the methods of the present disclosure, the subject may be treated with myeloreductive conditioning prior to infusion of the cell populations described herein. In some embodiments of the methods of the present disclosure, the subject may be treated with reduced intensity myeloablative conditioning prior to infusion of the cell populations described herein. In some embodiments of the methods of the present disclosure, the subject may be treated with non-myeloablative conditioning prior to administration of one or more cell populations described herein.

As used herein, the term conditioning regimen and the like applies to myeloablative conditioning, myeloablative therapy/conditioning of reduced intensity, and/or non-myeloablative conditioning. As used herein, the term myeloablative therapy/conditioning also includes myeloablative conditioning and myeloablative conditioning of reduced intensity.

In aspects and embodiments, the treatment and/or method further comprises a conditioning regimen, wherein the conditioning regimen is administered prior to administration of one of the following: (a) A solution comprising a first cd45+ cell population comprising Hematopoietic Stem and Progenitor Cells (HSPCs) and granulocytes, wherein up to about 10% of the first cd45+ cell population comprises granulocytes; (b) A solution comprising a population of cells enriched for regulatory T cells (tregs); and (c) a solution comprising a second population of cd45+ cells, wherein the second population of cd45+ cells is comprised toAbout 20% less cd3+ normal T cells (Tcon), at least about 10% monocytes and at least about 10% granulocytes; and (d) a solution comprising one or more doses of a Graft Versus Host Disease (GVHD) prophylaxis agent. In embodiments, the conditioning regimen is a myeloablative conditioning regimen. In some cases, the conditioning regimen comprises at least three conditioning agents, wherein at least one conditioning agent is thiotepa. In various embodiments, the myeloablative conditioning regimen comprises at least one dose of thiotepa, e.g., at least about 5 milligrams of thiotepa per kg of the actual or ideal body weight of a human subject, or at least about 10 milligrams of thiotepa per kg of the actual or ideal body weight of a human subject. In some embodiments, the conditioning regimen comprises one or more doses of busulfan, fludarabine, and thiotepa. In embodiments, the one or more doses comprise about 5 to about 12mg thiotepa/kg of the actual or ideal body weight of the human subject, about 7 to about 11mg busulfan/kg of the actual or ideal body weight of the human subject, and about 100 to about 200mg fludarabine/m, respectively ² Body surface area.

In various embodiments, the method further comprises administering to the patient a myeloablative conditioning regimen prior to administering any cell population, the conditioning regimen comprising administering to the patient at least one conditioning agent.

In some embodiments, the patient does not receive any radiation as part of the myeloablative conditioning regimen.

In embodiments, the at least one conditioning agent is administered from about two days to about ten days prior to administration of any of the cell populations. In some cases, the at least one conditioning agent is administered about five days prior to administration of any of the cell populations.

In various embodiments, the human subject has undergone a myeloablative conditioning regimen prior to administration of any cell population, and the adverse event is associated with myeloablative conditioning.

In embodiments, at least one modulationThe pharmaceutical agents include busulfan and fludarabine. In some cases, the dosages of thiotepa, busulfan and fludarabine administered to the patient include about 10mg/kg of the patient's actual or ideal body weight, about 9.6mg/kg of the patient's actual or ideal body weight, and about 150mg/m, respectively ² Body surface area.

In some embodiments, the subject has been conditioned with radiation, chemotherapy, recombinant proteins, antibodies, or toxin-conjugated antibodies, or any combination thereof, prior to administration of one or more cell populations described herein. In some embodiments, the subject is conditioned to be suitable for cell transplantation therapy by first treating the subject with myeloablative therapy. Exemplary myeloablative therapies include chemotherapy or radiation therapy. Myeloablative therapy is thought to provide therapeutic benefit by shrinking tumors and/or reducing the number of cancer cells. Myeloablative therapy eradicates sufficient numbers of HSCs that would otherwise increase the patient's chances of developing GVHD. When HSPCs are subsequently administered to a subject with bone marrow ablation, donor cells can further attack the cancer and/or reconstitute the subject's blood and immune system.

In some embodiments, myeloablative therapy comprises administration of thiotepa (TTP), busulfan, cyclophosphamide, systemic irradiation (TBI), fludarabine, etoposide, or any combination thereof. In some embodiments, myeloablative therapy comprises administration of an anti-cKIT antibody. In some embodiments, myeloablative therapy comprises administration of an antibody drug conjugate. The antibody drug conjugate may be, for example, an anti-CD 45-saporin or anti-cKit-saporin therapeutic antibody. In some embodiments, the myeloablative therapy is a reduced intensity conditioning therapy. An exemplary conditioning regimen is described in table 15.

The conditioning regimen of the present disclosure may include one or more doses of busulfan. The conditioning regimen of the present disclosure may include fludarabine. The conditioning regimen of the present disclosure may include one or more doses of cyclophosphamide. The conditioning regimen of the present invention may comprise one or more doses of melphalan. The conditioning regimen of the present disclosure may include one or more doses of etoposide.

The methods of the present disclosure may include administering a combination of conditioning agents prior to administering the cells. The conditioning regimen as described herein may comprise the administration of 1, 2, 3 or 4 different conditioning agents. The conditioning agent used herein may be an alkylating agent. Conditioning agents used herein may be myeloablative. Conditioning agents used herein may be non-myeloablative. Conditioning agents used herein may be myeloablative. Conditioning agents for use herein may be in the form of chemotherapy.

The conditioning regimen described herein may include administration of an alkylating agent such as thiotepa (TTP). The conditioning regimen of the present disclosure comprising TTP may comprise at least one or more conditioning agents. In addition to TTP, conditioning agents administered to a subject may include one or more agents selected from the group consisting of: busulfan, dimethyl Ma Lelan (dimethyl mylaran), prednisone, methylprednisolone, azathioprine, cyclophosphamide, cyclosporine, anti-T cell monoclonal antibodies, anti-lymphoglobulin and anti-thymocyte globulin, fludarabine, etoposide, radiation, whole body irradiation (TBI). Aspects and embodiments include any combination of TTP and one or more conditioning agents. In some embodiments, the subject is administered a conditioning regimen comprising thiotepa, busulfan, and fludarabine. In some embodiments, the subject is administered a conditioning regimen comprising thiotepa, fludarabine, and TBI (e.g., HFTBI).

The conditioning regimen described herein may include administration of an alkylating agent, such as TTP. In some cases, conditioning regimens of the disclosure may include TTP administration for more than one day. The conditioning regimen of the present disclosure may include administering to the subject from 2mg/kg to 14mg/kg of TTP. The conditioning regimen of the present disclosure may include administering at least 3mg/kg of TTP to the subject. The conditioning regimen of the present disclosure may include administering up to 14mg/kg of TTP to the subject. The conditioning regimen of the present disclosure may include administering 2mg/kg to 5mg/kg, 2mg/kg to 6mg/kg, 2mg/kg to 8mg/kg, 2mg/kg to 10mg/kg, 2mg/kg to 12mg/kg, 2mg/kg to 14mg/kg, 5mg/kg to 6mg/kg, 5mg/kg to 8mg/kg, 5mg/kg to 10mg/kg, 5mg/kg to 12mg/kg, 5mg/kg to 14mg/kg, 6mg/kg to 8mg/kg, 6mg/kg to 10mg/kg, 6mg/kg to 12mg/kg, 6mg/kg to 14mg/kg, 8mg/kg to 10mg/kg, 8mg/kg to 12mg/kg, 8mg/kg to 14mg/kg, 10mg/kg to 12mg/kg, or TTP of 12mg/kg to 14mg/kg to the subject. The conditioning regimen of the present disclosure may include administering to the subject 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 8mg/kg, 10mg/kg, 12mg/kg, or 14mg/kg of TTP. The conditioning regimen of the present disclosure may include administering up to 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 8mg/kg, 10mg/kg, or 12mg/kg of TTP to the subject. The conditioning regimen of the present disclosure may include administering at least 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 8mg/kg, 10mg/kg, 12mg/kg, or 14mg/kg of TTP to the subject.

As used herein, a recited dose, e.g., #mg/kg, may be relative to the actual body weight of the subject (in kg) or relative to the ideal body weight of the subject (in kg). Alternatively, if the actual body weight of the subject is greater than 120% of the Ideal Body Weight (IBW), the listed doses may be relative to the subjectBody Weight (ABW).

A subject administered one or more cell populations described herein can be administered one or more doses of TTP prior to cell transplantation. A subject receiving one or more cell populations described herein can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of TTP prior to cell transplantation. In some cases, each dose of TTP has the same concentration. In some cases, one or more doses of TTP have different concentrations. The subject may administer 1mg/kg to 10mg/kg of TTP in a single dose. The subject may administer at least 1mg/kg of TTP in a single dose. The subject may administer up to 10mg/kg of TTP in a single dose. The subject may be administered 1mg/kg to 2mg/kg, 1mg/kg to 3mg/kg, 1mg/kg to 4mg/kg, 1mg/kg to 5mg/kg, 1mg/kg to 6mg/kg, 1mg/kg to 7mg/kg, 1mg/kg to 8mg/kg, 1mg/kg to 9mg/kg, 1mg/kg to 10mg/kg, 2mg/kg to 3mg/kg, 2mg/kg to 4mg/kg, 2mg/kg to 5mg/kg, 2mg/kg to 6mg/kg, 2mg/kg to 7mg/kg, 2mg/kg to 8mg/kg, 2mg/kg to 9mg/kg, 2mg/kg to 10mg/kg, 3mg/kg to 4mg/kg, 3mg/kg to 5mg/kg, 3mg/kg to 6mg/kg, 3mg/kg to 7mg/kg, 2mg/kg to 5mg/kg 3mg/kg to 8mg/kg, 3mg/kg to 9mg/kg, 3mg/kg to 10mg/kg, 4mg/kg to 5mg/kg, 4mg/kg to 6mg/kg, 4mg/kg to 7mg/kg, 4mg/kg to 8mg/kg, 4mg/kg to 9mg/kg, 4mg/kg to 10mg/kg, 5mg/kg to 6mg/kg, 5mg/kg to 7mg/kg, 5mg/kg to 8mg/kg, 5mg/kg to 9mg/kg, 5mg/kg to 10mg/kg, 6mg/kg to 7mg/kg, 6mg/kg to 8mg/kg, 6mg/kg to 9mg/kg, 6mg/kg to 10mg/kg, 7mg/kg to 8mg/kg, 7mg/kg to 9mg/kg, 7mg/kg to 10mg/kg, 5mg/kg to 8mg/kg, TTP of 8mg/kg to 9mg/kg, 8mg/kg to 10mg/kg or 9mg/kg to 10 mg/kg. The subject may administer 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg or 10mg/kg of TTP in a single dose. The subject may administer up to 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg or 10mg/kg of TTP in a single dose. The subject may administer at least 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, or 10mg/kg of TTP in a single dose.

The conditioning regimen of the present disclosure may include one or more doses of busulfan. One or more doses of busulfan may be administered to a subject prior to administration of one or more doses of another conditioning agent, such as TTP. One or more doses of busulfan may be administered to a subject after one or more doses of another conditioning agent, such as TTP. One or more doses of busulfan can be administered to a subject with administration of one or more doses of another conditioning agent, such as TTP.

The conditioning regimen of the present disclosure may comprise administering to the subject from about 6mg/kg to about 12mg/kg busulfan. The conditioning regimen of the present disclosure may comprise administering at least about 6mg/kg busulfan to a subject. The conditioning regimen of the present disclosure may include administering to the subject up to about 12mg/kg busulfan. The conditioning regimen of the present disclosure may include administering to the subject about 6mg/kg to about 7mg/kg, about 6mg/kg to about 8mg/kg, about 6mg/kg to about 9mg/kg, about 6mg/kg to about 10mg/kg, about 6mg/kg to about 11mg/kg, about 6mg/kg to about 12mg/kg, about 7mg/kg to about 8mg/kg, about 7mg/kg to about 9mg/kg, about 7mg/kg to about 10mg/kg, about 7mg/kg to about 11mg/kg, about 7mg/kg to about 12mg/kg, about 8mg/kg to about 9mg/kg, about 8mg/kg to about 10mg/kg, about 8mg/kg to about 11mg/kg, about 8mg/kg to about 12mg/kg, about 9mg/kg to about 10mg/kg, about 9mg/kg to about 11mg/kg, about 9mg/kg to about 12mg/kg, about 10mg/kg to about 11mg/kg, about 11mg/kg to about 11mg/kg, about 10mg/kg to about 12 mg/kg. The conditioning regimen of the present disclosure may include administering to the subject 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, or 12mg/kg busulfan. The conditioning regimen of the present disclosure may comprise administering at least 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, or 11mg/kg of busulfan to the subject. The conditioning regimen of the present disclosure may include administering to the subject up to 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, or 12mg/kg of busulfan.

A subject receiving one or more cell populations described herein may be administered one or more doses of busulfan prior to cell transplantation. A subject receiving one or more of the cellular components described herein may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of busulfan prior to cell transplantation. In some cases, each dose of busulfan has the same concentration. In some cases, one or more doses of busulfan have different concentrations. The subject may administer 1mg/kg to 10mg/kg of busulfan in a single dose. The subject may administer at least 1mg/kg of busulfan in a single dose. The subject may administer at least 2mg/kg of busulfan in a single dose. The subject may administer at least 3mg/kg of busulfan in a single dose.

The conditioning regimen of the present disclosure may include one or more doses of fludarabine. One or more doses of fludarabine may be administered to the subject prior to administration of one or more doses of another conditioning agent, such as TTP. One or more doses of fludarabine may be administered to the subject after one or more doses of another conditioning agent, such as TTP. One or more doses of fludarabine may be administered to a subject together with administration of one or more doses of another conditioning agent, such as TTP.

The conditioning regimen of the present disclosure may include administering 20mg/m to the subject based on the surface area of the subject ² To 180mg/m ² Fludarabine of (c). The conditioning regimen of the present disclosure may comprise administering at least 20mg/m to the subject ² Fludarabine of (c). The conditioning regimen of the present disclosure may comprise administering up to 180mg/m to the subject ² Fludarabine of (c). The conditioning regimen of the present disclosure may comprise administering 20mg/m to the subject ² To 30mg/m ² 、20mg/m ² To 40mg/m ² 、20mg/m ² To 50mg/m ² 、20mg/m ² To 60mg/m ² 、20mg/m ² To 80mg/m ² 、20mg/m ² To 100mg/m ² 、20mg/m ² To 120mg/m ² 、20mg/m ² To 150mg/m ² 、20mg/m ² To 180mg/m ² 、30mg/m ² To 40mg/m ² 、30mg/m ² To 50mg/m ² 、30mg/m ² To 60mg/m ² 、30mg/m ² To 80mg/m ² 、30mg/m ² To 100mg/m ² 、30mg/m ² To 120mg/m ² 、30mg/m ² To 150mg/m ² 、30mg/m ² To 180mg/m ² 、40mg/m ² To 50mg/m ² 、40mg/m ² To 60mg/m ² 、40mg/m ² To 80mg/m ² 、40mg/m ² To 100mg/m ² 、40mg/m ² To 120mg/m ² 、40mg/m ² To 150mg/m ² 、40mg/m ² To 180mg/m ² 、50mg/m ² To 60mg/m ² 、50mg/m ² To 80mg/m ² 、50mg/m ² To 100mg/m ² 、50mg/m ² To 120mg/m ² 、50mg/m ² To 150mg/m ² 、50mg/m ² To 180mg/m ² 、60mg/m ² To 80mg/m ² 、60mg/m ² To 100mg/m ² 、60mg/m ² To 120mg/m ² 、60mg/m ² To 150mg/m ² 、60mg/m ² To 180mg/m ² 、80mg/m ² To 100mg/m ² 、80mg/m ² To 120mg/m ² 、80mg/m ² To 150mg/m ² 、80mg/m ² To 180mg/m ² 、100mg/m ² To 120mg/m ² 、100mg/m ² To 150mg/m ² 、100mg/m ² To 180mg/m ² 、120mg/m ² To 150mg/m ² 、120mg/m ² To 180mg/m ² Or 150mg/m ² To 180mg/m ² Fludarabine of (c). The conditioning regimen of the present disclosure may comprise administering 20mg/m to the subject ² 、30mg/m ² 、40mg/m ² 、50mg/m ² 、60mg/m ² 、80mg/m ² 、100mg/m ² 、120mg/m ² 、150mg/m ² Or 180mg/m ² Fludarabine of (c). The conditioning regimen of the present disclosure may comprise administering at least 20mg/m to the subject ² 、30mg/m ² 、40mg/m ² 、50mg/m ² 、60mg/m ² 、80mg/m ² 、100mg/m ² 、120mg/m ² Or 150mg/m ² Fludarabine of (c). The conditioning regimen of the present disclosure may comprise administering up to 30mg/m to the subject ² 、40mg/m ² 、50mg/m ² 、60mg/m ² 、80mg/m ² 、100mg/m ² 、120mg/m ² 、150mg/m ² Or 180mg/m ² Fludarabine of (c).

A subject receiving one or more of the cellular components described herein may be administered one or more doses of fludarabine prior to cell transplantation. A subject receiving one or more of the cellular components described herein may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of fludarabine prior to cell transplantation. In some cases, each dose of fludarabine has the same concentration. In some cases, one or more doses of fludarabine have different concentrations. The subject may be administered 20 to 60mg/m in a single dose ² The dose of fludarabine. The subject may be administered at least 30mg/m in a single dose ² Fludarabine of (c). The subject may administer at least 40mg/m in a single dose ² Fludarabine of (c). The subject may administer at least 50mg/m in a single dose ² Fludarabine of (c).

The conditioning regimen of the present disclosure may include administering 20mg/m to the subject based on the surface area of the subject ² To 180mg/m ² Melphalan of (a). The conditioning regimen of the present disclosure mayComprising administering to a subject at least 20mg/m ² Melphalan of (a). The conditioning regimen of the present disclosure may comprise administering up to 180mg/m to the subject ² Melphalan of (a). The conditioning regimen of the present disclosure may comprise administering 20mg/m to the subject ² To 30mg/m ² 、20mg/m ² To 40mg/m ² 、20mg/m ² To 50mg/m ² 、20mg/m ² To 60mg/m ² 、20mg/m ² To 80mg/m ² 、20mg/m ² To 100mg/m ² 、20mg/m ² To 120mg/m ² 、20mg/m ² To 150mg/m ² 、20mg/m ² To 180mg/m ² 、30mg/m ² To 40mg/m ² 、30mg/m ² To 50mg/m ² 、30mg/m ² To 60mg/m ² 、30mg/m ² To 80mg/m ² 、30mg/m ² To 100mg/m ² 、30mg/m ² To 120mg/m ² 、30mg/m ² To 150mg/m ² 、30mg/m ² To 180mg/m ² 、40mg/m ² To 50mg/m ² 、40mg/m ² To 60mg/m ² 、40mg/m ² To 80mg/m ² 、40mg/m ² To 100mg/m ² 、40mg/m ² To 120mg/m ² 、40mg/m ² To 150mg/m ² 、40mg/m ² To 180mg/m ² 、50mg/m ² To 60mg/m ² 、50mg/m ² To 80mg/m ² 、50mg/m ² To 100mg/m ² 、50mg/m ² To 120mg/m ² 、50mg/m ² To 150mg/m ² 、50mg/m ² To 180mg/m ² 、60mg/m ² To 80mg/m ² 、60mg/m ² To 100mg/m ² 、60mg/m ² To 120mg/m ² 、60mg/m ² To 150mg/m ² 、60mg/m ² To 180mg/m ² 、80mg/m ² To 100mg/m ² 、80mg/m ² To 120mg/m ² 、80mg/m ² To 150mg/m ² 、80mg/m ² To 180mg/m ² 、100mg/m ² To 120mg/m ² 、100mg/m ² To 150mg/m ² 、100mg/m ² To 180mg/m ² 、120mg/m ² To 150mg/m ² 、120mg/m ² To 180mg/m ² Or 150mg/m ² To 180mg/m ² Melphalan of (a). The conditioning regimen of the present disclosure may comprise administering 20mg/m to the subject ² 、30mg/m ² 、40mg/m ² 、50mg/m ² 、60mg/m ² 、80mg/m ² 、100mg/m ² 、120mg/m ² 、150mg/m ² Or 180mg/m ² Melphalan of (a). The conditioning regimen of the present disclosure may comprise administering at least 20mg/m to the subject ² 、30mg/m ² 、40mg/m ² 、50mg/m ² 、60mg/m ² 、80mg/m ² 、100mg/m ² 、120mg/m ² Or 150mg/m ² Melphalan of (a). The conditioning regimen of the present disclosure may comprise administering up to 30mg/m to the subject ² 、40mg/m ² 、50mg/m ² 、60mg/m ² 、80mg/m ² 、100mg/m ² 、120mg/m ² 、150mg/m ² Or 180mg/m ² Melphalan of (a).

A subject receiving one or more of the cellular components described herein may administer one or more doses of melphalan prior to cell transplantation. A subject receiving one or more of the cellular components described herein may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of melphalan prior to cell transplantation. In some cases, each dose of melphalan has the same concentration. In some cases, one or more doses of melphalan have different concentrations.

The conditioning regimen of the present disclosure may include administration of 100mg/kg to 140mg/kg cyclophosphamide. The conditioning regimen of the present disclosure may comprise administering at least 100mg/kg cyclophosphamide. The conditioning regimen of the present disclosure may include administration of up to 140mg/kg cyclophosphamide. The conditioning regimen of the present disclosure may include administering 100mg/kg to 110mg/kg, 100mg/kg to 120mg/kg, 100mg/kg to 130mg/kg, 100mg/kg to 140mg/kg, 110mg/kg to 120mg/kg, 110mg/kg to 130mg/kg, 110mg/kg to 140mg/kg, 120mg/kg to 130mg/kg, 120mg/kg to 140mg/kg, or 130mg/kg to 140mg/kg of cyclophosphamide. The conditioning regimen of the present disclosure may include administration of about 100mg/kg, 110mg/kg, 120mg/kg, 130mg/kg, or 140mg/kg cyclophosphamide. The conditioning regimen of the present disclosure may include administration of at least 100mg/kg, 110mg/kg, 120mg/kg, or 130mg/kg cyclophosphamide. The conditioning regimen of the present disclosure may include administration of up to 110mg/kg, 120mg/kg, 130mg/kg, or 140mg/kg cyclophosphamide.

A subject receiving one or more of the cellular components described herein may be administered one or more doses of cyclophosphamide prior to cell transplantation. A subject receiving one or more of the cellular components described herein may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of cyclophosphamide prior to cell transplantation. In some cases, each dose of cyclophosphamide has the same concentration. In some cases, one or more doses of cyclophosphamide have different concentrations.

The conditioning regimen of the present disclosure may include administration of 40mg/kg to 80mg/kg of etoposide. The conditioning regimen of the present disclosure may include administration of at least 40mg/kg of etoposide. The conditioning regimen of the present disclosure may include administration of up to 80mg/kg of etoposide. The conditioning regimen of the present disclosure may include administration of etoposide from 40mg/kg to 50mg/kg, 40mg/kg to 60mg/kg, 40mg/kg to 70mg/kg, 40mg/kg to 80mg/kg, 50mg/kg to 60mg/kg, 50mg/kg to 70mg/kg, 50mg/kg to 80mg/kg, 60mg/kg to 70mg/kg, 60mg/kg to 80mg/kg, or 70mg/kg to 80 mg/kg. The conditioning regimen of the present disclosure may include administration of about 40mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, or 80mg/kg of etoposide. The conditioning regimen of the present disclosure may include administration of at least 40mg/kg, 50mg/kg, 60mg/kg, or 70mg/kg of etoposide. The conditioning regimen of the present disclosure may include administration of up to 50mg/kg, 60mg/kg, 70mg/kg, or 80mg/kg of etoposide.

A subject receiving one or more of the cellular components described herein may be administered one or more doses of etoposide prior to cell transplantation. A subject receiving one or more of the cellular components described herein may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of etoposide prior to cell transplantation. In some cases, each dose of etoposide has the same concentration. In some cases, one or more doses of etoposide have different concentrations.

Conditioning protocols of the present disclosure including TTP may include one or more doses of whole body irradiation (TBI) such as superdivided TBI (HFTBI). One or more doses of HFTBI may be administered to a subject prior to administration of one or more doses of another conditioning agent, such as TTP. One or more doses of HFTBI may be administered to a subject after administration of one or more doses of another conditioning agent, such as TTP. One or more doses of HFTBI can be administered to a subject together with administration of one or more doses of another conditioning agent, such as TTP.

The conditioning regimen of the present disclosure may include administering 800cGy to 1,500cGy of HFTBI to the subject. The conditioning regimen of the present disclosure may comprise administering to the subject at least 800cGy of HFTBI. The conditioning regimen of the present disclosure may include administering to the subject up to 1,500cgy of HFTBI. The conditioning regimen of the present disclosure may include administering 800cGy to 900cGy, 800cGy to 1,000cGy, 800cGy to 1,100cGy, 800cGy to 1,200cGy, 800cGy to 1,300cGy, 800cGy to 1,375cGy, 800cGy to 1,400cGy, 800cGy to 1,500cGy, 900cGy to 1,000cGy, 900cGy to 1,100cGy, 900cGy to 1,200cGy, 900cGy to 1,300cGy, 900cGy to 1,375cGy, 900cGy to 1,400cGy, 900cGy to 1,500cGy, 1,000cGy to 1,100cGy, 1,000cGy to 1,200cGy 1,000cGy to 1,300cGy, 1,000cGy to 1,375cGy, 1,000cGy to 1,400cGy, 1,000cGy to 1,500cGy, 1,100cGy to 1,200cGy, 1,100cGy to 1,300cGy, 1,100cGy to 1,375cGy, 1,100cGy to 1,400cGy, 1,100cGy to 1,375cGy, 1,200cGy to 1,300cGy, 1,200cGy to 1,400cGy, 1,200cGy to 1,500cGy, 1,300cGy to 1,375cGy, 1,300cGy to 1,400cGy, 1,300cGy to 1,500cGy, 1,375cGy to 1,375cGy, or 1,400cGy to 1,400 cGy. The conditioning regimen of the present disclosure may comprise administering to the subject about 800cGy, 900cGy, 1,000cGy, 1,100cGy, 1,200cGy, 1,300cGy, 1,375cGy, 1,400cGy, or 1,500cGy of HFTBI. The conditioning regimen of the present disclosure may include administering to the subject at least 800cGy, 900cGy, 1,000cGy, 1,100cGy, 1,200cGy, 1,300cGy, 1,375cGy, or 1,400cGy of HFTBI. The conditioning regimen of the present disclosure may include administering to the subject an HFTBI of up to 900cGy, 1,000cGy, 1,100cGy, 1,200cGy, 1,300cGy, 1,375cGy, 1,400cGy, or 1,500 cGy.

A subject receiving one or more of the cellular components described herein may be administered one or more doses of HFTBI prior to cell transplantation. A subject receiving one or more of the cellular components described herein may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of HFTBI prior to cell transplantation. In some cases, each dose of HFTBI has the same concentration. In some cases, one or more doses of HFTBI have different concentrations. Subjects may administer 75 to 150cGy of HFTBI in a single dose. The subject may administer at least 75cGy of HFTBI in a single dose. The subject may administer at least 100cGy of HFTBI in a single dose. The subject may administer at least 125cGy of HFTBI in a single dose.

Exemplary conditioning regimen 1: in some embodiments, a conditioning regimen comprising thiotepa, busulfan, and fludarabine is administered to the subject. In some embodiments, thiotepa is administered to the subject at 5mg/kg of actual body weight or desired body weight. In some embodiments, thiotepa is administered to the subject at about 5mg/kg for two days (e.g., consecutive days). In some embodiments, busulfan is administered to a subject at about 3.2mg/kg of actual or desired body weight. In some embodiments, busulfan is administered to a subject at about 3.2mg/kg daily for three days (e.g., consecutive days). In some embodiments, at about 50mg/m ² (square meters-body surface area) fludarabine is administered to a subject. In some embodiments, at about 50mg/m ² Fludarabine is administered to the subject for three days (e.g., consecutive days). In some embodiments, the subject administers thiotepa at 5mg/kg of actual or desired body weight, busulfan at about 3.2mg/kg of actual or desired body weight, and fludarabine at about 50mg/m2 (square meter-body surface area). In some embodiments, the subject administers thiotepa at about 5mg/kg for two days (e.g., continuous days), busulfan at about 3.2mg/kg per day for three days (e.g., continuous days), and at about 50mg/m ² Fludarabine is administered for three days (e.g., consecutive days). In some embodiments, the subject administers thiotepa at about 5mg/kg on days-7 and-6, busulfan at about 3.2mg/kg daily on days-5 to-3, and busulfan at about 50mg/m on days-5 to-3 ² Fludarabine is administered.

Exemplary conditioning regimen 2: in some embodimentsIn (c) administering to the subject a conditioning regimen comprising thiotepa, fludarabine, and TBI (e.g., HFTBI). In some embodiments, thiotepa is administered to the subject at 5mg/kg of actual body weight or desired body weight. In some embodiments, thiotepa is administered to the subject at about 5mg/kg for two days (e.g., consecutive days). In some embodiments, at about 25mg/m ² (square meters-body surface area) fludarabine is administered to a subject. In some embodiments, at about 25mg/m ² Fludarabine is administered to the subject for three days (e.g., consecutive days). In some embodiments, 125cGy (centigray) of HFTBI is administered to a subject. In some embodiments, the HFTBI is administered to the subject in 11 fractions of 125cGy each. In some embodiments, the HFTBI is administered to the subject in 11 fractions of 125cGy each over 4 days. In some embodiments, the subject administers thiotepa at 5mg/kg of actual or desired body weight at about 25mg/m ² (square meters-body surface area) fludarabine was administered, and 125cGy of HFTBI was administered. In some embodiments, the subject administers thiotepa at about 5mg/kg for two days (e.g., consecutive days), at about 50mg/m ² Fludarabine was administered for three days (e.g., consecutive days) and HFTBI was administered in 11 fractions of 125cGy each. In some embodiments, the subject administers thiotepa at about 5mg/kg on days-7 and-6 and about 50mg/m on days-5 to-3 ² Fludarabine was administered and HFTBI was administered in 11 fractions of 125cGy each over 4 days.

The subject may receive one or more cell populations described herein 1 day after completion of the conditioning regimen. The subject may receive one or more cellular components described herein 2, 3, 4, 5, 6, 7, 8, 9, 10 days after completion of the conditioning regimen. The subject may receive one or more of the cellular components described herein 1 day after receiving the final dose of conditioning agent, such as TTP. The subject may receive one or more of the cellular components described herein 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after receiving the final dose of conditioning agent, such as TTP. The subject may receive one or more of the cellular components described herein 1 day after receiving the first dose of the conditioning agent, such as TTP. The subject may receive one or more cellular components described herein 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after receiving the first dose of the conditioning agent, such as TTP.

IV Graft Versus Host Disease (GVHD)

Graft Versus Host Disease (GVHD) is a significant cause of morbidity and mortality in hematopoietic stem cell transplantation (HCT) recipients. Aspects and embodiments herein provide compositions and methods for reducing the incidence of GVHD, reducing the severity of GVHD, reducing the relative risk of GVHD, preventing GVHD, or a combination thereof in HCT receptors.

Graft Versus Host Disease (GVHD) is an inflammatory disease that may occur in a xenograft environment. GVHD involves donor cells (grafts) attacking recipient cells (hosts). GVHD can be life threatening and can involve, for example, the skin, gut and/or liver. Morbidity and mortality associated with GVHD may be the primary factors limiting HCT success. GVHD may occur despite the use of HLA-matched sibling donors and the use of various GVHD prophylaxis/immunosuppression agents, such as the use of two or more of tacrolimus, sirolimus, cyclosporine, methotrexate, mycophenolate mofetil, anti-thymocyte globulin, and corticosteroids.

GVHD classification and ranking

GVHD can be classified into acute GVHD (aGVHD) and chronic GVHD (cGVHD). In some embodiments, GVHD occurring within the first 100 days after transplantation may be referred to as aGVHD, while GVHD after the first 100 days may be referred to as chronic GVHD (cGVHD). cGVHD is a major source of late treatment-related complications and can be life threatening. In addition to inflammation, cGVHD may also lead to the development of fibrosis, which may lead to dysfunction.

Another aspect provides any of the multicomponent drug treatments disclosed herein, wherein the risk and/or severity of an adverse event associated with the multicomponent drug treatment is reduced as compared to a similar drug treatment in which a human subject received Tcon but did not receive Treg, or any of the methods disclosed herein, wherein the risk and/or severity of an adverse event associated with the method is reduced as compared to a similar method in which a human subject received Tcon but did not receive Treg.

In various embodiments, the adverse event is acute GVHD (aGVHD).

In some embodiments, the adverse event is aGVHD in stage two or higher.

In embodiments, the adverse event is chronic GVHD (cGVHD).

In various embodiments, the human subject is free of cGVHD about one year after administration of the cell population.

In some embodiments, the adverse event is moderate to severe cGVHD.

In embodiments, the adverse event is acute graft versus host disease (aGVHD), such as aGVHD in stage two or higher. In some cases, the patient has no second-phase or higher aGVHD about 180 days after administration of the cell population.

In various embodiments, the adverse event is chronic graft versus host disease (cGVHD). In some cases, the patient has no cGVHD about one year after administration of the cell population.

In some embodiments, the adverse event is moderate to severe cGVHD. In some cases, the patient has no moderate to severe cGVHD about one year after administration of the cell population.

In embodiments, the patient does not develop GVHD within about 30 days of administering Tcon, does not develop GVHD within about 100 days of administering Tcon, does not develop GVHD within about 180 days of administering Tcon, and/or does not develop GVHD within about one year of administering Tcon.

In various embodiments, a human subject does not develop GVHD above stage 2 within about 100 days of said administering said second cd45+ cell population, said human subject does not develop GVHD above stage 2 within about 180 days or about 200 days of said administering said second cd45+ cell population, and said human subject does not develop GVHD above stage 2 within about 1 year of said administering said second cd45+ cell population.

GVHD and cGVHD can be ranked using a system that first evaluates GVHD stages of skin, liver and gut and then combines scores from organ stages to determine overall GVHD grade. Examples of GVHD staging criteria that can be used for individual organs are provided in table 1:

table 2 provides a set of criteria for assessing overall GVHD grade.

The aGVHD grade can also be determined based on the most severe target organ involvement as defined in the MAGIC normalization standard described in Harris et al, "International, multicenter standardization of acute graft-versus-host disease clinical data collection: a report from the Mount Sinai Acute GVHD International Consortium" Biology of Blood and Marrow Transplantation 22.1.22.1 (2016): 4-10. For example, aGVHD organ staging can be assessed according to table 3 and overall aGVHD grade can be assessed as follows:

level 0: no organs at stage 1-4.

Stage I: stage 1-2 skin, no liver, upper GI or lower GI involvement.

Stage II: stage 3 rash and/or stage 1 liver and/or stage 1 upper GI and/or stage 1 lower GI.

Class III: stage 2-3 liver and/or stage 2-3 lower GI, with stage 0-3 skin and/or stage 0-1 upper GI. Grade IV: stage 4 skin, liver or lower GI involvement, with stage 0-1 upper GI involvement.

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In some embodiments, GVHD stage and GVHD grade are synonyms.

cGVHD can also be assessed by the method described in Jagasia et al, "National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I.the 2014diagnosis and staging working group report"Biology of Blood and Marrow Transplantation 21.3 (2015): 389-401. To determine the diagnosis of cGVHD, these criteria may require, for example, at least one diagnostic manifestation of chronic GVHD or at least one unique manifestation plus related biopsies, laboratory or other tests (e.g., PFT, schirmer test), evaluation by a specialist (ophthalmologist, gynecologist) or radiological imaging showing chronic GVHD in the same organ or other organs.

Organ systems may be scored as described in Jagasia et al, and mild cGVHD may be present when one or both organs are affected and the score does not exceed 1 plus the lung score is zero; a moderate cGVHD may be present when three or more organs are affected and the score does not exceed 1, or when at least one non-lung organ has a score of 2, or when the lung has a score of 1; and severe cGVHD may be present when at least one organ has a score of 4 or the lung has a score of 2 or 3.

Table 4 provides diagnostic and unique manifestations of cGVHD. Infections, pharmaceutical effects, malignant tumors or other causes are excluded from the special manifestations. Bronchiolitis obliterans syndrome can be diagnosed as chronic GVHD of the lung only if a distinct indication or symptom appears in another organ. Diagnosis of chronic GVHD based on myositis or polymyositis may require biopsies.

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In some embodiments, GVHD severity may be graded using the Gluckberg scale (I-IV) or International Bone Marrow Transplant Registry (IBMTR) grading system (A-D). The severity of acute GVHD can be determined by assessing the extent of involvement of the skin, liver and gastrointestinal tract. The stage of organ involvement alone is combined with the (glucosberg) or not with the (IBMTR) patient's performance status to produce an overall grade.

GVHD prevention and treatment

Immunosuppressive agents may be used to reduce the likelihood of GVHD (GVHD preventive agents) or to treat when GVHD occurs (GVHD therapeutic agents). However, in some cases, the use of a GVHD preventing agent, a GVHD treating agent, or both is insufficient to effectively prevent or treat GVHD. For example, although tacrolimus, sirolimus, cyclosporine, methotrexate, mycophenolate mofetil, anti-thymocyte globulin, corticosteroids, or combinations thereof (e.g., two or more of the agents) are used, the incidence of GVHD in the graft recipient may be high.

Administration of multiple GVHD preventing and/or treating agents, high doses of GVHD preventing and/or treating agents, or both, may not be effective in treating GVHD in many alloHCT environments, or may result in increased susceptibility to infection and reduced graft anti-tumor therapeutic effects.

Non-limiting examples of GVHD preventing and/or GVHD therapeutic agents that may be used include calcineurin inhibitors (e.g., tacrolimus, cyclosporine a), sirolimus, monoclonal antibodies, methotrexate, mycophenolate mofetil, anti-thymocyte globulin, corticosteroids, azathioprine, and mycophenolate mofetil. Monoclonal antibodies useful as immunosuppressive agents include, for example, antagonist antibodies (e.g., antibodies that antagonize IL-2R, such as basiliximab (basiliximab) and daclizumab (daclizumab)), and antibodies that deplete immune cell populations by antibody-dependent cytotoxicity (e.g., anti-CD 3 antibodies for T cell depletion, such as moromiab-CD 3).

The compositions and methods described herein may include administering one or more GVHD preventing agents to the HCT receptor. In this case, GVHD prophylaxis should be considered to be different from GVHD treatment, such that GVHD prophylaxis agents will be administered to HCT receptors prior to assessing the incidence of GVHD. In some cases, HCT receptors may be administered one or more GVHD preventing agents, rather than GVHD therapeutic agents. In some cases, HCT receptors do not require GVHD treatment and/or do not receive GVHD treatment.

The compositions and methods disclosed herein can reduce the incidence of GVHD, reduce the severity of GVHD, reduce the relative risk of GVHD, prevent GVHD, or a combination thereof in HCT receptors. In some embodiments, such benefits are obtained despite the absence of administration of a GVHD preventing agent as disclosed herein. In some embodiments, such benefits are obtained despite administration of a reduced amount of a GVHD preventing agent (e.g., a single GVHD preventing agent), a low dose of a GVHD preventing agent, or a combination thereof, as disclosed herein. In some embodiments, 1 GVHD preventing agent is administered to a subject. In some embodiments, no more than a GVHD preventing agent is administered to the subject. In some embodiments, 2 GVHD preventative agents are administered to a subject. In some embodiments, no more than 2 GVHD preventing agents are administered to the subject.

In some embodiments, one or more GVHD prophylaxis agents may be administered to the HCT receptor for a duration of 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months after the transplantation of the one or more cell populations. One or more GVHD prophylaxis agents may be administered to the HCT receptor starting on the day of transplantation of one or more cell populations. For example, a GVHD prevention regimen may begin on the day of administration of a HSPC cell population and/or a Treg cell population to a recipient. Alternatively, the GVHD prevention regimen may begin on the day of administration of the population of Tcon cells to the patient.

In some embodiments, tacrolimus is not administered to the subject. In some embodiments, sirolimus is not administered to the subject. In some embodiments, cyclosporine is not administered to a subject. In some embodiments, methotrexate is not administered to the subject. In some embodiments, mycophenolate mofetil is not administered to the subject. In some embodiments, the anti-thymocyte globulin is not administered to a subject. In some embodiments, the corticosteroid is not administered to the subject.

In some embodiments, the GVHD preventing agent is tacrolimus.

Aspects and embodiments herein provide methods of transplanting a population of cells into a human patient as part of a treatment regimen for hematological malignancies. The method comprises administering to a patient a population of Hematopoietic Stem and Progenitor Cells (HSPCs) comprising HSPCs and a liquid suspending the HSPCs; administering to a patient a population of regulatory T cells (tregs) to be administered to the patient, the population of tregs comprising tregs and a liquid suspending the tregs; and administering to the patient a heterologous cell population to be administered to the patient, the heterologous cell population comprising lymphocytes, granulocytes, monocytes and a liquid suspending said cells, wherein at least about 30% of said lymphocytes comprise conventional T cells (Tcon); and administering to the patient a single Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) comprising tacrolimus (tacrolimus GHVDPA) over a period of up to about 180 days, wherein tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the patient's blood above a threshold level during said period of time; and wherein the risk and/or severity of GHVD associated with a treatment regimen for hematological malignancy is significantly reduced.

In cases where aGVHD occurs, the responsiveness of aGVHD to a GVHD therapeutic agent (e.g., a corticosteroid) can be assessed by the criteria of table 5.

In some embodiments, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cases in which aGVHD occurs in a subject receiving a composition of the present disclosure exhibit a complete response to a GVHD therapeutic agent.

In some embodiments, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cases in which aGVHD occurs in a subject receiving a composition of the present disclosure exhibit a partial response to a GVHD therapeutic agent.

In some embodiments, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cases in which aGVHD occurs in a subject receiving a composition of the present disclosure exhibit a very good partial response to a GVHD therapeutic agent.

In cases where cGVHD occurs, the responsiveness of cGVHD to a GVHD therapeutic agent (e.g., corticosteroid) can be assessed by the criteria of table 6. Abbreviations used: ALT, alanine aminotransferase; FEV1, forced breathing volume in the first second; OMRS, oral mucosa grade scale; PFT, pulmonary function test; P-ROM, photographic movement range; ULN, upper normal limit.

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In some embodiments, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cases in which cGVHD occurs in a subject receiving a composition of the present disclosure exhibit a complete response to a GVHD therapeutic agent.

In some embodiments, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cases in which cGVHD occurs in a subject receiving a composition of the present disclosure exhibit a partial response to a GVHD therapeutic agent.

C. Incidence of acute GVHD

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit low incidence of ≡1 aGVHD, e.g., lower incidence of ≡1 aGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of the subjects administered the compositions of the present disclosure develop ≡1 aGVHD, for example, within 30 days after transplantation, within 100 days after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

As used herein, an alternative composition lacks one or more of the cell populations and/or prophylactic agents disclosed herein and/or recited in the claims. For example, the replacement composition lacks one or more of a first cd45+ cell population comprising at least HSPCs, a Treg-enriched cell population, a second cd45+ cell population comprising at least Tcon, and a prophylactic agent.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit low incidence of ≡2 aGVHD, e.g., lower incidence of ≡2 aGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the suitable amount of GV 2 as disclosed herein within 30 days, 100 days after transplantation, or another suitable amount of GV 2.

In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure develop ≡2 aGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure develop ≡2 aGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop ≡2 aGVHD. In some embodiments, less than about 8% of subjects administered the compositions of the present disclosure develop ≡2 aGVHD. In some embodiments, less than about 7% of subjects administered the compositions of the present disclosure develop ≡2 aGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit low incidence of ≡3 aGVHD, e.g., lower incidence of ≡3 aGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the suitable amount of GV 3 as disclosed herein, for example, within 30 days after transplantation, or within 100 days after another suitable amount of GV 3.

In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure develop ≡3 grade aGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure develop ≡3 grade aGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop ≡3 grade aGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure develop ≡3 grade aGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure develop ≡3 grade aGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure develop ≡3 grade aGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure develop ≡3 grade aGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit low incidence of aGVHD at > 4, e.g., lower incidence of aGVHD at > 4 compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, or less than about 30% of the subjects administered the compositions of the present disclosure develop ≡4 grade aGVHD, for example, within 30 days after transplantation, within 100 days after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop ≡4 grade aGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure develop ≡4 grade aGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure develop ≡4 grade aGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure develop ≡4 grade aGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure develop ≡4 grade aGVHD.

The incidence of aGVHD may be assessed after implantation for a suitable amount of time, for example, about 20 days, about 21 days, about 25 days, about 28 days, about 30 days, about 35 days, about 40 days, about 42 days, about 45 days, about 49 days, about 50 days, about 55 days, about 56 days, about 60 days, about 63 days, about 65 days, about 70 days, about 75 days, about 77 days, about 80 days, about 84 days, about 85 days, about 90 days, about 91 days, about 95 days, about 98 days, about 100 days, about 105 days, about 110 days, about 112 days, about 115 days, about 119 days, about 120 days, after implantation.

The incidence of aGVHD may be calculated based on a population of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 subjects.

1. GVHD-free prophylaxis

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) in the absence of a GVHD prophylaxis agent exhibit low incidence of ≡1 aGVHD, e.g., lower incidence of ≡1 aGVHD compared to subjects administered the alternative composition. In some embodiments, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD preventing agent develop ≡1 grade aGVHD, for example, within 30 days after transplantation, within 100 days after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) in the absence of a GVHD prophylaxis agent exhibit low incidence of ≡2 aGVHD, e.g., lower incidence of ≡2 aGVHD compared to subjects administered the alternative composition. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the suitable amount of GVHD in a subject is not present in the subject in the absence of GVHD preventing agent.

In some embodiments, less than about 50% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 aGVHD. In some embodiments, less than about 40% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 aGVHD. In some embodiments, less than about 30% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 aGVHD. In some embodiments, less than about 25% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 aGVHD. In some embodiments, less than about 20% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 aGVHD. In some embodiments, less than about 10% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 aGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) in the absence of a GVHD prophylaxis agent exhibit low incidence of ≡3 aGVHD, e.g., lower incidence of ≡3 aGVHD compared to subjects administered the alternative composition. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the suitable amount of GVHD in a subject is not present in the subject in the absence of GVHD preventing agent in the composition of the present disclosure, such as is present.

In some embodiments, less than about 40% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 aGVHD. In some embodiments, less than about 30% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 aGVHD. In some embodiments, less than about 20% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 aGVHD. In some embodiments, less than about 10% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 aGVHD. In some embodiments, less than about 5% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 aGVHD. In some embodiments, less than about 3% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 aGVHD. In some embodiments, less than about 2% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 aGVHD. In some embodiments, less than about 1% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 aGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) in the absence of a GVHD prophylaxis agent exhibit a low incidence of ≡4 aGVHD, e.g., a lower incidence of ≡4 aGVHD compared to subjects administered the alternative composition. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, or less than about 30% of the subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD preventing agent develop ≡grade 4 aGVHD, for example, within 30 days post-transplant, 100 days, or another suitable amount of time post-transplant as disclosed herein.

In some embodiments, less than about 20% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade-4 aGVHD. In some embodiments, less than about 15% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 aGVHD. In some embodiments, less than about 10% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 aGVHD. In some embodiments, less than about 5% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade-4 aGVHD. In some embodiments, less than about 3% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 aGVHD. In some embodiments, less than about 2% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 aGVHD. In some embodiments, less than about 1% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 aGVHD.

The absence of a GVHD preventing agent may refer to a situation in which no GVHD preventing agent is administered to the subject for the first 20, 21, 25, 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 65, 70, 75, 77, 80, 84, 85, 90, 91, 95, 98, 100, 105, 110, 112, 115, 119 or 120 days after the transplantation.

2. Single agent GVHD prevention

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit a low incidence of ≡1 aGVHD, e.g., a lower incidence of ≡1 aGVHD than subjects administered alternative compositions. In some embodiments, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop ≡1, for example, within 30 days after transplantation, within 100 days after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit a low incidence of ≡2 aGVHD, e.g., a lower incidence of ≡2 aGVHD than subjects administered alternative compositions. In some embodiments, the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) are administered to a subject in less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, less than about 50%, or less than about 50% of the amount of the following a suitable amount of GVHD after a further day, such as disclosed herein after a further day of transplantation.

In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 aGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 aGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 aGVHD. In some embodiments, less than about 8% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 aGVHD. In some embodiments, less than about 7% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 aGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit a low incidence of ≡3 grade aGVHD, e.g., a lower incidence of ≡3 grade aGVHD than subjects administered alternative compositions. In some embodiments, the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) are administered to a subject in less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, less than about 50%, or less than about 50% of the amount of the following a suitable amount of GVHD after a further day of transplantation, such as disclosed herein after a further day of 100.

In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 3 aGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 3 aGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 3 aGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 3 aGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade aGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 3 aGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 3 aGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit a low incidence of ≡4 aGVHD, e.g., a lower incidence of ≡4 aGVHD compared to subjects administered the alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, or less than about 30% of the subjects to whom a GVHD preventing agent (e.g., a single GVHD preventing agent) is administered develop ≡4 grade aGVHD, e.g., within 30 days after transplantation, 100 days after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 4 aGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 4 aGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 4 aGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 4 aGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 4 aGVHD.

The single GVHD prophylaxis agent may be tacrolimus.

The single GVHD prophylaxis agent may be sirolimus.

No more than one GVHD preventing agent (e.g., a single GVHD preventing agent) may be administered to a subject for the first 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 200 days, 365 days, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years after transplantation. In some embodiments, no more than about 20 days, less than about 30 days, less than about 40 days, less than about 50 days, less than about 60 days, less than about 70 days, less than about 80 days, less than about 90 days, less than about 100 days, less than about 110 days, less than about 120 days, less than about 150 days, less than about 200 days, less than about 365 days, less than about 13 months, less than about 14 months, less than about 15 months, less than about 16 months, less than about 17 months, less than about 18 months, less than about 19 months, less than about 20 months, less than about 21 months, less than about 22 months, less than about 23 months, less than about 2 years, less than about 2.5 years, less than about 3 years, less than about 3.5 years, less than about 4 years, less than about 4.5 years, or less than about 5 years after continuous transplantation is administered to a subject.

3. Low dose GVHD prevention

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of ≡1 aGVHD, e.g., lower incidence of ≡1 aGVHD than subjects administered alternative compositions. In some embodiments, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of the subjects administered the compositions of the present disclosure and low dose of a GVHD preventing agent (e.g., a low dose of a single GVHD preventing agent) develop ≡1 GVHD, for example, within 30 days post-transplant, 100 days post-transplant, or within another suitable amount of time post-transplant as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of ≡2 aGVHD, e.g., lower incidence of ≡2 aGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, or less than about 50% of the amount of post-transplant, e.g., within about 100% of another suitable amount of post-day after-transplant period, e.g., after an additional days of development of hd, as disclosed herein, in a subject.

In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 aGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 aGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 aGVHD. In some embodiments, less than about 8% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 aGVHD. In some embodiments, less than about 7% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 aGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of ≡3 grade aGVHD, e.g., lower incidence of ≡3 grade aGVHD than subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, or less than about 50% of the amount of post-transplant, e.g., within about 100% of another suitable amount of post-day after-transplant stage, such as disclosed herein after an additional days of development of hd in a subject.

In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 aGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 aGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 aGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 aGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 aGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 aGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 aGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of ≡4 aGVHD, e.g., lower incidence of ≡4 aGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29% or less than about 30% of the subjects administered the compositions of the present disclosure (e.g., low dose single GVHD preventative agents) develop ≡4, for example, within 30 days after transplantation, within 100 days after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 aGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 aGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 aGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 aGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 aGVHD.

The low dose GVHD prophylactic agent can be, for example, a target trough level of less than about 25ng/mL, less than about 20ng/mL, less than about 15ng/mL, less than about 12ng/mL, less than about 11ng/mL, less than about 10ng/mL, less than about 9ng/mL, less than about 8ng/mL, less than about 7ng/mL, less than about 6ng/mL, less than about 5ng/mL, less than about 4ng/mL, less than about 3ng/mL, less than about 2ng/mL, or less than about 1 ng/mL.

In some embodiments of the present invention, in some embodiments, the low dose GVHD preventive agent is about 1-25ng/mL, about 1-20ng/mL, about 1-15ng/mL, about 1-12ng/mL, about 1-11ng/mL, about 1-10ng/mL, about 1-9ng/mL, about 1-8ng/mL, about 1-7ng/mL, about 1-6ng/mL, about 1-5ng/mL, about 1-4ng/mL, about 1-3ng/mL, about 1-2ng/mL, about 2-25ng/mL, about 2-20ng/mL, about 2-15ng/mL, about 2-12ng/mL, about 2-11ng/mL, about 2-10ng/mL, about 2-9ng/mL, about 2-8ng/mL, about about 2-7ng/mL, about 2-6ng/mL, about 2-5ng/mL, about 2-4ng/mL, about 2-3ng/mL, about 3-25ng/mL, about 3-20ng/mL, about 3-15ng/mL, about 3-12ng/mL, about 3-11ng/mL, about 3-10ng/mL, about 3-9ng/mL, about 3-8ng/mL, about 3-7ng/mL, about 3-6ng/mL, about 3-5ng/mL, about 3-4ng/mL, about 4-25ng/mL, about 4-20ng/mL, about 4-15ng/mL, about 4-12ng/mL, about 4-11ng/mL, about 4-10ng/mL, about, about 4-9ng/mL, about 4-8ng/mL, about 4-7ng/mL, about 4-6ng/mL, about 4-5ng/mL, about 5-25ng/mL, about 5-20ng/mL, about 5-15ng/mL, about 5-12ng/mL, about 5-11ng/mL, about 5-10ng/mL, about 5-9ng/mL, about 5-8ng/mL, about 5-7ng/mL, about 5-6ng/mL, about 6-25g/mL, about 6-20ng/mL, about 6-15ng/mL, about 6-12ng/mL, about 6-11ng/mL, about 6-10ng/mL, about 6-9ng/mL, about 6-8 mL, about 6-7ng/mL, about 8-25ng/mL, about 8-20ng/mL, about 8-15ng/mL, about 8-12ng/mL, about 8-11ng/mL, about 8-10ng, about 8-10ng/mL, about 10 ng/mL.

In some embodiments, the low dose GVHD prophylaxis agent is tacrolimus at a target trough level of about 5ng/mL to about 10 ng/mL. In some embodiments, the low dose GVHD prophylaxis agent is tacrolimus at a target trough level of about 4ng/mL to about 6 ng/mL.

In some embodiments, the low dose GVHD prophylaxis agent is sirolimus having a target trough level of about 3ng/mL to about 8 ng/mL. In some embodiments, the low dose GVHD prophylaxis agent is sirolimus having a target trough level of about 4ng/mL to about 8 ng/mL.

The subject may be administered a low dose of a GVHD preventing agent (e.g., a low dose of a single GVHD preventing agent) for the first 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 200 days, 365 days, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years after the transplantation. In some embodiments, a low dose of a GVHD preventing agent (e.g., a low dose of a single GVHD preventing agent) is administered to a subject for less than about 20 days, less than about 30 days, less than about 40 days, less than about 50 days, less than about 60 days, less than about 70 days, less than about 80 days, less than about 90 days, less than about 100 days, less than about 110 days, less than about 120 days, less than about 150 days, less than about 200 days, less than about 365 days, less than about 13 months, less than about 14 months, less than about 15 months, less than about 16 months, less than about 17 months, less than about 18 months, less than about 19 months, less than about 20 months, less than about 21 months, less than about 22 months, less than about 23 months, less than about 2 years, less than about 2.5 years, less than about 3 years, less than about 3.5 years, less than about 4 years, less than about 4.5 years, or less than about 5 years after transplantation.

D. Incidence of chronic GVHD

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low incidence of cGVHD of ≡1, e.g., a lower incidence of cGVHD of ≡1 compared to subjects administered alternative compositions. In some embodiments, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of the subjects administered the compositions of the present disclosure develop ≡1 cGVHD, for example, within 365 days post-transplant, two years post-transplant, or within another suitable amount of time post-transplant as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low incidence of cGVHD of ≡2, e.g., a lower incidence of cGVHD of ≡2 compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of another suitable amount of hd as disclosed herein within 365 days, two years or more after transplantation, or another suitable amount of cGV 2 in a subject to whom the compositions of the present disclosure are administered.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure develop ≡2 grade cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure develop ≡2 cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure develop ≡2 grade cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure develop ≡2 grade cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop ≡2 grade cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure develop ≡2 grade cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low incidence of cGVHD of ≡3, e.g., a lower incidence of cGVHD of ≡3 compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of another suitable amount of hd as disclosed herein within 365 days, two years or more after transplantation, or another suitable amount of cGV 3 in a subject to whom the compositions of the present disclosure are administered.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure develop ≡3 grade cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low incidence of cGVHD of ≡4, e.g., a lower incidence of cGVHD of ≡4 compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, or less than about 30% of the subjects administered the compositions of the present disclosure develop ≡4 grade cGVHD, for example, within 365 days after transplantation, within two years after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure develop ≡4 grade cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure develop ≡4 grade cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure develop ≡4 grade cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop ≡4 grade cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure develop ≡4 grade cGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure develop ≡4 grade cGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure develop ≡4 grade cGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure develop ≡4 grade cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit low incidence of mild to severe cGVHD, e.g., lower incidence of mild to severe cGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of subjects administered the compositions of the present disclosure develop mild to severe cGVHD, for example, within 365 days post-transplant, two years post-transplant, or within another suitable amount of time post-transplant as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit low incidence of moderate to severe cGVHD, e.g., lower incidence of moderate to severe cGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the appropriate amount of hd in a subject to another suitable degree of gv disclosed herein within 365 days, two years after transplantation, or within another time after transplantation.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit low incidence of severe cGVHD, e.g., lower incidence of severe cGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of another suitable amount of hd as disclosed herein within 365 days, two years after transplantation, or within another suitable amount of gv.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure develop severe cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure develop severe cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure develop severe cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure develop severe cGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure develop severe cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop severe cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure develop severe cGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure develop severe cGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure develop severe cGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure develop severe cGVHD.

The incidence of cGVHD can be assessed after implantation for a suitable amount of time, for example, about 150 days, about 200 days, about 365 days, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, or about 5 years after implantation.

The incidence of cGVHD may be calculated based on a population of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 subjects.

4. GVHD-free prophylaxis

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) in the absence of a GVHD prophylaxis agent exhibit a low incidence of cGVHD grade 1 or greater, e.g., a lower incidence of cGVHD grade 1 or greater than the subjects administered the alternative composition. In some embodiments, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD preventing agent develop ≡grade 1 cGVHD, for example, within 365 days post-transplant, two years post-transplant, or within another suitable amount of time post-transplant as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) in the absence of a GVHD prophylaxis agent exhibit a low incidence of ≡2 grade cGVHD, e.g., a lower incidence of ≡2 grade cGVHD compared to subjects administered the alternative composition. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the suitable amounts of GVHD after, for example, within two days after transplantation, or within another suitable amount of cHD of the present disclosure in a subject administered the composition of the present disclosure in the absence of GVHD preventive agent.

In some embodiments, less than about 50% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 cGVHD. In some embodiments, less than about 40% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 cGVHD. In some embodiments, less than about 30% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 cGVHD. In some embodiments, less than about 25% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 cGVHD. In some embodiments, less than about 20% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 cGVHD. In some embodiments, less than about 10% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 2 cGVHD. In some embodiments, less than about 5% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop ≡2 grade cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) in the absence of a GVHD prophylaxis agent exhibit a low incidence of ≡3 grade cGVHD, e.g., a lower incidence of ≡3 grade cGVHD compared to subjects administered the alternative composition. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the suitable amounts of GVHD in a subject to be transplanted after another day or after another period of time of development of GVHD as disclosed herein after transplantation, e.g., within 3 years.

In some embodiments, less than about 50% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 cGVHD. In some embodiments, less than about 40% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 cGVHD. In some embodiments, less than about 30% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 cGVHD. In some embodiments, less than about 20% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 cGVHD. In some embodiments, less than about 10% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 cGVHD. In some embodiments, less than about 5% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop ≡3 grade cGVHD. In some embodiments, less than about 3% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop ≡3 grade cGVHD. In some embodiments, less than about 2% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 cGVHD. In some embodiments, less than about 1% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 3 cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) in the absence of a GVHD prophylaxis agent exhibit a low incidence of cGVHD of ≡4, e.g., a lower incidence of cGVHD of ≡4 compared to subjects administered the alternative composition. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, or less than about 50% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD preventing agent develop ≡grade 4 cGVHD, for example, within 365 days post-transplant, two years post-transplant, or within another suitable amount of time post-transplant as disclosed herein.

In some embodiments, less than about 30% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 cGVHD. In some embodiments, less than about 20% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 cGVHD. In some embodiments, less than about 15% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 cGVHD. In some embodiments, less than about 10% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 cGVHD. In some embodiments, less than about 5% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 cGVHD. In some embodiments, less than about 3% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 cGVHD. In some embodiments, less than about 2% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 cGVHD. In some embodiments, less than about 1% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylactic agent develop grade 4 cGVHD.

Subjects administered a composition of the present disclosure (e.g., a cell population as described herein) in the absence of a GVHD prophylaxis agent exhibit low incidence of mild to severe cGVHD, e.g., lower incidence of mild to severe cGVHD compared to subjects administered an alternative composition. In some embodiments, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of subjects to whom the compositions of the present disclosure are administered in the absence of a GVHD prophylaxis agent develop mild to severe cGVHD, for example, within 365 days post-transplant, two years post-transplant, or within another suitable amount of time post-transplant as disclosed herein.

Subjects administered a composition of the present disclosure (e.g., a cell population as described herein) in the absence of a GVHD prophylaxis agent exhibit low incidence of moderate to severe cGVHD, e.g., lower incidence of moderate to severe cGVHD, as compared to subjects administered an alternative composition. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the total number of the appropriate amount of GVHD in a subject's post-transplant, e.g., within two days or another suitable time period of development of post-graft as disclosed herein after a further cycle of hd in a subject who has been reached in the absence of a composition of the present.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 25% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure develop moderate to severe cGVHD in the absence of a GVHD prophylaxis agent.

Subjects administered a composition of the present disclosure (e.g., a cell population as described herein) in the absence of a GVHD prophylactic agent exhibit a low incidence of severe cGVHD, e.g., a lower incidence of severe cGVHD compared to subjects administered an alternative composition. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the appropriate amount of the following, for example, within 365% of a further day, or another suitable amount of hd after a further day after a transplant has occurred in a subject who has developed a composition of the present disclosure in the absence of a GVHD.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure develop severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure develop severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure develop severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure develop severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure develop severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure develop severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure develop severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure develop severe cGVHD in the absence of a GVHD prophylaxis agent. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure develop severe cGVHD in the absence of a GVHD prophylaxis agent.

The absence of a GVHD prophylactic agent may refer to the situation where no GVHD prophylactic agent is administered to the subject within the first 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 200 days, 365 days, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years after the transplantation.

5. Single agent GVHD prevention

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit a low incidence of ≡1 grade cGVHD, e.g., a lower incidence of ≡1 grade cGVHD than subjects administered alternative compositions. In some embodiments, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop ≡1, for example, within 365 days post-transplant, two years post-transplant, or another suitable amount of time post-transplant as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit a low incidence of ≡2 grade cGVHD, e.g., a lower incidence of ≡2 grade cGVHD compared to subjects administered the alternative compositions. In some embodiments, the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) are administered to a subject in less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, less than about 50% or less than about 50% of the amount of GVHD after another suitable amount of post-graft in two days or after a further cycle of 2.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop grade 2 cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit a low incidence of ≡3 grade cGVHD, e.g., a lower incidence of ≡3 grade cGVHD compared to subjects administered the alternative compositions. In some embodiments, the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) are administered to a subject in less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, less than about 50% or less than about 50% of the amount of GVHD after another suitable amount of post-graft in two days or after a suitable post-graft stage of GVHD is disclosed herein.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡3 grade cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit a low incidence of ≡4 grade cGVHD, e.g., a lower incidence of ≡4 grade cGVHD compared to subjects administered the alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, or less than about 30% of the subjects to whom the compositions of the present disclosure are administered (e.g., single GVHD preventative agent) develop ≡grade 4 cGVHD, e.g., within 365 days after transplantation, two years after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop ≡4 cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡4 cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡4 cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop ≡4 cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop ≡4 cGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡4 cGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop ≡4 cGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure and no more than one GVHD prophylactic agent (e.g., a single GVHD prophylactic agent) develop ≡4 cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit low incidence of mild to severe cGVHD, e.g., lower incidence of mild to severe cGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop mild to severe cGVHD, e.g., within 365 days after transplantation, two years after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit low incidence of moderate to severe cGVHD, e.g., lower incidence of moderate to severe cGVHD, as compared to subjects administered alternative compositions. In some embodiments, the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) are administered to a subject in less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the total after another suitable amount of GVHD after a graft is disclosed herein in two days.

In some embodiments, less than about 50% of subjects administered a composition of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop moderate to severe cGVHD. In some embodiments, less than about 40% of subjects administered a composition of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop moderate to severe cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop moderate to severe cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop moderate to severe cGVHD. In some embodiments, less than about 10% of subjects administered a composition of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop moderate to severe cGVHD. In some embodiments, less than about 5% of subjects administered a composition of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop moderate to severe cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and no more than one GVHD prophylaxis agent (e.g., a single GVHD prophylaxis agent) exhibit a low incidence of severe cGVHD, e.g., a lower incidence of severe cGVHD, as compared to subjects administered alternative compositions. In some embodiments, the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) are administered to a subject in less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, or less than about 50% of the total after another suitable amount of hd after two days of post-transplant.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD. In some embodiments, less than about 5% of subjects administered a composition of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD. In some embodiments, less than about 3% of subjects administered a composition of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD. In some embodiments, less than about 2% of subjects administered a composition of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD. In some embodiments, less than about 1% of subjects administered a composition of the present disclosure and no more than one GVHD preventing agent (e.g., a single GVHD preventing agent) develop severe cGVHD.

The single GVHD prophylaxis agent may be tacrolimus.

The single GVHD prophylaxis agent may be sirolimus.

6. Low dose GVHD prevention

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of ≡1 grade cGVHD, e.g., lower incidence of ≡1 grade cGVHD than subjects administered alternative compositions. In some embodiments, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of the subjects administered the compositions of the present disclosure and low dose GVHD prophylaxis agents (e.g., low dose single GVHD prophylaxis agents) develop ≡grade 1 cGVHD, for example, within 365 days post-transplant, two years post-transplant, or within another suitable amount of time post-transplant as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of ≡2 grade cGVHD, e.g., lower incidence of ≡2 grade cGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, or less than about 50% of the amount of post-transplant after another suitable amount of GVHD is disclosed herein after a further cycle of 2% or more after a suitable period of time of development of a period of, such as, for example, in a second or more than about 2% of a post-day.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 cGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡2 cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of ≡3 grade cGVHD, e.g., lower incidence of ≡3 grade cGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, or less than about 50% of the amount of post-transplant after another suitable amount of GVHD is disclosed herein within, for example, within three days or more than about 365% of another suitable post-cycle after the period of transplantation.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡3 grade cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit a low incidence of cGVHD grade No. 4, e.g., a lower incidence of cGVHD grade No. 4 compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29% or less than about 30% of the subjects administered the compositions of the present disclosure (e.g., low dose single GVHD preventative agent) develop ≡4 grade cGVHD, e.g., within 365 days after transplantation, within two years after transplantation, or within another suitable amount of time after transplantation as disclosed herein.

In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 cGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 cGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 cGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop ≡4 cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of mild to severe cGVHD, e.g., lower incidence of mild to severe cGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of the subjects administered the compositions of the present disclosure and low dose GVHD prophylaxis agents (e.g., low dose single GVHD prophylaxis agents) develop mild to severe cGVHD, for example, within 365 days post-transplant, two years post-transplant, or within another suitable amount of time post-transplant as disclosed herein.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of moderate to severe cGVHD, e.g., lower incidence of moderate to severe cGVHD, as compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, or less than about 50% of the total amount of post-heat transfer in another suitable post-transplant period, such as disclosed herein after a further cycle of time of two days.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop moderate to severe cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop moderate to severe cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop moderate to severe cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop moderate to severe cGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop moderate to severe cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop moderate to severe cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop moderate to severe cGVHD.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) exhibit low incidence of severe cGVHD, e.g., lower incidence of severe cGVHD compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, or less than about 50% of the amount of post-severe after another, e.g., within 365% of a suitable amount of post-day after a graft, or another amount of post-transplant period of time as disclosed herein in a subject to receive a graft.

In some embodiments, less than about 50% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD. In some embodiments, less than about 40% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD. In some embodiments, less than about 30% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD. In some embodiments, less than about 20% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD. In some embodiments, less than about 15% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD. In some embodiments, less than about 10% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD. In some embodiments, less than about 5% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD. In some embodiments, less than about 3% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD. In some embodiments, less than about 2% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD. In some embodiments, less than about 1% of subjects administered the compositions of the present disclosure and low doses of GVHD prophylaxis agents (e.g., low doses of single GVHD prophylaxis agents) develop severe cGVHD.

E. Organ-specific GVHD stage morbidity

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low incidence of ≡1 phase GVHD indications of skin, liver, gut, or a combination thereof, e.g., lower incidence compared to subjects administered alternative compositions. In some embodiments, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, or less than about 85% of subjects administered the compositions of the present disclosure exhibit an ≡1 GVHD indication of skin, liver, intestinal tract.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low incidence of ≡2 GVHD indications of skin, liver, gut, or a combination thereof, e.g., lower incidence compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, or less than about 50% of the subjects administered the compositions of the present disclosure exhibit an or equal to 1% GVHD of the GVHD phase of the skin, liver, intestinal tract, or a combination thereof.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low incidence of ≡3 phase GVHD indications of skin, liver, gut, or a combination thereof, e.g., lower incidence compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49% or less than about 50% of the subjects administered the compositions of the present disclosure exhibit GVHD of 3.

Subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low incidence of ≡4 GVHD indications of skin, liver, gut, or a combination thereof, e.g., lower incidence compared to subjects administered alternative compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 21%, less than about 22%, less than about 23%, less than about 24%, less than about 25%, less than about 26%, less than about 27%, less than about 28%, less than about 29%, less than about 30%, less than about 31%, less than about 32%, less than about 33%, less than about 34%, less than about 35%, less than about 36%, less than about 37%, less than about 38%, less than about 39%, less than about 40%, less than about 41%, less than about 42%, less than about 43%, less than about 44%, less than about 45%, less than about 46%, less than about 47%, less than about 48%, less than about 49%, or less than about 50% of the subjects administered the compositions of the present an indication of GVHD of the skin, liver, intestinal tract, or a combination thereof.

The organ specific GVHD indication may be assessed after implantation for a suitable amount of time, for example, about 20 days, about 21 days, about 25 days, about 28 days, about 30 days, about 35 days, about 40 days, about 42 days, about 45 days, about 49 days, about 50 days, about 55 days, about 56 days, about 60 days, about 63 days, about 65 days, about 70 days, about 75 days, about 77 days, about 80 days, about 84 days, about 85 days, about 90 days, about 91 days, about 95 days, about 98 days, about 100 days, about 105 days, about 110 days, about 112 days, about 115 days, about 119 days, about 120 days, about 150 days, about 200 days, about 365 days, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years or about 5 years.

The incidence of an organ-specific GVHD indication may be calculated based on a population of, for example, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 subjects.

In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the subjects have a CD4: CD 8T cell ratio of ≡1.5 when assessed after a suitable amount of time after transplantation, e.g., about 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, or 100 days after transplantation.

V. other clinical results

A. Survival rate, hospitalization and recurrence

In some embodiments, a patient treated with or provided with a composition, multicomponent drug treatment, cell population, solution, formulation, kit, and/or method has a reduced risk of at least one of malignancy recurrence, infection, or renal failure.

The population of subjects treated with or provided with the compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods of the present disclosure exhibit high overall survival, e.g., higher overall survival than the subjects administered the replacement compositions. In some embodiments, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the subjects to whom the compositions of the present disclosure are administered remain viable after about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 7 years, about 10 years, about 15 years, about 20 years, about 30 years post-transplantation.

The population of subjects treated with or provided with the compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods of the present disclosure exhibit low treatment-related mortality, e.g., lower treatment-related mortality as compared to subjects administered the replacement compositions. In some embodiments, when assessed after a suitable amount of time after implantation, e.g., about 6 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, or about 5 years, the population of subjects administered the compositions of the present disclosure has a therapeutically relevant mortality rate of less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, or less than about 50%. In some embodiments, the treatment-related mortality of a population of subjects administered the compositions of the present disclosure is less than about 5% when assessed 1 year after transplantation. In some embodiments, the treatment-related mortality of the population of subjects administered the compositions of the present disclosure is less than about 1% when assessed 1 year after transplantation.

The population of subjects treated with or provided with the compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods of the present disclosure, e.g., cell populations comprising a population of cells described herein, exhibit GVHD-free and relapse-free survival (GRFS) rates, e.g., higher GRFS rates, as compared to subjects administered the replacement compositions. In some embodiments, GRFS may refer to recurrence-free or survival rate of grade ≡3aGVHD or broad (e.g., severe) cGVHD. In some embodiments, GRFS may refer to recurrence-free or survival rate of grade ≡2aGVHD or broad (e.g., moderate to severe) cGVHD. In some embodiments, GRFS may refer to survival without symptoms of GVHD. In some embodiments, the GRFS ratio of the population of subjects administered the compositions of the present disclosure is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% when assessed after a suitable amount of time after transplantation, e.g., about 6 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, or about 5 years after transplantation. In some embodiments, the GRFS ratio of the population of subjects administered the compositions of the present disclosure is at least about 60% when assessed 1 year after transplantation. In some embodiments, the GRFS ratio of the population of subjects administered the compositions of the present disclosure is at least about 75% when assessed 1 year after transplantation. In some embodiments, the GRFS rate of the population of subjects administered the compositions of the present disclosure is greater than 75% when assessed 3 years after transplantation. In some embodiments, the GRFS rate of the population of subjects administered the compositions of the present disclosure is greater than 75% when assessed 5 years after transplantation. In some embodiments, the GRFS rate of the population of subjects administered the compositions of the present disclosure is greater than 75% when assessed 7 years after implantation. In some embodiments, the GRFS rate of the population of subjects administered the compositions of the present disclosure is greater than 75% when assessed 10 years after transplantation.

The population of subjects treated with or provided with the compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods of the present disclosure exhibit short discharge times, e.g., shorter discharge times, than subjects administered the replacement compositions. In some embodiments, the average discharge time after day 0 of the transplant regimen is less than about 20 days, less than about 19 days, less than about 18 days, less than about 17 days, less than about 16 days, less than about 15 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, or less than about 8 days. In some embodiments, the average discharge time after day 0 of the transplant regimen is less than about 17 days. In some embodiments, the average discharge time after day 0 of the transplant regimen is less than about 18 days. In embodiments, a human subject treated with or provided with a composition, multicomponent drug treatment, cell population, solution, formulation, kit, and/or method as disclosed herein has no recurrence of its malignancy about one year after administration of the drug dosing regimen.

The population of subjects treated with or provided with the compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods of the present disclosure exhibit low recurrence rates, e.g., lower recurrence rates compared to subjects administered in lieu of the compositions, cell populations, solutions, formulations, kits, and/or methods of the present disclosure (e.g., cell populations comprising the population of cells described herein). In some embodiments, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, or less than about 60% of the subjects to whom the compositions of the present disclosure are administered relapse within a suitable amount of time after implantation, such as relapse within about 90 days, about 100 days, about 120 days, about 6 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, or about 5 years after implantation. In some embodiments, less than about 35% of the subjects administered the compositions of the present disclosure relapse within 1 year after transplantation. In some embodiments, less than about 25% of the subjects administered the compositions of the present disclosure relapse within 1 year after transplantation.

Populations of subjects without active disease when treated or provided with the compositions, multicomponent drug treatments, cell populations, solutions, formulations, kits, and/or methods of the present disclosure exhibit low recurrence rates, e.g., lower recurrence rates, compared to subjects administered the replacement compositions. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, or less than about 60% of the subjects without active disease when the compositions of the present disclosure are administered relapse within a suitable amount of time after implantation, such as relapse within about 90 days, about 100 days, about 120 days, about 6 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, or about 5 years after implantation.

In some cases, a patient treated with or provided with a composition, multicomponent drug treatment, cell population, solution, formulation, kit, and/or method has no recurrence of its malignancy about one year after administration of the cell population.

The population of subjects in complete remission when the compositions of the present disclosure (e.g., cell populations as described herein) are administered exhibit a low recurrence rate, e.g., a lower recurrence rate as compared to subjects administered the replacement composition. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, or less than about 40% of the subject in complete remission when administered the compositions of the present disclosure recur within a suitable amount of time after implantation, e.g., about 90 days, about 100 days, about 120 days, about 6 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, or about 5 years recur.

In some cases, the patient has no GHVD or no recurrence of its malignancy one year after administration of the cell population.

B. Implantation and immune reconstitution

7. Graft failure

The population of subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low primary graft failure rate, e.g., a lower primary graft failure rate, as compared to subjects administered the replacement composition. Primary graft failure may be failure to achieve an absolute neutrophil count of >500 cells/μl after day 30 post-implantation. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 20%, less than about 30%, or less than about 40% of the subjects administered the compositions of the present disclosure exhibit primary graft failure. In some embodiments, less than about 1% of the subjects administered the compositions of the present disclosure exhibit primary graft failure. In some embodiments, less than about 3% of the subjects administered the compositions of the present disclosure exhibit primary graft failure.

The population of subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a low secondary graft failure rate, e.g., a lower secondary graft failure rate as compared to subjects administered the replacement composition. Secondary graft failure may be a sustained loss of hematopoietic function following implantation. In some embodiments, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 20%, less than about 30%, or less than about 40% of subjects administered the compositions of the present disclosure exhibit secondary graft failure when assessed at a suitable amount of time after implantation, e.g., about 90 days, about 100 days, about 120 days, about 6 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, or about 5 years after implantation. In some embodiments, less than about 1% of the subjects administered the compositions of the present disclosure exhibit secondary graft failure within 1 year after transplantation. In some embodiments, less than about 5% of the subjects administered the compositions of the present disclosure exhibit secondary graft failure within 1 year after transplantation.

8. Neutrophil implantation

The population of subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit rapid neutrophil engraftment, e.g., faster neutrophil engraftment as compared to subjects administered the replacement composition. Neutrophil implantation may be indicated by a sustained neutrophil count of >500 cells/μl in the peripheral blood of the recipient. In some embodiments, the population of subjects administered the compositions of the present disclosure achieves neutrophil engraftment to a median of about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 14 days, or about 15 days after transplantation. In some embodiments, the population of subjects administered the compositions of the present disclosure achieves neutrophil implantation to a median of 13 days post-implantation. In some embodiments, the population of subjects administered the compositions of the present disclosure achieves neutrophil implantation to a median value of 12 days post-implantation. In some embodiments, the population of subjects administered the compositions of the present disclosure achieves neutrophil implantation to a median value of 11 days post-implantation.

9. Platelet implantation

The population of subjects administered the compositions of the present disclosure (e.g., a population of cells comprising a population of cells described herein) exhibit rapid platelet implantation, e.g., faster platelet implantation as compared to subjects administered the replacement composition. Platelet implantation may be by platelet count in peripheral blood of the recipient without platelet infusion >20,000/mm ³ Indicated for 3 consecutive days. In some embodiments, the population of subjects administered the compositions of the present disclosure achieves platelet implantation to a median of about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 14 days, or about 15 days after implantation. In some embodiments, a population of subjects of a composition of the present disclosure is administered to post-transplant 1The median of 3 days achieved platelet implantation. In some embodiments, platelet implantation is achieved from a population of subjects administered a composition of the present disclosure to a median value of 12 days post-implantation. In some embodiments, platelet implantation is achieved from a population of subjects administered a composition of the present disclosure to a median value of 11 days post-implantation.

T cell implantation

The population of subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a high proportion of circulating tregs, e.g., a higher proportion of circulating tregs than the subjects administered the replacement composition. In some embodiments, when the subject is evaluated at a suitable amount of time after implantation, e.g., about 14, 15, 20, 21, 25, 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 65, 70, 75, 77, 80, 84, 85, 90, 91, 95, 98, 100, 110, 120, 130, 140, 150, 160, 170, or 180 days after implantation, at least about 5, at least about 7.5, at least about 10, at least about 12.5, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25% of the circulating cd4+ T cells are Treg. In some embodiments, when the subject is evaluated 28 days after transplantation, an average of at least about 15% of circulating cd4+ T cells are tregs.

The population of subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a normal CD4 to CD 8T cell ratio, e.g., a higher CD4 to CD 8T cell ratio as compared to subjects administered the replacement composition or normal healthy control subjects.

In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the subjects have a CD4: CD 8T cell ratio of ≡0.8 when assessed after a suitable amount of time after transplantation, e.g., about 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, or 100 days after transplantation.

In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the subjects have a CD4: CD 8T cell ratio of ≡1 when assessed after a suitable amount of time after transplantation, e.g., about 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, or 100 days after transplantation. In some embodiments, at least 50% of the subjects have a CD4 to CD 8T cell ratio of ≡1 when assessed 28 days post-transplantation.

In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the subjects have a CD4: CD 8T cell ratio of ≡1.2 when assessed after a suitable amount of time after transplantation, e.g., about 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, or 100 days after transplantation.

The population of subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a high proportion of donor-derived circulating T cells at an early point in time after transplantation, e.g., a higher proportion of donor-derived circulating T cells than the subjects administered the replacement composition.

In some embodiments, more than 50% of the circulating T cells are donor-derived after a suitable amount of time following transplantation, e.g., in at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed at about 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, or 100 days following transplantation.

In some embodiments, more than 60% of the circulating T cells are donor-derived after a suitable amount of time after transplantation, e.g., in at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed at about 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, or 100 days after transplantation.

In some embodiments, more than 70% of the circulating T cells are donor-derived after a suitable amount of time after transplantation, e.g., in at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed at about 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, or 100 days after transplantation.

In some embodiments, more than 80% of the circulating T cells are donor-derived after a suitable amount of time after transplantation, e.g., in at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed at about 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, or 100 days after transplantation.

In some embodiments, more than 90% of the circulating T cells are donor-derived after a suitable amount of time following transplantation, e.g., in at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed at about 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, or 100 days following transplantation.

In some embodiments, more than 50% of the circulating T cells are donor-derived in at least about 70% of subjects assessed 30 days post-transplantation. In some embodiments, more than 70% of the circulating T cells are donor-derived in at least about 60% of subjects assessed 30 days post-transplantation.

B cell implantation

The population of subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a high concentration of circulating B cells at an early point in time after transplantation, e.g., a higher concentration of circulating B cells as compared to subjects administered the replacement composition. Achieving a high concentration of circulating B cells at an early point in time after transplantation may be important for immunocompetence and may allow the vaccine to elicit a protective immune response at an earlier point in time after transplantation.

In some embodiments, more than about 50B cells/μl are present in the blood of at least about 50, at least about 55, at least about 60, at least about 65, at least about 75, at least about 80, at least about 85, at least about 90% or at least about 95% of the subject assessed after a suitable amount of time after transplantation, e.g., at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90% or at least about 95% of the subject is assessed after transplantation, e.g., at least about 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 65, 70, 75, 98, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133 or 140. In some embodiments, more than 50B cells/μl are present in the blood in at least 75% of the subjects assessed about 60 days post-transplantation.

In some embodiments, more than about 60B cells/μl are present in the blood of at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 85%, at least about 90%, or at least about 95% of the subject assessed after a suitable amount of time after transplantation, e.g., at least about 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 65, 70, 75, 77, 80, 84, 85, 90, 91, 95, 98, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation. In some embodiments, more than 60B cells/μl are present in the blood in at least 75% of the subjects assessed about 60 days post-transplantation.

In some embodiments, more than about 70B cells/μl are present in at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the blood of a subject assessed after a suitable amount of time after transplantation, e.g., after about 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days post-transplantation. In some embodiments, more than 70B cells/μl are present in the blood in at least 75% of the subjects assessed about 60 days post-transplantation.

In some embodiments, more than about 80B cells/μl are present in blood of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the subject assessed after a suitable amount of time after transplantation, e.g., after about 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days.

In some embodiments, more than about 90B cells/μl are present in at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% of the blood of a subject assessed after a suitable amount of time after transplantation, e.g., after about 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133 or 140 days after transplantation. In some embodiments, more than 90B cells/μl are present in the blood in at least 75% of the subjects assessed about 180 days post-transplantation.

In some embodiments, more than about 100B cells/μl are present in blood of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the subject assessed after a suitable amount of time after transplantation, e.g., after about 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days. In some embodiments, more than 100B cells/μl are present in the blood in at least 75% of the subjects assessed about 180 days post-transplantation.

In some embodiments, more than about 110B cells/μl are present in at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the blood of a subject assessed after a suitable amount of time after transplantation, e.g., after about 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation. In some embodiments, more than 110B cells/μl are present in the blood in at least 75% of the subjects assessed about 180 days post-transplantation.

The population of subjects administered the compositions of the present disclosure (e.g., cell populations as described herein) exhibit a high proportion of mature B cells at an early point in time after transplantation, e.g., a higher proportion of circulating B cells as mature B cells (e.g., igd+ and/or cd27+) than the subjects administered the replacement compositions. Achieving a high proportion of mature B cells at an early point in time after transplantation may be important for immunocompetence and may allow the vaccine to elicit a protective immune response at an early point in time after transplantation.

In some embodiments, at least about 50% of the circulating cd19+ B cells are igd+ in at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed after a suitable amount of time after transplantation, e.g., after about 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation.

In some embodiments, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the circulating cd19+ B cells are igd+ in at least about 60% of the subjects assessed after a suitable amount of time after transplantation, e.g., about 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 65, 70, 75, 77, 80, 84, 85, 90, 91, 95, 98, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation.

In some embodiments, at least about 70% of the circulating cd19+ B cells are igd+ in at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed after a suitable amount of time after transplantation, e.g., about 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation.

In some embodiments, at least about 80% of the circulating cd19+ B cells are igd+ in at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed after a suitable amount of time after transplantation, e.g., about 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 65, 70, 75, 77, 80, 84, 85, 90, 91, 95, 98, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation. In some embodiments, at least about 80% of the circulating cd19+ B cells are igd+ cells in at least about 75% of subjects assessed about 100 days post-transplantation.

In some embodiments, at least about 90% of the circulating cd19+ B cells are igd+ in at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed after a suitable amount of time after transplantation, e.g., about 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 65, 70, 75, 77, 80, 84, 85, 90, 91, 95, 98, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation. In some embodiments, at least about 90% of the circulating cd19+ B cells are igd+ cells in at least about 75% of subjects assessed about 100 days post-transplantation.

In some embodiments, at least about 25% of the circulating cd19+ B cells are cd27+ in a subject assessed after a suitable amount of time following transplantation, e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the subjects assessed at about 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days following transplantation.

In some embodiments, at least about 30% of the circulating cd19+ B cells in a subject assessed at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the circulating cd19+ B cells are cd27+ after a suitable amount of time after transplantation, e.g., about 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 65, 70, 75, 77, 80, 84, 85, 90, 91, 95, 98, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation. In some embodiments, at least about 30% of circulating cd19+ B cells are cd27+ in at least about 75% of subjects assessed 100 days post-transplantation.

In some embodiments, at least about 35% of the circulating cd19+ B cells in a subject assessed at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the circulating cd19+ B cells are cd27+ after a suitable amount of time after transplantation, e.g., about 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 65, 70, 75, 77, 80, 84, 85, 90, 91, 95, 98, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation. In some embodiments, at least about 35% of circulating cd19+ B cells are cd27+ in at least about 75% of subjects assessed 100 days post-transplantation.

In some embodiments, at least about 40% of the circulating cd19+ B cells are cd27+ in at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of subjects assessed after a suitable amount of time after transplantation, e.g., about 28 days, 30 days, 35 days, 40 days, 42 days, 45 days, 49 days, 50 days, 55 days, 56 days, 60 days, 63 days, 65 days, 70 days, 75 days, 77 days, 80 days, 84 days, 85 days, 90 days, 91 days, 95 days, 98 days, 100, 105, 110, 112, 115, 119, 120, 125, 126, 130, 133, or 140 days after transplantation. In some embodiments, at least about 40% of circulating cd19+ B cells are cd27+ in at least about 75% of subjects assessed 100 days post-transplantation.

The clinical outcome disclosed herein may be calculated based on a population of, for example, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 subjects.

VI kit

In another aspect, a kit comprising a solution is provided, the kit comprising a first container comprising a first cd45+ cell population, a second container comprising a solution comprising a second cd45+ cell population, and a third container comprising a solution comprising a cell population enriched for regulatory T cells (tregs). In this aspect, the solution comprising the first cd45+ cell population, the solution comprising the second cd45+ cell population, and the solution comprising the Treg-enriched cell population are as defined according to any multicomponent drug treatment or method disclosed herein. In various embodiments, the kit further comprises a fourth container comprising a GVHD preventing agent. In some cases, instructions for performing any of the methods disclosed herein are also included.

A further aspect provides a kit comprising: (a) One or more agents that sort cd34+ cells from the mobilized peripheral blood composition; (b) One or more agents that sort regulatory T cells (tregs) from the mobilized peripheral blood composition; (c) One or more reagents for detecting the number of cd3+ conventional T cells in mobilized peripheral blood; and (d) a solution comprising one or more doses of a Graft Versus Host Disease (GVHD) prophylaxis agent. In some embodiments, the kit further comprises instructions for performing any of the methods disclosed herein.

Various embodiments provide a kit comprising one or more reagents for sorting HSPCs, e.g., the kit may comprise reagents for enriching a cd34+ cell population from a mobilized peripheral blood donation.

Some embodiments provide a kit comprising one or more reagents for sorting tregs, e.g., the kit may comprise reagents that enrich a population of Treg cells (using markers as described elsewhere herein) from a mobilized peripheral blood donation.

Embodiments provide a kit comprising one or more conditioning agents for a conditioning regimen, e.g., the kit may comprise an agent for bone marrow ablation or bone marrow reduction of a subject.

Some embodiments provide a kit comprising one or more GVHD prophylaxis agents.

In any of the methods disclosed herein, the method further comprises providing instructions for use (IFU) comprising instructions for administering the cell population to the patient. In some cases, the IFU further comprises instructions for administering one or more pharmaceutical agents or compositions to the patient.

Definition of the definition

The terminology used herein is for the purpose of describing particular situations only and is not intended to be limiting.

As used herein, unless otherwise indicated, the terms "a," "an," and "the" are intended to include both the plural and singular forms unless the context clearly dictates otherwise.

The terms "comprise," comprising, "" contains, "" containing, "" including, "" containing, "" having, "" with "(or variants thereof) as used in the specification and/or claims are intended to be inclusive in a manner similar to the term" comprising.

The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. For example, "about" may mean greater or less than 10% of the stated value. In another example, "about" may mean within 1 or more than 1 standard deviation depending on the practice of the given value. In the event that a particular value is described in the disclosure and claims, the term "about" should be assumed to mean an acceptable error range for that particular value, unless otherwise specified.

The term "substantially" in most cases means to a substantial extent; or essentially. In other words, the term substantially may mean almost exactly to the desired property or slightly different from the exact property. May be substantially indistinguishable from the desired properties. Can be substantially distinguished from the desired properties, but the differences are not important or negligible.

The term "one or more" or "at least one" means at least one, e.g., one, two, three, four, five, six, seven, eight, nine, ten or more.

The term "similar" should be understood to be similar to and include the same as up to and including the same. Thus, two (or more) items may be identical, substantially identical, include equivalent components, include alternative components, include similar components, include comparable components, include complementary components, include related components, include similar components, and/or the differences between the two (or more) items are not substantial and/or result in compositions having equivalent properties, identical properties, substantially identical properties, etc.

The terms "patient" and "subject" are synonymous.

Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein.

Additional aspects

Aspects provide a method of transplanting a population of conventional T cells (Tcon) into a human subject without eliciting a phase 2 or higher Graft Versus Host Disease (GVHD) response up to about 100 days after the transplantation. The method comprises (i) administering a heterogeneous population of cells comprising lymphocytes, granulocytes and monocytes, wherein at least about 30% of said lymphocytes comprise conventional T cells (Tcon); and (ii) administering a population of regulatory T cells (tregs). In this method, the heterologous cell fraction and/or Treg population comprises less than about 5EU/ml endotoxin.

A further aspect provides a method of treating a human subject, the method comprising the step (a) of administering a plurality of cell populations, wherein the plurality of cell populations comprises: (i) a population of Hematopoietic Stem and Progenitor Cells (HSPCs); (ii) a population of cells comprising regulatory T cells (tregs); and (iii) a population of conventional T cells (Tcon); and administering no more than one Graft Versus Host Disease (GVHD) prophylactic agent for less than about 120 days. In this method, the HSPC population comprises less than about 2% cd3+ cells.

One aspect is a method of treating a human subject in need thereof, the method comprising administering to the human subject at least two pharmaceutical compositions, wherein the pharmaceutical compositions are selected from the group consisting of (a) a pharmaceutical composition comprising a population of hematopoietic stem cells and progenitor cells (HSPCs); (b) A pharmaceutical composition comprising a population of regulatory T cells (tregs); and (c) a pharmaceutical composition comprising a population of conventional T cells (Tcon). In this method, each of pharmaceutical compositions (a), (b) and (c) comprises less than about 5EU/ml endotoxin; and less than 15 human subjects in a group of 100 human subjects administered two or more pharmaceutical compositions develop a stage 2 or higher Graft Versus Host Disease (GVHD) response within about 30 days after administration of the pharmaceutical composition comprising the Tcon population.

An additional aspect is a method of transplanting a population of conventional T cells (Tcon) into a human subject without eliciting a phase 2 or higher Graft Versus Host Disease (GVHD) response up to about 100 days after the transplantation. The method comprises the following steps: (i) administering a solution comprising a population of conventional T cells (Tcon); and (ii) administering a solution comprising a population of regulatory T cells (tregs). In this method, the Tcon population 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 5EU endotoxin/ml solution.

In another aspect, there is provided a method of treating hematological malignancy in a human subject in need thereof, the method comprising administering to the human subject: (a) a population of Hematopoietic Stem and Progenitor Cells (HSPCs); (b) a population of regulatory T cells (tregs); and (c) a population of conventional T cells (Tcon). In this method, the HSPC population and Treg population are administered prior to the Tcon population; and the peripheral blood of the human subject showed an elevated Treg count as compared to a healthy human subject not administered the three cell populations until about 100 days after administration of the three cell populations.

VIII. Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Example 1: clinical study

C. Study design

Clinical studies were performed in subjects with advanced hematological malignancies undergoing myeloablative allogeneic hematopoietic cell transplantation (alloHCT).

The composition and manufacturing process of the implant are detailed below.

The primary endpoints of this study included the incidence of primary graft failure; and incidence, severity and timing of grade III-V acute GVHD.

Secondary endpoints for all groups included neutrophil implantation, platelet implantation, incidence of secondary graft failure, incidence and severity of treatment-induced adverse events (TEAE), incidence and severity of steroid refractory acute GVHD (e.g., grade 3-4 steroid refractory acute GVHD), incidence and severity of chronic GVHD, incidence of post-transplant lymphoproliferative disorder (PTLD), non-recurrent mortality (NRM), disease recurrence (groups I and III), no recurrence survival), GVHD and no recurrence survival (GRFS), total survival, incidence of severe infection, and T cell immune reconstruction parameters.

D. Study population

Objects are eligible to receive the compositions of the present disclosure if they meet all of the following criteria:

(1) The recruitment time age is greater than or equal to 18 years and less than or equal to 65 years.

(2) Is diagnosed with any one of the following histopathologically confirmed diseases: (a) Acute myeloid leukemia, acute lymphoid leukemia or mixed phenotype leukemia in Complete Remission (CR) or CR with incomplete hematological recovery (CRi), and no known minimal residual disease; or (b) acute myeloid leukemia, acute lymphoid leukemia, or mixed phenotype leukemia, which is: (i) Not in morphological CR, wherein bone marrow infiltration by leukemia blasts is less than or equal to 10%, or (ii) in morphological CR, wherein residual positives are minimal by multiparameter flow cytometry analysis or by nucleic acid-based techniques; (c) High or very high risk myelodysplastic syndrome; (d) Myelofibrosis (MF) that meets the transplantation qualification of the national integrated cancer network guidelines. In particular, the patient should be diagnosed with MF, which is: (i) Medium-2 or high risk according to IPSS, DIPSS, or DIPSS-plus scoring systems; or (ii) moderate-1-risk disease associated with high risk features such as high symptomatic burden, low platelet count, or complex cytogenetics, according to NCCN guidelines and the discretion of the researcher; (e) myeloproliferative syndrome; (f) Non-hodgkin lymphomas with poor risk characteristics that are not suitable for autologous HCT.

(3) The subject is planned to undergo myeloablative allogeneic hematopoietic cell transplantation (MA-alloHCT), including a suitable myeloablative conditioning regimen.

(4) Matching with the following donors: (a) Matched sibling donors for 8/8 matches for HLA-A, -B, -C and-DRB 1 (all typed using DNA-based high resolution methods); or (B) matched unrelated donors that match 8/8 at HLA-A, -B, -C and-DRB 1 (all typed using DNA-based high resolution methods).

(5) Estimated glomerular filtration rate (effr) >30 mL/min; or >50 mL/min for patients scheduled to prevent GVHD with tacrolimus.

(6) Resting heart ejection fraction greater than or equal to 45% or shortening fraction greater than or equal to 27% as measured by echocardiography or radionuclide scan (MUGA).

(7) The carbon monoxide Dispersion (DLCO) of the lung (regulated for hemoglobin) is more than or equal to 50 percent.

(8) Negative serum or urine beta-HCG test for women with fertility within 3 weeks after enrollment.

(9) Total bilirubin < 2 times the Upper Limit of Normal (ULN) (where hemolysis has been excluded and approved by a medical supervisor, patients with gilbert syndrome may be included).

(10) ALT/AST < 3 times the upper limit of normal value (ULN).

Subjects were not eligible to receive the compositions of the present disclosure if they met any of the following exclusion criteria:

(1) The previous heterologous HCT was accepted.

(2) Candidates for autograft.

(3) Corticosteroids or other immunosuppressive therapies are currently being accepted. A local corticosteroid or oral systemic corticosteroid dosage of less than or equal to 10 mg/day is allowed.

(4) Planned Donor Lymphocyte Infusions (DLI) recipients.

(5) In vivo or ex vivo T cells deplete planned recipients of drugs, such as cyclophosphamide (Cy) after transplantation, pre-transplantation anti-thymocyte globulin (ATG) or alemtuzumab. For patients who have been previously exposed to T cell depleting agents, 5 half-life elution of the agent must be performed before the planned day 0 (day of infusion of Treg and HSPC components of the graft).

(6) Anti-donor HLA antibodies against mismatched alleles in selected donors are positive, as determined by any of the following: (a) a positive cross-match test of any titer; or (b) the presence of anti-donor HLA antibodies directed against any HLA locus.

(7) Karnofsky performance score <70%.

(8) Hematopoietic cell transplantation specific co-disease index (HCT-CI) >4.

(9) Uncontrolled bacterial, viral or fungal infection at enrollment (currently undergoing antimicrobial therapy with or without clinical improvement).

(10) Is seropositive for HIV-1 or-2, HTLV-1 or-2, hepatitis B surface antigen or hepatitis C antibody.

(11) It is known to have allergic or hypersensitivity reactions or intolerance to tacrolimus (or tacrolimus and sirolimus if administered in combination with either of the single agents).

(12) Recorded allergic or hypersensitivity reactions to iron dextran or bovine, murine, algal or streptavidin proteins.

(13) Any uncontrolled autoimmune disease requiring active immunosuppressive therapy.

(14) Malignant tumors or active diseases are complicated within 1 year, except for non-melanoma skin cancers that have been healed resected.

(15) Psychological and social situations that prevent patients from performing transplantation or from being responsible for participating in follow-up care.

(16) Pregnant or lactating women.

(17) Women with fertility (WOCBP) or men who are in sexual contact with WOCBP but do not wish to adopt an effective form of birth control or desire control within one year after implantation.

(18) Any serious medical condition or abnormality in clinical laboratory tests, at the discretion of the researcher or medical supervisor, prevents safe participation of the recipient and completion of the study, or may affect compliance with the regimen or interpretation of the results.

The objects are parts of the following groups:

group 1: a subject intended to undergo myeloablative allogeneic hematopoietic cell transplantation (MA-alloHCT) for treatment of acute myeloid leukemia, acute lymphoid leukemia, or mixed phenotype leukemia at Complete Remission (CR) or CR (CRi) with incomplete hematologic recovery intended to receive MA-alloHCT and no known minimal residual disease positive.

Group 2: a subject intended to experience MA-alloHCT of acute myeloid leukemia, acute lymphoid leukemia or mixed phenotype leukemia, which is: (i) Morphological CR which is not at < 10% of bone marrow infiltration by leukemic blast cells, or (ii) morphological CR which is at least residual positive as evidenced by multiparameter flow cytometry analysis or by nucleic acid-based techniques.

Group 3: subjects who are scheduled to experience high or very high risk myelodysplastic syndrome (MDS) myelodysplastic syndrome or myelofibrosis (primary myelofibrosis or myelofibrosis evolving from other myeloproliferative neoplasms).

Subjects sensitive to iron dextran, chemical products derived from cyanine dyes, and protein products derived from murine, bovine, algal, and streptavidin sources were excluded.

Figures 1A-B show schematic representations of transplants according to the present study (identified as high precision Orca-T or Orca T) and differences from standard of care (SOC) queues (identified as regular transplants or SOCs). Fig. 1C shows a schematic representation of graft generation and administration.

Figure 2A shows the body weight of the patient enrolled in the study. Table 7 shows demographic data for a representative subset of subjects, primary disease, characteristics determining disease risk status, disease status at the time of implantation, and implantation details. Abbreviations: DLBCL, diffuse large B-cell lymphoma; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; MDS, myelodysplastic syndrome; MF, myelofibrosis; MPAL, mixed phenotype acute leukemia; CML, chronic myelogenous leukemia; CR1, 1 st complete remission; CR2, 2 nd complete remission; active, active disease (not at CR as defined for the disease entity); mrd+, minimal residual disease positive; MRD, matched relevant donors; URD, unrelated donor; MF, myelofibrosis. FTBI, fractionated whole body irradiation; cy, cyclophosphamide; VP-16, etoposide; bu, busulfan; flt3+, FMS-like tyrosine kinase 3; c-kit, ASXL1, additional comb-like 1. The days of follow-up represent the number of days per patient from day 0 of transplantation at the time of reporting.

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Standard care control queue

To compare clinical results, a standard of care (SOC) control cohort was retrospectively determined from subjects receiving SOC myeloablative alloHCT at one of the same clinical sites during the same time period; also shown in fig. 1A-B are schematic diagrams of the protocol followed by the SOC patient. In order to be incorporated into an SOC queue, an object must meet all of the following criteria: (a) They were diagnosed as having hematological malignancy eligible to receive alloHCT treatment; (b) They received mobilized peripheral blood xenografts (i.e., grafts that were not bone marrow-derived); (c) Their donors are HLA-perfect matched relevant donors (unrelated donors are excluded due to the limited number of donors in treatment cohorts so far); (d) they received a myeloablative conditioning regimen; and (e) they were not included in the survey plan. The person determining the subject for the SOC queue takes a blind approach to his clinical outcome.

All patients in the SOC cohort received myeloablative conditioning regimen and dual agent GVHD prevention with tacrolimus and methotrexate.

Clinical characteristics of the subjects in the SOC queue are provided in table 8.

Table 8: features of a representative subset of subjects in a standard of care (SOC) cohort. Abbreviations used: DLBCL, diffuse large B-cell lymphoma; AML, acute myeloid leukemia; ALL, acute myelogenous leukemia; MDS, myelodysplastic syndrome; MF, myelofibrosis; MPAL, mixed phenotype acute leukemia; CML, chronic myelogenous leukemia; CR1, 1 st complete remission; CR2, 2 nd complete remission; "active disease (not at CR as defined for each disease entity); MRD, minimal residual disease; MRD, matched relevant donors; MF, myelofibrosis. FTBI, fractionated whole body irradiation; cy, cyclophosphamide; VP-16, etoposide; bu, busulfan; flt3+, FMS-like tyrosine kinase 3; tac, tacrolimus; MTX, methotrexate.

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E. Donor(s)

Either related donors or unrelated donors with the same HLA are used.

Donors meeting all of the following inclusion criteria were used:

(1) The registration age is more than or equal to 16 years old and less than or equal to 75 years old

(2) Matching to the patient is as follows: any one of the following: (i) Matched relevant donors, 8/8 matched for HLA-A, -B, -C and-DRB 1, all typed using DNA-based high resolution methods; (ii) Matched unrelated donors, which match 8/8 at HLA-A, -B, -C and-DRB 1, were all typed using a DNA-based high resolution method.

(3) Site donation to be able to be performed after mobilization with the Spectra Optia apheresis system;

(4) The federal qualification criteria for living, leukocyte-enriched cells or Tissues donors as defined by 21CFR 1271 2018 and all relevant FDA industry guidelines (Eligibility Determination for Donors of Human Cells, tissue, 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 (synphilis), 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;Donor Screening Recommendations to Reduce the Risk of Transmission of Zika Virus by Human Cells,Tissues,and Cellular and Tissue-Based Products, 2018).

(5) Meets any other donation standard as specified by standard NMDP guidelines (NMDP donors) or institutional standards (non-NMDP donors).

(6) Female donors with fertility must have a negative serum or urine βhcg test within 3 weeks of mobilization.

(7) Can perform a leukopenia, has a sufficient venous access, and is willing to perform an insertion of a central catheter if the leukopenia performed via the peripheral vein is insufficient.

According to 21CFR 1271.65 2018, based on the results of the required testing and/or screening, the donors determined to be failed (if any apply) are still included: (a) the donor is a primary or secondary blood relative of the recipient; or (b) urgent medical need, meaning that no comparable human cell product is available, and as demonstrated by researchers, the recipient may suffer from death or severe morbidity without human cell product.

Donors meeting any of the following exclusion criteria were unacceptable:

(1) Evidence of active infection

(2) Seropositive for HIV-1 or-2, HTLV-1 or-2

(3) Positive against Hepatitis C (HCV) antibodies or HCV NAT.

(4) Positive serological or PCR test results indicating acute or chronic HBV infection. Donors whose HBV infection status cannot be determined ultimately by serological test results must be negative by PCR to be eligible for study participation.

(5) The likelihood of a Zika virus infection, as defined by any one of the following: (i) Medical diagnosis of Zika virus infection in the past 6 months; (ii) Living or traveling in the past 6 months in the region where active Zika virus was transmitted; (iii) Unprotected behavior occurred with persons known to have any one of risk factors (i) or (ii) over the past 6 months. Donors determined to be failed based on the results of the Zika virus screening may be determined to be eligible if the following conditions are met: (a) The donor had no indication or symptoms consistent with active zika virus infection; and (b) the donor is a primary or secondary ancestor of the recipient, or ii) in the event of urgent medical need, meaning that no comparable human cell product is available, and as demonstrated by the researcher, the recipient may suffer from death or severe morbidity in the absence of the human cell product.

(6) Pregnant or lactating women.

F. Production of cellular components

The donor received daily mobilization therapy for G-CSF. The recommended dose is 10 μg/kg/day SQ (rounded to the nearest 300 or 480 μg vial size). The mobilization phase starts on the first day of G-CSF administration and continues until the last day of leukapheresis. A schematic of graft generation and administration of this protocol is provided in fig. 1C.

High volume apheresis begins on day 4 of G-CSF administration, which is typically day-3 relative to the infusion of CD34 (HSPC) and Treg enriched products (defined as day 0) into the subject. So that the selection is more than or equal to 3 multiplied by 10 ⁶ The individual CD34+ cells/kg receptor body weight was targeted to initiate apheresis. In order to have enough cells available for CD34 enrichment and Treg sorting procedures, the donor was harvested on consecutive 2 days (e.g., day-3 and day-2) by apheresis. Where possible, day 1 apheresis (day-3) harvest was scheduled in the afternoon and day 2 (day-2) was scheduled in the early morning, limiting the time from the end of the first harvest to the infusion of cell products to less than 72 hours. If the researcher and/or attending physician recommends the use of Plerixafor (e.g., 0.24mg/kg SC, once) to achieve HSPC dose objectives,it can be used prior to the second apheresis procedure.

The CD34 reduced (flow-through) fraction was retained and used to isolate donor tregs. For cell selection, clinical grade reagents are used under Good Manufacturing Practice (GMP) conditions in the BMT cell therapy institution.

Cd25+ cells were then selected from the CD34 depleted fractions using bead purification (Miltenyi). Tcon was obtained from the negative fraction and the positive fraction was used for Treg purification. Cd4+cd25+cd127dim cells were further selected by FACS using a BD influx cell sorter (BD Biosciences, san Jose CA). Tregs are enriched after cd34+ cell depletion by immunomagnetic selection, cd25+ selection by immunomagnetic selection and purification by FACS sorting of cd4+cd127lowcd25+ cells. Tregs of high purity are obtained. These cells are highly inhibited in Mixed Lymphocyte Reaction (MLR).

When lower than expected Treg dose levels are reached, for example, the final Treg yield is<2×10 ⁶ Weight of the recipient per kg, recipient 1-2×10 ⁶ A dose of Treg/kg (if this dose can be reached) and a reduced dose of Tcon is received such that the ratio of Treg to Tcon administered is 1:1.

G. Treatment of

Subject receiving the cellular components shown in Table 9

In embodiments, the term "HSPC" as transplantation, as graft, as cell dose, as product, as pharmaceutical product, as component of a graft or as component of a graft, etc., corresponds to a "first cd45+ cell composition" or the like as disclosed elsewhere in the present application (including the claims); the term "Treg" as transplantation, as graft, as cell dose, as product, as pharmaceutical product, as component of a graft or as component of a graft, etc., corresponds to "Treg-enriched cell composition" as disclosed elsewhere in the present application (including the claims), etc.; and the term "Tcon" as transplantation, as graft, as cell dose, as product, as pharmaceutical product, as component of a graft or as component of a graft, etc. corresponds to a "second cd45+ cell composition" as disclosed elsewhere in the present application (including the claims), etc.

Patients received Treg cells with a purity higher than 90%, with a median Treg purity of 93.8% +/-3.1%. Figures 2B-D show the cell dose of HSPC and Treg cells administered to a patient. Table 10 below shows an analysis of HSPC drug products (e.g., first cd45+ cell populations) from a representative subset of 20 subjects enrolled in the study. Table 11 below shows an analysis of Treg drug products (e.g., treg-enriched cell populations) from a representative subset of 20 subjects enrolled in the study. The cell fraction was provided in the form of single dose transfer bags, with a fill volume of about 100mL for both Treg and HSPC fractions. For each of tables 10-11, samples from 20 subjects from the multi-center clinical trial were analyzed. The mean and Standard Deviation (SD) are shown in each table. The average (SD) patient weight was 71.8kg (11.2 kg). The impurity cell dose was calculated by multiplying the% impurity per patient by the reported cell dose.

The Tcon fraction (e.g., second cd45+ cell population) was provided by freezing in a volume of about 15mL after storage in a gas phase liquid nitrogen tank. CD3+Tcon cells of the pooled apheresis products were evaluated and the volume of apheresis frozen product containing 3.0E+06Tcon was calculated and administered to the subject. Table 12 below shows an analysis of Tcon drug products from a representative subset of 20 subjects enrolled in the study. Analysis of Tcon drug product from 20 grafts (n=20) in a multicenter clinical trial. Mean and Standard Deviation (SD) are shown. The cell dose of the cell population was calculated based on the target CD3+ T cell dose of 3M/kg. Biomarker data (BM), manufacturing data (mfg); the average (SD) patient weight was 71.8kg (11.2 kg).

Each cell therapy product contained cells, plasma-Lyte a and human serum albumin, at a pH of about 7.4. The bag and/or primary bag container has a label with the appropriate label text as required by the regulatory authorities.

The amount of time from when the cell product was received by the donor to when it was administered to the recipient was less than 60 hours, as shown in table 13 for the subset of patients.

Dosing is rounded to the nearest 1/10 kg based on the actual body weight of the subject as assessed during screening, or if the actual body weight of the subject is greater than 120% of the Ideal Body Weight (IBW)Weight (ABW), calculated as ABW [ in kg ]]= [ (actual body weight-IBW) ×0.40]+IBW。

Table 14 shows endotoxin levels in each cell population for a subset of patients.

Table 10: HSPC product analysis

Table 11: treg product analysis

Table 12: tcon product analysis

Table 13: duration between collection and application

Table 14: endotoxin levels

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The recipient placed the appropriate long-term central venous access prior to initiating the conditioning regimen. Unless contraindicated, subjects were administered acetaminophen or paracetamol (e.g., 500-1000 mg) and diphenhydramine (e.g., 25-50 mg) prior to administration of each cell component.

Cd34+ HSPC cell fraction (e.g., first cd45+ cell population), followed by Treg cell fraction (e.g., treg-enriched cell population), was administered Intravenously (IV) via central venous catheter on study day 0.

The subject received a myeloablative conditioning (MA) regimen prior to administration of the cellular component. Examples of MA schemes are provided in table 15. Busulfan may also be administered for maintenance of a daily average AUC of 4,800-6,000. Mu.M-min.

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The subject receives tacrolimus or sirolimus as a single agent for GVHD prevention, starting the next day (typically day +3) after Tcon infusion, e.g., a second cd45+ cell population.

Tacrolimus begins at 0.03 mg/kg/day IV with a target trough blood level of 5-10ng/mL. Oral (PO) administration is allowed if the patient is able to tolerate food.

For subjects with MA with TBI/Cy/thiotepa, cy/TBI, TBI/etoposide/Cy (i.e. a regimen that does not include busulfan), sirolimus started as a single loading dose of 6mg PO for target blood levels of 3-8ng/mL, followed by 2mg daily.

GVHD prophylaxis can be reduced, for example, by a dose of about 20% per month, for subjects not showing an indication of grade 2 acute GVHD by day +60. For subjects exhibiting an indication of ≡2 grade acute GVHD, GVHD prevention may be gradually reduced after a suitable period of time (e.g., a suitable period of time after cessation of administration of any GVHD therapeutic agent, and no indication of ≡2 grade GVHD is observed) no indication of GVHD is observed.

H. Clinical results

The following paragraphs provide data and explanation relating to clinical results from the clinical studies described herein. As an introductory matter of the interpretation data, it should be appreciated that graft failure may be a complication of HCT disease and may be associated with significant mortality.

Neutrophil engraftment by day +28: neutrophil implantation was defined as reaching Absolute Neutrophil Count (ANC) 500/mm or more within 3 consecutive days to day +28 ³ . The first of the three days is designated as the date of implantation. If ANC never drops below 500/mm ³ Day +1 is designated as the date of implantation. Study group patients showed early neutrophils (median 11 days versus 14 days, by Mann-Whitney U, p<0.0001)。

Platelet implantation by day +50: platelet implantation was defined as continuous 3 days of platelet count by day +50 without platelet infusion for the previous 7 days>20,000/mm ³ . The first of the three days is designated as the date of implantation. If the platelet count never drops below 20,000/mm ³ Day +1 is designated as the date of implantation. Study group patients showed earlier platelet implantation (11 days versus 17 days, p <0.0001)。

Secondary graft failure by day +100: secondary graft failure was defined as neutrophil implantation by day +100, followed by a subsequent decrease in absolute neutrophil count <500 cells/μl, no response to growth factor therapy.

Failure to achieve an absolute neutrophil count of >500 cells/μl after day +30 can indicate primary graft failure. No subjects in the study group experienced primary graft failure.

Continued loss of hematopoietic function after implantation may indicate secondary graft failure. No subjects in the study group experienced secondary graft failure.

As part of the follow-up procedure, a chimeric analysis was also performed on peripheral blood samples from subjects. The blood samples are processed to select various cell populations using standard markers for each cell population. On day +28, day +56, day +100, day +180, and day +365 post-implantation, blood was drawn from subjects receiving the compositions of the present disclosure, and the frequency of peripheral blood cells was quantified by flow cytometry (markers for analysis are described in table 16).

Platelet count for 3 consecutive days without platelet infusion >20,000/mm ³ Platelet implantation may be indicated. As shown in fig. 3A, subjects in the study group showed faster platelet implantation than subjects in the SOC cohort, platelet implantation was achieved after a median of 11 days compared to 17 days in the SOC cohort (p<0.0001). Figure 3B shows platelet counts in the donor before and after mobilization and in the recipient patient before and after transplantation. Box line graph display: the bins show the 75 th, 50 th and 25 th percentiles; the bin whisker shows the 90 th and 10 th percentiles. X-axis naming: leading digits (e.g., 01, 02, 025 … …) are used for ordering; after underlining, dsrn=g-CSF healthy donor before mobilization, rsrn=acceptor within 1 month prior to conditioning, apher=healthy donor blood withdrawn at apheresis, d 028=acceptor on day 28 post-transplant, d056-d 365=acceptor on days post-transplant. N shown represents the sample amount at each time point. The symbols represent the individual measured values.

Sustained neutrophil counts up to >500 cells/μl may indicate neutrophil engraftment. Figure 3C shows platelet counts in the donor before and after mobilization and in the recipient patient before and after transplantation. The legend of the figure is similar to the legend described in fig. 3B.

Figures 3D-E show monocyte and lymphocyte implantation in a patient. Figures 3M and N show lymphocyte and monocyte counts for representative patients compared to representative SOC queues.

Fig. 3F shows B cell implantation in a recipient of a composition of the present disclosure, and mature B cells are present by day +100 post-implantation. The excellent engraftment and/or early functionality of transplanted B cells may represent significant advantages over standard care grafts, e.g., enhanced immunity and allowing post-transplantation vaccination.

FIG. 3G shows CD3+ T cell implantation; fig. 3H shows NK cell engraftment, fig. 3I shows cd4+ T cell engraftment, and fig. 3J shows cd8+ T cell engraftment in a recipient of the composition of the present disclosure. FIG. 3K shows the ratio of CD4 to CD 8T cells in the receptor.

Figure 3L provides a comparison of the proportion of cd3+cd4+ T cells as tregs in healthy donors versus in graft recipients several days post-implantation. These data indicate that the receptors of the compositions of the present disclosure exhibit high frequencies of circulating cd4+ tregs. Without wishing to be bound by theory, lower Treg frequencies may help prevent GVHD, reduce the risk of developing GVHD, reduce the incidence of GVHD, reduce the severity of GVHD, or a combination thereof.

Fig. 3O shows representative data from two subjects compared to healthy controls. In healthy controls, 3.72% of circulating cd3+cd4+ T cells are tregs (cd25+cd127 dim). Of the two graft recipients, 28.1% and 23.7% of cd3+cd4+ T cells were tregs at day +28, 32.3% and 17.8% of cd3+cd4+ T cells were tregs at day +56, and 19.2% and 20.7% of cd3+cd4+ T cells were tregs at day +100.

Blood was drawn from the graft recipient at day +100 post-implantation, and samples were processed for flow cytometry evaluation of various B cell markers, including CD19, CD20, igD, and CD 27. Figure 3P compares a scatter plot of graft recipients versus healthy controls. In all cases, the Y-axis represents cd19+ staining. The left panel shows the gating of lymphocytes to identify B cells (cd19+) and T cells (cd3+). 13.4% of lymphocytes in the graft recipient were B cells, while 9.84% of lymphocytes in the healthy control were B cells. The lower panel shows that 98.3-100% of cells gated to CD19+ are also CD20+. The second panel on the right shows the fraction of B cells as igd+ which can be used to identify mature B cells. 92.1% of B cells in the transplant recipients were IgD+, while 89.5% of B cells in the healthy controls were IgD+. The right-most panel shows staining of CD27, which can be used, for example, to identify memory B cells, late plasmablasts, and plasma cells. 43.6% of B cells in the transplant recipients were CD27+, while 67.1% of B cells in the healthy controls were CD27+.

These results indicate that B cells were implanted into the recipient of the composition of the present disclosure and that mature B cells were present by day +100 post-transplantation. The excellent engraftment and/or early functionality of transplanted B cells may represent significant advantages over standard care grafts, e.g., enhanced immunity and allowing post-transplantation vaccination.

GVHD assessment

Acute GVHD was staged and graded according to MAGIC normalization criteria.

Chronic GVHD was diagnosed, staged and graded according to international NIH chronic GVHD diagnostic and staging consensus working group criteria.

Clinically significant manifestations of both acute and chronic GVHD are first treated by local (local), local (topical), and/or systemic corticosteroids (e.g., prednisone). Any patient refractory or resistant, dependent or intolerant to corticosteroids should be considered to receive the second line therapy, as defined by the BMT-NIH-CIBMTR working group.

Adverse events occurring in Treatment (TEAE): using MedDRA version 21.0, TEAE is classified according to system organ category and preferred terminology and CTCAE version 5.0.

Acute GVHD: acute GVHD (aGVHD) is an important driver of morbidity and mortality associated with alloHCT, and reducing the severity and morbidity of aGVHD is likely to be greatly beneficial to the graft recipient. Acute GVHD was staged and graded according to the international association for acute GVHD of Kanesian (Mount Sinai Acute GvHD International Consortium) (MAGIC) standardization standard.

A subject is considered to be rated aGVHD if the subject develops symptoms of aGVHD before day +100 (day 100 post-transplant), or does not develop symptoms of aGVHD after day +100. Using these criteria, 17 patients could be assessed as aGVHD at the time of reporting.

On reporting, an AGVHD of 16% was observed at > 2. This is advantageous compared to published rates in similar populations and is lower than patients in the SOC cohort. The onset of grade 2 aGVHD is shown in FIG. 4A compared to the SOC queue. One subject developed aGVHD of the upper digestive (GI) tract, manifested as nausea and cachexia. Symptoms of the subject are relieved after short-term corticosteroid treatment, followed by follow-up.

Severe (grade 3-4) aGVHD is the leading cause of non-recurrent mortality after alloHCT and can be observed in 10-20% of patients after HLA-matched related donor transplantation. 5% of patients in the study group developed grade 3-4 aGVHD, while 20% of patients in the SOC cohort developed grade 3-4 aGVHD. The onset of ≡3 grade aGVHD by day +120 compared to SOC cohort is shown in fig. 4B.

As shown in table 7, four patients in the study group did not receive any GVHD prophylaxis. Of these patients, only one developed symptoms of aGVHD (cases of aGVHD in the upper GI tract as described above). The results reported thus far indicate that the compositions of the present disclosure can be safely administered to patients without GVHD prevention or with minimal prevention. For example, these strategies may significantly benefit patients due to increased Graft Versus Tumor (GVT), increased Graft Versus Infection (GVI), and reduced adverse effects (e.g., nephrotoxicity and hepatotoxicity) that may be associated with immunosuppressive agents.

These results demonstrate that the compositions of the present disclosure can reduce the incidence and severity of aGVHD in a subject, as compared to standard care, for example, even in the absence of a GVHD preventative agent or with reduced GVHD prevention.

Steroid refractory acute GVHD: steroid refractory acute GVHD is defined according to the EBMT-NIH-CIBMTR working group position statement.

Chronic GVHD: since chronic GVHD (cGVHD) is associated with significant morbidity and reduced survival, reducing the severity or incidence of cGVHD is likely to be greatly beneficial to the graft recipient. Chronic GVHD was diagnosed according to 2014 international NIH chronic GVHD diagnostic and staging consensus working group criteria.

At the time of reporting, no subjects in the study group developed moderate or severe cGVHD. One subject in the study group developed transient steroid responsive mild skin cGVHD.

Analysis was performed using an expiration date of 365 days (i.e., excluding cGVHD events occurring after day +365). As shown in fig. 4C, subjects in the study group experienced significantly fewer moderate or severe cGVHD events than patients in the SOC cohort (5% versus 43%, respectively; p < 0.0001). While receiving less or no prevention of GVHD, the data shows that the compositions of the present disclosure can significantly reduce the incidence of cGVHD compared to subjects in the SOC cohort. In addition, several subjects in the study group were followed for more than 2.5 years, with no symptoms of cGVHD reported.

Since cGVHD is associated with reduced overall survival and significant long-term morbidity, these results indicate that the compositions of the present disclosure can improve long-term survival and quality of life of recipient subjects compared to standard care.

Post-transplant lymphoproliferative disorder (PTLD): PTLD is defined as a biopsy conforming to the 2017 World Health Organization (WHO) PTLD classification (non-destructive [ plasma cell hyperplasia, infectious mononucleosis-like and floral follicular hyperplasia ], polymorphic, monomorphic or hodgkin's lymphoma-like), as well as a suitable staging procedure for lymphoma types, such as Computed Tomography (CT) with or without 18F-fluorodeoxyglucose positron emission tomography (FDG-PET).

Morbidity of non-recurrent mortality (NRM). NRM is defined as death with no sign of disease recurrence. Disease recurrence/progression is considered a competitive event.

Incidence of disease recurrence: for acute leukemia, recurrence is defined as any of the following (mrd+ alone is insufficient): (i) more than or equal to 5% of mother cells in bone marrow or peripheral blood; or (ii) the recurrence of cytogenetic abnormalities prior to transplantation; or (iii) new evidence or recurrence of an extramedullary disease. For MDS, recurrence is defined as any of the following: (i) meets criteria for evolving into acute leukemia; (ii) Reproduction of preimplantation morphological abnormalities detected in bone marrow samples; alternatively, (iii) in each of two independent consecutive examinations separated by at least one month, in at least one intermediate stage, the reproduction of cytogenetic abnormalities before transplantation, irrespective of the number of intermediate stages of analysis.

Treatment-related mortality includes death caused by complications or toxicity associated with therapy (such as infection, GVHD, or organ failure). At the time of reporting, the treatment-related mortality in the study group was 5% for one year after the transplantation, in contrast to 13% for the SOC cohort, as shown in fig. 4D. No subjects in the study group died from complications other than disease recurrence.

The survival and recurrence of the subject are monitored. To compare clinical results, a standard of care (SOC) control cohort of subjects receiving SOC myeloablative alloHCT from mobilized peripheral blood of HLA-matched donors at one of the same clinical sites during the same time period was retrospectively identified. All subjects in the SOC cohort received myeloablative conditioning regimen and dual agent GVHD prevention with tacrolimus and methotrexate.

Figures 4E-H show recurrence, GVHD and recurrence-free survival, chronic GVHD-free survival and overall survival of subjects as recipients of standard care grafts compared to subjects receiving grafts as described in this example. These data demonstrate that the compositions described herein increase the recurrence-free survival of subjects experiencing myeloablative alloHCT.

GVHD and recurrence-free survival are composite readings, which may refer to survival without recurrence or grade 3 acute or broadly chronic GVHD. At the time of reporting, the percentage of GVHD-free and relapse-free survival for one year after transplantation was 74% for the study group, compared to 34% for the SOC cohort, as shown in fig. 4F. This difference is statistically significant (p=0.0001 by log rank test).

Total lifetime: OS is defined as the time from the day of transplantation to the day of death due to any cause, or for surviving patients, to the day of the last follow-up. At the time of reporting, the percentage of total survival for one year after the transplantation was 90% for the study group, in contrast to 78% for the SOC cohort, as shown in fig. 4H. This difference is statistically significant (p=0.0242 by log rank test).

H. Hospitalization time

Standard of care for HCT may require long stay hospitalized. Compared to patients in the SOC cohort, the median time from implantation (day 0) to discharge was shortened by 2.5 days (from day 18.5 to day 16, by Mann-Whitney U test, p < 0.01). Fig. 4I shows hospitalization times for a representative subset of patients.

I. Disease condition following therapy

Subjects in the study were evaluated for first relapse on day +90 post-implantation. At the time of reporting, only 16% of patients relapsed, in contrast to 19% of patients in the SOC cohort.

Table 16: object status for a representative subset of objects on day +90 in the past. Abbreviations used: DLBCL, diffuse large B-cell lymphoma; AML, acute myeloid leukemia; ALL, acute myelogenous leukemia; MDS, myelodysplastic syndrome; MF, myelofibrosis; MPAL, mixed phenotype acute leukemia; CML, chronic myelogenous leukemia; CR: complete remission; CR1, 1 st complete remission; CR2, 2 nd complete remission; "active disease (not at CR as defined for a given disease entity).

The risk of recurrence may be associated with the disease state at the time of implantation. For example, if a subject is not fully relieved at the time of transplantation, the prognosis of AML or ALL subjects will be significantly worse (i.e., if the subject suffers from active leukemia or detectable minimal residual disease at the time of transplantation, the prognosis is worse). In another example, the prognosis of a subject with a detectable minimal residual disease is worse than a patient without a detectable minimal residual disease. Subjects with active disease or minimal residual disease may represent an unmet critical medical need.

Figure 5 summarizes the disease states of a subset of subjects in the study group prior to and at day +90, day +180, and day +356 post-implantation. Of the 10 subjects receiving the compositions of the present disclosure, 7 achieved durable remissions, showing graft anti-tumor effects. Two relapsing subjects had active disease at the time of transplantation and one subject had detectable Minimal Residual Disease (MRD) at the time of transplantation.

J. Characteristics of transplanted cells

A schematic of graft generation and administration for the sorting protocol is provided in fig. 1A. Evaluating the cell products from day 2 of apheresis collection and administering to the subject a portion of the apheresis product comprising a heterologous cell component (e.g., a second cd45+ cell population) consisting of 3 x 10 ⁶ Individual Tcon cells. After staining the various cell populations, a portion of the heterogeneous cell components were analyzed for different cell components, as described in table 17.

Table 18 below shows the data collected from a few patients from the study and identified as containing 3X 10 ⁶ Transplantation of a portion of the heterologous cellular component of an individual Tcon cell into various cell populations in a subject.

Table 18: heterologous cellular component

Example 2: analysis of patients receiving different management protocols

Clinical outcome data for subjects receiving different conditioning regimens is further quantified. Figure 6A compares acute GVHD of patients receiving Bu/Cy conditioning (see table 14) versus conditioning regimen comprising thiotepa (BFT-busulfan, fludarabine, and thiotepa; regimen described in table 14). Acute GVHD rates were similar for both regimens, but lower in patients receiving thiotepa. A higher difference in chronic GVHD was observed in the patient receiving thiotepa (fig. 6B). Surprisingly, the recurrence rate of patients was significantly reduced in patients receiving BFT regimen (p < 0.03), wherein patients receiving BFT regimen did not relapse (see fig. 6C and D). GVHD and relapse free survival (GRF) were also significantly lower in BFT regimen patients (see fig. 6E). Overall survival was also improved in patients receiving BFT (see figure 6F). These data indicate that conditioning regimens comprising thiotepa provide distinct and surprising advantages.

Example 3: analysis of patients receiving different GVHD preventive agent regimens

Clinical trial data of clinical outcome of subjects receiving different GVHD preventative agents (sirolimus or tacrolimus) were further quantified. Fig. 7A compares acute GVHD of patients receiving sirolimus alone or tacrolimus alone versus standard of care (SOC) patients receiving a combination of methotrexate and tacrolimus (see fig. 1B for SOC regimens). Patients receiving only tacrolimus showed higher GVHD rates than patients receiving sirolimus (fig. 7A-C). However, survival (fig. 7E), recurrence (fig. 7F), GRFS rate (fig. 7G), and overall survival (fig. 7H) were improved in patients with tacrolimus alone relative to patients with tacrolimus alone. As shown in fig. 7A, multiple agents can be used as GHVD prophylaxis with significantly reduced rates of acute GVHD compared to standard care.

After further analysis, patients were observed to have different serum trough levels. As shown in fig. 8A-9G, serum tacrolimus levels had a direct effect on the clinical outcome of the patient population. As shown in fig. 8A, patients who maintained average serum tacrolimus Mo Sigu levels above 4ng/ml for the first 30 days post-transplantation had lower GVHD rates. Fig. 8B-C are derived from the data in fig. 8A and further present data for acute GVHD and chronic GVHD rates. They show the calculation of the probability of developing GVHD (y-axis) if tacrolimus levels fall below the threshold (x-axis).

Figures 9A-9G show a direct comparison of clinical results for patients with serum levels of tacrolimus Mo Sigu above or below 4 ng/ml. The legend of each figure depicts the number of patients and the period of time when their serum tacks Mo Sigu level is above or below 4 ng/ml. Acute GVHD rates showed significant improvement with increasing levels of tacrolimus (fig. 9A), and chronic GVHD rates also showed improvement (fig. 9B).

Patients receiving the same conditioning regimen (Bu/Cy for FIGS. 9C-D; TBI/BFT for FIGS. 9E-G) but with different serum levels of tacrolimus Mo Sigu (above or below 4 ng/ml) were also analyzed for GVHD rate and the results are shown in FIGS. 9C-G. In all cases, higher levels of tacks Mo Sigu showed improvement in GVHD rate.

Significant correlation was found when comparing T cell chimerism to the average tacrolimus Mo Sigu level at day +30 post-transplantation; lower levels of tacrolimus Mo Sigu correlate with increased engraftment of donor T cells (p=0.0011). Figure 9H shows the average tacks Mo Sigu levels for a small subset of patients to day +30 post-transplant, plotted against the proportion of donor-derived cd3+ cells at day +30 (except that the chimerism data is from day 90, which is denoted by "D90"). These data indicate that maintaining lower circulating levels of tacrolimus may contribute to improved engraftment of donor T cells and improved chimerism of donor T cells, which may contribute to improved relapse-free survival of allogeneic hematopoietic stem cell transplant recipients. Without wishing to be bound by theory, it is hypothesized that improved chimerism due to higher tacrolimus levels may lead to improved GVHD and survival outcome in patients.

Conclusion(s)

While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will now be apparent to those skilled in the art without departing from the present disclosure. For example, various embodiments of the therapeutic compositions, methods may be suitable for use in a variety of pediatric, geriatric, and veterinary applications, including one or more of feline, canine, and equine applications. Specific adaptations to this may include one or more of the cell types in the therapeutic composition administered to the patient and the dosage of cells in the therapeutic composition. They may also be suitable for the treatment of any number of hematologic cancers and/or stem cell-based cancers, including leukemia, lymphoma, myeloma, and many t-cell mediated and other autoimmune diseases, including one or more of multiple sclerosis, IBD, celiac disease, crohn's disease, ulcerative colitis, ankylosing spondylitis, myasthenia gravis, and diabetes. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

Elements, features, or acts from one embodiment may be readily recombined with or replaced by one or more elements, features, or acts from other embodiments to form additional embodiments within the scope of the present invention. Furthermore, in various embodiments, elements shown or described as combined with other elements, features, steps, or acts may be present as separate elements, features, steps, or acts. Furthermore, the various embodiments expressly contemplate any negative statement of any element, feature, step, act, or the like shown or described in one or more embodiments. Accordingly, the scope of the invention is not to be limited by the details of the described embodiments, but only by the appended claims.

Claims

1. A multi-component drug therapy to be administered to a human subject in need thereof, the multi-component therapy comprising:

(a) A solution comprising a first cd45+ cell population comprising Hematopoietic Stem and Progenitor Cells (HSPCs) and granulocytes, wherein up to about 10% of the first cd45+ cell population comprises granulocytes;

(b) A solution comprising a population of cells enriched for regulatory T cells (tregs);

(c) A solution comprising a second population of cd45+ cells, wherein the second population of cd45+ cells comprises at least about 20% cd3+ normal T cells (Tcon), at least about 10% monocytes and at least about 10% granulocytes; and

(d) A solution comprising one or more doses of a Graft Versus Host Disease (GVHD) prophylaxis agent.

2. The multicomponent drug treatment of claim 1, wherein the GVHD preventing agent is tacrolimus.

3. The multicomponent drug treatment of claim 1 or claim 2, wherein the tacrolimus is formulated for oral administration or for intravenous administration.

4. The multicomponent drug treatment of claim 2, wherein the tacrolimus is administered in an amount that maintains a target blood level of at least about 3ng/ml for at least about 20 days after administration of the second cd45+ cell population.

5. The multicomponent drug treatment of any one of claims 2-4, wherein the tacrolimus is administered in an amount that maintains a target blood level of about 4ng/ml or greater for at least about 40 days after administration of the second cd45+ cell population.

6. The multicomponent drug treatment of any one of claims 2-5, wherein the tacrolimus is administered in an amount that maintains a target blood level of about 4ng/ml or greater for at least about 40 days after administration of the second cd45+ cell population.

7. The multicomponent drug treatment of any one of claims 2-6, wherein the tacrolimus is administered in an amount that maintains a target blood level of up to about 10ng/ml for at least 30 days after administration of the second cd45+ cell population.

8. The multicomponent drug treatment of any one of claims 2-7, wherein the tacrolimus is administered for at least about 60 days after administration of the second cd45+ cell population.

9. The multicomponent drug treatment of any one of claims 2-8, wherein the tacrolimus is administered for at least about 90 days after administration of the second cd45+ cell population.

10. The multicomponent drug treatment of any one of claims 2-9, wherein the tacrolimus is administered up to about 150 days after administration of the second cd45+ cell population.

11. The multicomponent drug treatment of any one of claims 2-9, wherein the tacrolimus is administered up to about 120 days after administration of the second cd45+ cell population.

12. The multicomponent drug treatment of any one of claims 2-9, wherein the tacrolimus is formulated for oral or intravenous delivery.

13. The multi-component drug therapy of any one of the preceding claims, wherein the first population of cd45+ cells comprises at least about 0.5% granulocytes.

14. The multi-component drug therapy of any one of the preceding claims, wherein the first population of cd45+ cells comprises at least about 1% granulocytes.

15. The multi-component drug therapy of any one of the preceding claims, wherein the first population of cd45+ cells comprises up to about 5% granulocytes.

16. The multi-component drug therapy of any one of the preceding claims, wherein the first population of cd45+ cells comprises up to about 3% granulocytes.

17. The multicomponent drug treatment of any one of the preceding claims, wherein the first cd45+ cell population comprises up to about 3% monocytes.

18. The multicomponent drug treatment of any one of the preceding claims, wherein the first cd45+ cell population comprises up to about 2% monocytes.

19. The multi-component drug therapy of any one of the preceding claims, wherein the first population of cd45+ cells comprises up to about 0.5% lymphocytes.

20. The multi-component drug therapy of any one of the preceding claims, wherein the first population of cd45+ cells comprises up to about 2% lymphocytes.

21. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises at least about 15% granulocytes.

22. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises at least about 20% granulocytes.

23. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises up to about 35% granulocytes.

24. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises up to about 30% granulocytes.

25. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises up to about 25% granulocytes.

26. The multicomponent drug treatment of any one of the preceding claims, wherein the second cd45+ cell population comprises at least about 15% monocytes.

27. The multicomponent drug treatment of any one of the preceding claims, wherein the second cd45+ cell population comprises at least about 20% monocytes.

28. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises up to about 35% monocytes.

29. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises up to about 30% monocytes.

30. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises up to about 25% monocytes.

31. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises at least about 0.5% NK cells.

32. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises at least about 2% NK cells.

33. The multi-component drug therapy of any one of the preceding claims, wherein the second population of cd45+ cells comprises at least about 0.1% cd34+ cells or about 0.2% to about 20% cd34+ cells.

34. The multicomponent drug treatment of any one of the preceding claims, wherein the second cd45+ cell population comprises at least about 0.1% tregs.

35. The multicomponent drug treatment of any one of the preceding claims, wherein the treatment further comprises a conditioning regimen, wherein the conditioning regimen is administered prior to any one of (a) to (d).

36. The multicomponent drug treatment of any one of the preceding claims, wherein the conditioning regimen is administered from about two days to about ten days prior to any one of (a) to (d).

37. The multicomponent drug treatment of claim 35, wherein the conditioning regimen is a myeloablative conditioning regimen.

38. The multicomponent drug treatment of any one of claims 35-36, wherein the conditioning regimen comprises at least three conditioning agents, wherein at least one conditioning agent comprises thiotepa.

39. The multicomponent drug treatment of any one of claim 36, wherein the myeloablative conditioning regimen comprises at least one dose of thiotepa.

40. The multicomponent drug treatment of claim 39 wherein the at least one dose of thiotepa comprises at least about 5 milligrams of thiotepa per kg of actual or ideal body weight of the human subject.

41. The multicomponent drug treatment of claim 40 wherein the at least one dose of thiotepa comprises at least about 10 milligrams of thiotepa per kg of actual or ideal body weight of the human subject.

42. The multicomponent drug treatment of any one of claims 35-41 wherein the conditioning regimen comprises one or more doses of busulfan, fludarabine, and thiotepa.

43. The multicomponent drug treatment of claim 42 wherein the one or more doses of busulfan, fludarabine, and thiotepa each comprise about 5 to about 12mg thiotepa per kg of the actual or desired body weight of the human subject, about 7 to about 11mg busulfan per kg of the actual or desired body weight of the human subject, and about 100 to about 200mg fludarabine per m, respectively ² Body surface area.

44. The multicomponent drug treatment of any one of the preceding claims, wherein the first cd45+ cell population comprises less than about 5EU endotoxin/ml solution.

45. The multicomponent drug treatment of any one of the preceding claims, wherein the first cd45+ cell population comprises less than about 1EU endotoxin/ml solution.

46. The multicomponent drug treatment of any one of the preceding claims, wherein the first cd45+ cell population comprises less than about 0.5EU endotoxin/ml solution.

47. The multicomponent drug treatment of any one of the preceding claims, wherein the Treg-enriched cell population comprises less than about 5EU endotoxin/ml solution.

48. The multicomponent drug treatment of any one of the preceding claims, wherein the Treg-enriched cell population comprises less than about 1EU endotoxin/ml solution.

49. The multicomponent drug treatment of any one of the preceding claims, wherein the Treg-enriched cell population comprises less than about 0.5EU endotoxin/ml solution.

50. The multicomponent drug treatment of any one of the preceding claims, wherein the second cd45+ cell population comprises less than about 5EU endotoxin/ml solution.

51. The multicomponent drug treatment of any one of the preceding claims, wherein the second cd45+ cell population comprises less than about 1EU endotoxin/ml solution.

52. The multicomponent drug treatment of any one of the preceding claims, wherein the second cd45+ cell population comprises less than about 0.5EU endotoxin/ml solution.

53. The multi-component drug therapy of any one of the preceding claims, wherein the HSPCs are cd34+.

54. The multicomponent drug treatment of any one of the preceding claims, wherein the Treg is cd4+cd25+cd127dim.

55. The multicomponent drug treatment of any one of the preceding claims, wherein the population of Treg-enriched cells comprises cd45+ cells, wherein more than about 90% of the cd45+ cells are tregs.

56. The multicomponent drug treatment of any one of the preceding claims, wherein the Treg is cd4+cd25+cd127dim or cd4+foxp3+.

57. The multicomponent drug treatment according to any one of the preceding claims, wherein the first cd45+ fines comprising HSPCsThe cell population comprises about 5X 10 ⁵ Up to about 2X 10 ⁷ Individual HSPCs/kg of the ideal body of the human subject.

58. The multicomponent drug treatment of any one of the preceding claims, wherein the population of Treg-enriched cells comprises about 1 x 10 ⁵ Up to about 1X 10 ⁷ Individual tregs/kg the actual or ideal body weight of the human subject.

59. The multicomponent drug treatment of any one of the preceding claims, wherein the population of Treg-enriched cells comprises about 5 x 10 ⁵ Up to about 4X 10 ⁶ Individual tregs/kg the actual or ideal body weight of the human subject.

60. The multicomponent drug treatment of any one of the preceding claims, wherein the second cd45+ cell population comprises about 1 x 10 cells ⁵ Up to about 1X 10 ⁷ Tcon/kg the actual or ideal body weight of the human subject.

61. The multicomponent drug treatment of any one of the preceding claims, wherein the second cd45+ cell population comprises about 5 x 10 cells ⁵ Up to about 5X 10 ⁶ Tcon/kg the actual or ideal body weight of the human subject.

62. The multicomponent drug treatment of any one of the preceding claims, wherein the first cd45+ cell population and the Treg-enriched cell population are administered before the second cd45+ cell population.

63. The multicomponent drug treatment of any one of the preceding claims, wherein a first dose of the one or more doses of the GVHD preventing agent is administered after administration of the second cd45+ cell population.

64. A method of treating a human subject diagnosed with a hematological malignancy, the method comprising:

administering to the human subject a solution comprising a first cd45+ cell population, a solution comprising a cell population enriched for regulatory T cells (tregs), a solution comprising a second cd45+ cell population, and a solution comprising one or more doses of a GVHD prophylaxis agent; and is also provided with

Wherein the solution comprising the first cd45+ cell population, the solution comprising the cell population enriched for regulatory tregs, the solution comprising the second cd45+ cell population and the solution comprising one or more doses of a GVHD prophylaxis agent are as defined in any one of claims 1-63.

65. The method of claim 64, wherein the hematological malignancy is selected from the group consisting of acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, multiple myeloma, lymphoma, hodgkin's lymphoma, and non-hodgkin's lymphoma.

66. The method of any one of claims 64-65, wherein the administering comprises infusing the first cd45+ cell population, the Treg-enriched cell population, and the second cd45+ cell population into the human subject.

67. The method of any one of claims 64-66, wherein the second population of cd45+ cells is administered at least about 12 hours after the first population of cd45+ cells.

68. The method of any one of claims 64-67, wherein the second population of cd45+ cells is administered about 24 to about 96 hours after the first population of cd45+ cells.

69. The method of any one of claims 64-68, wherein the second population of cd45+ cells is administered about 36 to about 60 hours after the first population of cd45+ cells.

70. The method of any one of claims 64-66, wherein the second cd45+ cell population is administered at least about 12 hours after the Treg-enriched cell population.

71. The method of any one of claims 64-66, wherein the second cd45+ cell population is administered about 24 to about 96 hours after the Treg-enriched cell population.

72. The method of any one of claims 64-66, wherein the second cd45+ cell population is administered about 36 to about 60 hours after the Treg-enriched cell population.

73. The method of any one of claims 64-72, wherein the human subject does not develop greater than phase 2 GVHD within about 100 days of the administering the second cd45+ cell population.

74. The method of any one of claims 64-73, wherein the human subject does not develop higher than stage 2 GVHD within about 180 days or about 200 days of the administering the second cd45+ cell population.

75. The method of any one of claims 64-74, wherein the human subject does not develop greater than stage 2 GVHD within about 1 year of the administering the second cd45+ cell population.

76. The method of any one of claims 64-75, wherein the human subject has been previously or is being treated for the hematological malignancy simultaneously.

77. The method of any one of claims 64-76, wherein the GVHD prophylaxis agent is tacrolimus and is initially administered to the human subject at about 0.03mg/kg of actual or ideal body weight per day of the human subject.

78. The method of any one of claims 64-76, wherein the administration of tacrolimus begins about 12 hours to about 24 hours after the administration of the second cd45+ cell population.

79. The method of claim 77, wherein the dose of tacrolimus begins to taper about 90 days after administration of the first dose to the human subject.

80. The method of claim 77, wherein the dose of tacrolimus begins to taper about 45 days after administration of the first dose to the human subject.

81. The method of any one of claims 64-80, wherein the first population of cd45+ cells, the population of Treg-enriched cells, and the second population of cd45+ cells are obtained from a single donor.

82. The method of claim 81, wherein the method further comprises collecting at least one mobilized peripheral blood donation from the donor.

83. The method of any one of claims 81-82 wherein the method further comprises collecting at most two mobilized peripheral blood donations from the donor.

84. The method of claim 83, wherein at least one of the mobilized peripheral blood donations is processed and sorted to enrich for cd34+ cells and tregs.

85. The method of claim 84, wherein the one or more of the mobilized peripheral blood donations are processed and sorted for less than about 35 hours.

86. The method of claim 84, wherein the one or more of the mobilized peripheral blood donations are processed and sorted for less than about 30 hours.

87. The method of claim 84, wherein the one or more of the mobilized peripheral blood donations are processed and sorted for less than about 25 hours.

88. The method of claim 84, wherein the one or more of the mobilized peripheral blood donations are processed and sorted for a time of up to about 35 hours.

89. The method of claim 84, wherein the one or more of the mobilized peripheral blood donations are processed and sorted for a time of up to about 25 hours.

90. The method of any one of claims 84-89, wherein one or more of the mobilized peripheral blood donations are processed and sorted using one or more immunoseparation particles (ISPs).

91. The method of claim 90, wherein the one or more ISPs comprise an affinity reagent.

92. The method of claim 91, wherein the affinity reagent is an immunomagnetically separating particle.

93. The method of claim 92, wherein the immunomagnetic separation particles are antibodies each conjugated to an iron-containing particle.

94. The method of any one of claims 91 to 93, wherein the affinity reagent comprises a plurality of CD34 reagents that bind to one or more CD34 receptors on HSPCs.

95. The method of any claim 94, wherein the CD34 agent comprises an anti-CD 34 antibody.

96. The method according to claim 94, wherein the average number of ISPs per HSPC in the population of HSPC cells is less than about 20,000.

97. The method according to claim 96, wherein the average number of ISPs per HSPC in the population of HSPC cells is equal to or less than about 20,000.

98. The method according to claim 97, wherein the average number of ISPs per HSPC in the population of HSPC cells is from about 1000 to about 20,000.

99. The method of claim 91, wherein the affinity reagent comprises a plurality of CD25 reagents that bind to one or more CD25 receptors on tregs, optionally wherein the CD25 reagents comprise anti-CD 25 antibodies.

100. The method of claim 99, wherein the average number of ISPs per T-reg cell in the population of tregs is equal to or less than about 4000.

101. The method of claim 100, wherein the average number of ISPs per T-reg cell in the population of tregs is about 1500 to about 2500.

102. The method of any one of claims 84-101, wherein the mobilized peripheral blood-donated cells are sorted such that the first cd45+ cell population comprises up to about 10% granulocytes.

103. The method of claim 102, wherein the mobilized peripheral blood-donated cells are sorted such that the first cd45+ cell population comprises up to about 7% granulocytes.

104. The method of any one of claims 84-103, wherein the mobilized donor peripheral blood-donated cells are sorted such that the first cd45+ cell population comprises up to about 4% monocytes.

105. The method of claim 104, wherein the mobilized donor peripheral blood-donated cells are sorted such that the first cd45+ cell population comprises at least about 0.1% monocytes.

106. The method of any one of claims 84-105, wherein the mobilized donor peripheral blood-donated cells are sorted such that the Treg-enriched population comprises up to about 10% CD25 cells.

107. The method of any one of claims 64-106, wherein the first population of cd45+ cells, the population of Treg-enriched cells, and/or the second population of cd45+ cells are allogeneic with respect to the human subject.

108. The method of any one of claims 64-106, wherein the first population of cd45+ cells, the population of Treg-enriched cells, and/or the second population of cd45+ cells are obtained from a donor that is HLA-matched relative to the human subject.

109. The method of any one of claims 64-106, wherein the first population of cd45+ cells, the population of Treg-enriched cells, and/or the second population of cd45+ cells are obtained from a donor that is HLA-mismatched relative to the human subject.

110. The method of any one of claims 64-106, wherein the first population of cd45+ cells, the population of Treg-enriched cells, and/or the second population of cd45+ cells are obtained from a donor that is haploid matched relative to the human subject.

111. The method of any one of claims 64-110, wherein the second population of cd45+ cells comprises a population of constant natural killer T cells (iNKT).

112. The method of claim 111, wherein the iNKT is cd3+vα24jα18+.

113. The method of any one of claims 111-112, wherein the iNKT population comprises more than about 5 x 10 ² Individual iNKT/kg ideal physical reality or ideal body weight of the human subject.

114. The method of any one of claims 111-113, wherein the iNKT population comprises about 5 x 10 ² Up to about 1X 10 ⁷ Individual iNKT/kg ideal physical reality or ideal body weight of the human subject.

115. The method of any one of claims 64-114, wherein the second population of cd45+ cells comprises a population of memory T cells (Tmem).

116. The method of claim 115, wherein the Tmem is cd3+cd45ra-cd45ro+.

117. The method of any one of claims 115-116, wherein the population of tmems comprises more than about 3 x 10 ⁵ Tmem/kg of the ideal physical reality or ideal weight of the human subject.

118. The method of any one of claims 115-117, wherein the population of tmems comprises about 3 x 10 ⁵ Up to about 1X 10 ⁹ Tmem/kg of the ideal physical reality or ideal weight of the human subject.

119. The multicomponent drug treatment of any one of claims 1-63, or the method of any one of claims 64-118, wherein the risk and/or severity of an adverse event associated with the multicomponent drug treatment or the method is reduced as compared to a similar drug treatment or method in which a human subject received Tcon but did not receive Treg.

120. The multicomponent drug treatment or method of claim 119, wherein the adverse event is acute GVHD (aGVHD).

121. The multicomponent drug treatment or method of claim 120, wherein the adverse event is aGVHD in stage two or higher.

122. The multicomponent drug treatment or method of claim 119, wherein the adverse event is chronic GVHD (cGVHD).

123. The multicomponent drug treatment or method of claim 122, wherein the human subject is free of cGVHD about one year after administration of the cell population.

124. The multicomponent drug treatment or method of claim 122, wherein the adverse event is moderate to severe cGVHD.

125. The multicomponent drug treatment or method of claim 119, wherein the adverse event is recurrence of malignancy in the human subject.

126. The multicomponent drug treatment or method of claim 125, wherein the human subject has no recurrence of its malignancy about one year after administration of the drug regimen.

127. The multicomponent drug treatment or method of claim 119, wherein the human subject has undergone a myeloablative conditioning regimen prior to administration of any cell population, and the adverse event is associated with the myeloablative conditioning.

128. The multicomponent drug treatment or method of any one of claims 64-127 wherein the method further comprises providing instructions for use (IFU) that includes instructions for administering the cell population to a patient.

129. The multi-component drug therapy or method of claim 128, wherein the IFU further comprises instructions for administering one or more pharmaceutical agents or compositions to the patient.

130. A method of transplanting a population of conventional T cells (Tcon) as part of a treatment regimen for hematological malignancies, the method reducing the risk and/or severity of adverse events associated with the treatment regimen, the method comprising:

administering to a patient a population of regulatory T cells (tregs) comprising tregs and a liquid suspending the tregs;

administering to the patient a heterogeneous population of cells comprising lymphocytes, granulocytes, monocytes and a liquid suspending said cells, wherein at least about 30% of said lymphocytes comprise Tcon; and is also provided with

Wherein after administration of the population of cells, the patient has a reduced risk and/or severity of the adverse event compared to a hematological malignancy patient who receives Tcon but does not receive Treg.

131. A method of transplanting a population of cells into a human patient as part of a treatment regimen for hematological malignancy, the method reducing the risk and/or severity of adverse events associated with the treatment regimen, the method comprising:

providing a population of Hematopoietic Stem and Progenitor Cells (HSPCs) to be administered to the patient, the population of HSPCs comprising HSPCs and a liquid suspending the HSPCs;

Providing a population of regulatory T cells (tregs) to be administered to the patient, the population of tregs comprising tregs and a liquid suspending the tregs; and

providing a heterogeneous population of cells to be administered to the patient, the heterogeneous population of cells comprising lymphocytes, granulocytes, monocytes and a liquid suspending said cells, wherein at least about 30% of said lymphocytes comprise conventional T cells (Tcon); and is also provided with

Wherein after administration of the cell population, the patient has a reduced risk and/or severity of the adverse event compared to a hematological malignancy patient that receives a population of Tcon cells but does not receive a population of T-reg cells.

132. The method of any one of claims 130-131, wherein the population of cells is administered to the patient by intravenous infusion.

133. The method of any one of claims 130-132, wherein the respective cell populations are provided as separate cell populations and are derived from a single human blood donor.

134. The method of any one of claims 130-133, wherein the adverse event is acute graft versus host disease (aGVHD).

135. The method of claim 134, wherein the adverse event is aGVHD in stage two or higher.

136. The method of claim 135, wherein the patient has no second-phase or higher aGVHD about 180 days after administration of the cell population.

137. The method of any one of claims 130-136, wherein the adverse event is chronic graft versus host disease (cGVHD).

138. The method of claim 137, wherein the patient is free of cGVHD about one year after administration of the population of cells.

139. The method of claim 137, wherein the adverse event is moderate to severe cGVHD.

140. The method of claim 139, wherein the patient has no moderate to severe cGVHD about one year after administration of the cell population.

141. The method of any one of claims 130-140, wherein the adverse event is recurrence of malignancy in the patient.

142. The method of claim 141, wherein the patient has no recurrence of his malignancy about one year after administration of the population of cells.

143. The method of any one of claims 130-142, wherein the adverse event comprises Graft Versus Host Disease (GVHD) and recurrence of malignancy in the patient.

144. The method of claim 143, wherein the patient has no recurrence of GHVD or malignancy thereof one year after administration of the population of cells.

145. The method of any one of claims 130-144, wherein at least one of the cell populations comprises less than about 5EU endotoxin/ml of corresponding suspension.

146. The method of any of claims 130-145, wherein prior to administration of the population of cells, the patient has undergone a myeloablative conditioning regimen and the adverse event is associated with the myeloablative conditioning.

147. The method of claim 146, wherein the adverse event comprises a recurrence or infection of a malignancy of the patient.

148. The method of any one of claims 130-147, wherein the heterologous cell population comprises from about 0.2% to about 2.0% hematopoietic stem cells and progenitor cells.

149. The method of any one of claims 130-148, wherein the hematological malignancy is acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, multiple myeloma, lymphoma, hodgkin's lymphoma, and non-hodgkin's lymphoma.

150. The method of any one of claims 130-149, wherein the gene expression level of the T-reg cells is correlated with cells harvested from the donor within about 60 hours prior to administration to the patient.

151. The method of any one of claims 130-150, wherein the number of T-reg cells in the T-reg population is about equal to the number of T-con cells in the heterologous cell population.

152. The method of claim 151, wherein the T-reg cells in the T-reg population inhibit activation of conventional T cells in the heterologous cell population by an amount of up to about 20% by healthy tissue of the patient.

153. The method of any one of claims 130-152, wherein the patient's peripheral blood exhibits an elevated ratio of Treg to cd4+ T cells up to about 100 days after administration of the cell population compared to a healthy human subject not administered the cell population.

154. The method according to any of the claims 130-153, wherein at least about 50% of the cells in the population of HSPC cells are colony forming units.

155. The method of any one of claims 130-154, wherein at least one of the cell populations has an elevated amount of granulocyte colony stimulating factor as compared to non-mobilized blood.

156. The method of claim 155, wherein the at least one cell population is a heterologous cell population.

157. The method of any one of claims 130-156, wherein at least one of the cell populations has a plurality of immunoisolating particles (ISPs) attached to receptors on the cells of the cell populations.

158. The method of claim 157, wherein the plurality of ISPs are immunomagnetically separated particles.

159. The method of claim 158, wherein the plurality of ISPs comprise antibodies conjugated to iron-containing particles.

160. The method according to claim 157, wherein at least a portion of the plurality of ISPs are attached to cd34+ receptors on the HPSCs of the HSPC cell population.

161. The method according to claim 160, wherein the average number of ISPs per HSPC in the population of HSPC cells is less than about 20,000.

162. The method according to claim 161, wherein the average number of ISPs per HSPC in the population of HSPC cells is equal to or less than about 10,000.

163. The method according to claim 162, wherein the average number of ISPs per HSPC in the population of HSPC cells is from about 1500 to about 20,000.

164. The method of claim 157, wherein at least a portion of the plurality of ISPs are attached to cd25+ receptors on the cells of the population of Treg cells.

165. The method of claim 164, wherein the average number of ISPs per T-reg cell in the population of tregs is equal to or less than about 4000.

166. The method of claim 165, wherein the average number of ISPs per T-reg cell in the population of tregs is about 1500 to about 2500.

167. The method of claim 157, wherein at least a portion of the plurality of ISPs are attached to cd3+ receptors on the cells of the heterologous cell population.

168. The method of claim 167, wherein the average number of ISPs per cell in the heterologous T population is less than about 1,000.

169. The method of any of claims 130-168, wherein the population of tcons is administered at least about 12 hours after the population of HSPCs.

170. The method of claim 169, wherein the population of tcons is administered from about 24 to about 96 hours after the population of HSPCs.

171. The method of claim 170, wherein the population of tcons is administered from about 36 to about 60 hours after the population of HSPCs.

172. The method of any of claims 130-171, wherein the population of Tcon is administered at least about 12 hours after the population of cells comprising tregs.

173. The method of claim 172, wherein the population of Tcon is administered about 24 to about 96 hours after the population of cells comprising tregs.

174. The method of claim 173, wherein the population of Tcon is administered about 36 to about 60 hours after the population of cells comprising tregs.

175. The method of any one of claims 130-174, further comprising:

administering to the patient a single Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) comprising tacrolimus (tacrolimus GHVDPA) over a period of up to about 180 days; wherein the tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the blood of the patient above a threshold level during the period of time; and is also provided with

Wherein the risk and/or severity of GHVD is significantly reduced.

176. The method of claim 175, wherein the threshold level is greater than about 4ng tacrolimus/ml of patient blood.

177. The method of claim 176, wherein the threshold level is greater than about 5ng tacrolimus/ml of patient blood.

178. The method of claim 175, wherein the tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the blood of the patient below an upper threshold level during the period of time.

179. The method of claim 178, wherein the upper threshold level is less than about 10ng tacrolimus/ml of patient blood.

180. The method of claim 178, wherein the patient has a reduced risk of at least one of malignancy recurrence, infection, or renal failure.

181. The method of claim 175, wherein the patient does not develop GVHD within about 30 days of administration of the Tcon.

182. The method of claim 175, wherein the patient does not develop GVHD within about 100 days of administration of the Tcon.

183. The method of claim 175, wherein the patient does not develop GVHD within about 180 days of administration of the Tcon.

184. The method of claim 175, wherein the patient does not develop GVHD within about one year of administration of the Tcon.

185. The method of claim 175, wherein the tacrolimus GHVDPA is administered intravenously.

186. The method of claim 175, wherein the tacrolimus GHVDPA is administered orally.

187. The method of claim 175, wherein administration of the tacrolimus GHVDPA begins about 12 to about 24 hours after administration of the T-con.

188. The method of claim 175 wherein the tacrolimus GHVDPA is administered for a period of up to about 90 days.

189. The method of claim 175 wherein the tacrolimus GHVDPA is administered for a period of up to about 60 days.

190. The method of claim 175 wherein the tacrolimus GHVDPA is initially administered to the patient at about 0.03mg/kg of patient's actual or ideal body weight per day.

191. The method of claim 175, wherein the dose of tacrolimus gvhpa administered to the patient begins to taper about 90 days after the first dose is administered to the patient.

192. The method of claim 175, wherein the dose of tacrolimus gvhpa administered to the patient begins to taper about 45 days after the first dose is administered to the patient.

193. The method of any of claims 130-190, further comprising administering to the patient a myeloablative conditioning regimen prior to administering any cell populations, the conditioning regimen comprising administering to the patient at least one conditioning agent.

194. The method of claim 193, wherein the patient does not receive any radiation as part of the myeloablative conditioning regimen.

195. The method of claim 193, wherein the at least one conditioning agent is administered about two days to about ten days prior to administration of any of the cell populations.

196. The method of claim 194, wherein the at least one conditioning agent is administered about five days prior to administration of any of the cell populations.

197. The method of claim 193, wherein the at least one conditioning agent comprises thiotepa.

198. The method of claim 197, wherein the dose of thiotepa administered to the patient is in the range of about 5 to about 10mg/kg actual or desired body weight.

199. The method of claim 197, wherein the at least one conditioning agent comprises busulfan and fludarabine.

200. The method of claim 199, wherein the doses of thiotepa, busulfan and fludarabine administered to the patient comprise about 10mg/kg of the patient's actual or ideal body weight, about 9.6mg/kg of the patient's actual or ideal body weight, and about 150mg/m, respectively ² Body surface area.

201. The method of any one of claims 130-200, further comprising:

instructions for use (IFU) are provided, the IFU comprising instructions for administering the population of cells to the patient.

202. The method of claim 201, wherein the IFU further comprises instructions for administering one or more pharmaceutical agents or compositions to the patient.

203. A method of transplanting a population of cells into a human patient as part of a treatment regimen for hematological malignancy, the method comprising:

administering to the patient a population of Hematopoietic Stem and Progenitor Cells (HSPCs) comprising HSPCs and a liquid suspending the HSPCs;

administering to the patient a population of regulatory T cells (tregs) to be administered to the patient, the population of tregs comprising tregs and a liquid suspending the tregs; and

administering to the patient a heterologous cell population to be administered to the patient, the heterologous cell population comprising lymphocytes, granulocytes, monocytes and a liquid suspending said cells, wherein at least about 30% of said lymphocytes comprise conventional T cells (Tcon); and

administering to the patient a single Graft Versus Host Disease (GVHD) prophylactic agent (gvhpa) comprising tacrolimus (tacrolimus GHVDPA) over a period of up to about 180 days, wherein the tacrolimus GHVDPA is administered to maintain the concentration of tacrolimus in the patient's blood above a threshold level during the period of time; and is also provided with

Wherein the risk and/or severity of GHVD associated with the treatment regimen of the hematological malignancy is significantly reduced.

204. A kit comprising a solution, the kit comprising:

a first container comprising a first population of CD45+ cells,

a second container comprising a solution comprising a second population of CD45+ cells, and

a third container comprising a solution comprising a population of cells enriched for regulatory T cells (tregs),

wherein the solution comprising the first cd45+ cell population, the solution comprising the second cd45+ cell population and the solution comprising the Treg-enriched cell population are as defined in any one of claims 1-63.

205. The kit of claim 119, further comprising a fourth container comprising the GVHD preventing agent.

206. The kit of claims 119-205, further comprising instructions for performing the method of any one of claims 64-118.

207. A kit, comprising:

a) One or more agents that sort cd34+ cells from the mobilized peripheral blood composition;

b) One or more agents that sort regulatory T cells (tregs) from the mobilized peripheral blood composition;

c) One or more reagents for detecting the number of cd3+ conventional T cells in the mobilized peripheral blood; and

d) A solution comprising one or more doses of a Graft Versus Host Disease (GVHD) prophylaxis agent.

208. The kit of claim 207, further comprising instructions for performing the method of any one of claims 64-118.

209. A method of transplanting a population of conventional T cells (Tcon) into a human subject without eliciting a phase 2 or higher Graft Versus Host Disease (GVHD) response up to about 100 days after the transplantation, the method comprising:

i. administering a heterogeneous population of cells comprising lymphocytes, granulocytes, and monocytes, wherein at least about 30% of said lymphocytes comprise conventional T cells (Tcon); and

administering a population of regulatory T cells (tregs);

wherein the heterologous cell component and/or the population of tregs comprises less than about 5EU/ml endotoxin.

210. A method of treating a human subject, the method comprising:

b) Administering a plurality of cell populations, wherein the plurality of cell populations comprises:

i. hematopoietic Stem and Progenitor (HSPC) populations;

a population of cells comprising regulatory T cells (tregs); and

a population of conventional T cells (Tcon); and

c) No more than one Graft Versus Host Disease (GVHD) prophylactic agent is administered for less than about 120 days,

wherein the population of HSPCs comprises less than about 2% cd3+ cells.

211. A method of treating a human subject in need thereof, the method comprising administering to the human subject at least two pharmaceutical compositions, wherein the pharmaceutical compositions are selected from the group consisting of

a) A pharmaceutical composition comprising a population of Hematopoietic Stem and Progenitor Cells (HSPCs);

b) A pharmaceutical composition comprising a population of regulatory T cells (tregs); and

c) A pharmaceutical composition comprising a population of conventional T cells (Tcon);

wherein each of pharmaceutical compositions a), b) and c) comprises less than about 5EU/ml endotoxin; and is also provided with

Wherein fewer than 15 human subjects in a group of 100 human subjects administered two or more pharmaceutical compositions develop a stage 2 or higher Graft Versus Host Disease (GVHD) response within about 30 days after administration of the pharmaceutical composition comprising the Tcon population.

212. A method of transplanting a population of conventional T cells (Tcon) into a human subject without eliciting a phase 2 or higher Graft Versus Host Disease (GVHD) response up to about 100 days after the transplantation, the method comprising:

i. administering a solution comprising a population of conventional T cells (Tcon); and

Administering a solution comprising a population of regulatory T cells (tregs);

wherein the population of Tcon is cryopreserved for at least about 4 hours; and is also provided with

Wherein the solution comprising a population of Tcon and the solution comprising a population of tregs comprise less than about 5EU endotoxin/ml solution.

213. A method of treating hematological malignancy in a human subject in need thereof, the method comprising administering to the human subject:

a) Hematopoietic Stem and Progenitor (HSPC) populations;

b) A population of regulatory T cells (tregs); and

c) A population of conventional T cells (Tcon);

wherein the HSPC population and the Treg population are administered prior to the Tcon population; and is also provided with

Wherein the peripheral blood of the human subject exhibits an elevated Treg count as compared to a healthy human subject not administered the three cell populations until about 100 days after administration of the three cell populations.

214. A method of transplanting a population of conventional T cells (Tcon) into a human subject without eliciting a phase 2 or higher Graft Versus Host Disease (GVHD) response up to about 100 days after the transplantation, the method comprising:

i. administering a population of conventional T cells (Tcon); and

administering a population of regulatory T cells (tregs);

wherein the population of Tcon is administered at least about 12 hours after administration of the population of tregs; and is also provided with

Wherein the population of Tcon and the population of tregs comprise less than about 5EU/ml endotoxin.