Enriched population of regulatory T cells and method for producing same

Abstract

Provided herein are populations of ex vivo expanded cord blood derived regulatory T cells, as well as methods for making and using same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 166,012, filed March 25, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] The present disclosure relates generally to the field of immunoregulatory T cells (Tregs) and methods for producing enriched populations of Tregs. [Background technology]

[0003] Dysregulation of the CXCL12 / CXCR4 pathway leads to disorders such as primary myelofibrosis, and there is a need for effective treatments for such disorders. Summary of the Invention

[0004] From cryopreserved human umbilical cord blood units, CXCR4 + 1. A method for producing an expanded population of human regulatory T (Treg) cells enriched for Treg cells, the method comprising: (a) thawing a cryopreserved human umbilical cord blood unit; (b) diluting and washing the thawed cord blood unit in a functionally closed system; and (c) detecting CD25 + (d) isolating Treg cells using a double selection method based on cell surface expression; and + The Treg cells are expanded ex vivo in a gas-permeable culture vessel in medium in the presence of (1) an effective amount of interleukin-2 (IL-2), (2) a reagent that specifically binds to CD3 and CD28, and (3) anti-CXCR4 magnetic microbeads for up to 10 days or up to 12 days, with the medium being changed about every 48 hours to detect CD25 and CD28. + (e) generating a CXCR4-enriched culture of Treg cells; and + CXCR4 + Cells were harvested from the culture and stained for CXCR4 +producing an expanded population of human Treg cells enriched for Treg cells.

[0005] In some embodiments, in step (d), (1) ex vivo expansion is initiated on day 0. (2) an effective amount of IL-2 is administered to isolated CD25 + (3) adding a reagent that specifically binds CD3 and CD28 to the isolated CD25 + Treg cells are added on day 0.

[0006] In some embodiments, the reagents that specifically bind to CD3 and CD28 are removed from the culture medium before the anti-CXCR4 magnetic microbeads are added to the culture medium.

[0007] In some embodiments, in step (d), the anti-CXCR4 magnetic microbeads are added to the medium 3 or 4 days after ex vivo expansion has commenced. In some embodiments, in step (d), the anti-CXCR4 magnetic microbeads are added to the medium 3 or 4 days after ex vivo expansion has commenced. + The isolated CD25 + Treg cells are enriched for CXCR4 at the third feed of ex vivo expansion.

[0008] In some embodiments, step (d) is carried out for 4 or 5 days.

[0009] In some embodiments, the reagent that specifically binds to CD25 is an anti-CD25 antibody or an antigen-binding fragment thereof. In some embodiments, the reagent that specifically binds to CD25 is conjugated to a solid support. In some embodiments, the solid support is a magnetic microbead.

[0010] In some embodiments, the reagent that specifically binds to CD3 and CD28 comprises an anti-CD3 antibody or antigen-binding fragment thereof, and an anti-CD28 antibody or antigen-binding fragment thereof, hi some embodiments, the reagent that specifically binds to CD3 and CD28 comprises an anti-CD3 coated bead and an anti-CD28 coated bead.

[0011] In some embodiments, the effective amount of IL-2 is about 1000 IU / ml.

[0012] CXCR4 from cryopreserved human umbilical cord blood units + 1. A method for producing an expanded population of human Treg cells enriched for Treg cells, the method comprising: (a) thawing a cryopreserved human umbilical cord blood unit; (b) diluting and washing the thawed cord blood unit in a functionally closed system; and (c) detecting CD25 + (d) ex vivo expansion of the isolated CD25+ Treg cells in a gas-permeable culture vessel in a medium, the ex vivo expansion step comprising: (1) on day 0, injecting anti-CD3 and anti-CD28 coated beads into the CD25+ Treg cells in the medium; + (1) on day 2, adding about 1000 IU / mL of IL-2 to the medium; (2) on day 2, removing the anti-CD3 and anti-CD28 coated beads from the medium and adding anti-CXCR4 magnetic microbeads to the medium; and (3) on day 3 or 4, removing the anti-CD3 and anti-CD28 coated beads from the medium and adding anti-CXCR4 magnetic microbeads to the medium. + The anti-CXCR4 magnetic microbeads attached to the Treg cells were removed from the medium, and fresh anti-CD3 and anti-CD28 coated beads were added to the CXCR4 magnetic microbeads. + adding to the Treg cells, ex vivo expansion for up to 10 days or up to 12 days, changing the medium about every 48 hours, and + (e) producing a CXCR4 enriched culture of Treg cells; and + CXCR4 + Cells were harvested from the culture medium and then subjected to immunofluorescence assay using CXCR4 +generating an expanded population of human Treg cells enriched for Treg cells.

[0013] In some embodiments, in step (d), IL-2 is administered to isolated CD25 + The medium containing the Treg cells is added approximately every 48 hours.

[0014] In some embodiments, the anti-CD3 coated beads and the anti-CD28 coated beads are in a 1:1 ratio. + The cells and anti-CD3 and anti-CD28 coated beads are in a 1:1 ratio.

[0015] In some embodiments, in step (e), about 1×10 6 CD25 + Cells / mL are cultured.

[0016] In some embodiments, in step (e), the cells are initially cultured at 10 cm 2 In some embodiments, the culture is then cultured in a gas permeable incubator having a membrane surface area of ​​100 cm. 2 In some embodiments, the culture is not rocked or agitated when IL-2 is added in step (d).

[0017] In some embodiments, in step (a), the cryopreserved human umbilical cord blood unit is thawed in a single step in a water bath.

[0018] In some embodiments, step (b) does not include manual washing. In some embodiments, step (b) is performed in a solution comprising PBS, EDTA, and about 0.5% human serum albumin.

[0019] In some embodiments, in step (c), CD25 is isolated using a dual ferromagnetic column method. + Isolate Treg cells.

[0020] In some embodiments, the method comprises administering to a subject a subject comprising administering to said subject a CXCR4 + The method further comprises cryopreserving the expanded population of human Treg cells enriched for Treg cells.

[0021] In some embodiments, CXCR4 + The expanded population of human Treg cells enriched for Treg cells has (i) ≥ 60% CD4 + CD25 + , (ii) ≥ 60% CD4 + CD25 + CXCR4 + and (iii) ≦10% CD4 - CD8 + It is.

[0022] Provided herein is a CXCR4 antibody produced by the methods disclosed herein. + An expanded population of human Treg cells enriched for Treg cells.

[0023] Also provided herein are methods for treating or preventing a bone marrow failure syndrome in a subject, comprising administering to the subject an effective amount of a population of activated human Treg cells produced by a method disclosed herein or an effective amount of a population disclosed herein, in some embodiments, the bone marrow failure syndrome is aplastic anemia, primary myelofibrosis, or myelodysplastic syndrome.

[0024] Provided herein is a method for treating or preventing primary myelofibrosis in a subject, the method comprising administering to the subject an effective amount of a population of activated human Treg cells produced by a method disclosed herein or a population disclosed herein, wherein a dose of 100 million Treg cells is administered to the subject on day 1 of a 28-day cycle for up to 6 cycles. [Brief description of the drawings]

[0025] [Figure 1]1 is a line graph showing the results of an assay measuring the viability (7AAD) of freshly activated Treg cells stored at room temperature (15-30° C.) or 4° C. (N=9). [Figure 2A] 1 shows a series of graphs showing that expanded activated Treg cells are immunosuppressive. [Figure 2B] Figure 2 shows a series of graphs demonstrating that expanded activated Treg cells are immunosuppressive. For suppression assays, conventional T cells (Tcon) (CD4+CD25-) cells were thawed and stained with CellTrace™ Violet (ThermoFisher) according to the manufacturer's instructions. Cord blood Tregs and Tcon were placed at various ratios in the presence of continued activation with CD3 / CD28 beads and analyzed after 3 days using flow cytometry. Figure 2A shows the significant suppression of expanding conventional T cells when co-cultured with Tregs at different ratios. Figure 2B shows the significantly increased suppressive capacity of activated expanded cord blood Tregs harvested on day 14 when compared to freshly isolated cord blood Tregs on day 0 in HLA matched pairs (p=0.03) and HLA mismatched pairs (p=0.03, 2-sided t-test). (n=2) [Diagram 3] 1 is a line graph showing that activated Treg cells can be immunosuppressive across HLA barriers. Using a xenograft-versus-host disease (GVHD) model (Parmar et al., Cytotherapy 16(10:90-100(2013)), non-SCID gamma-null (NSG) mice were sublethally irradiated and subsequently injected with peripheral blood mononuclear cells (PBMCs) from HLA A2-positive donors at a dose of 1×107 cells to induce GVHD. In the treatment group, cord blood Tregs from HLA A2-negative donors were injected at a dose of 1×106 cells one day prior to PBMC injection. Mice were followed for survival. Even at a 1 log lower dose, CB Tregs were able to rescue the deleterious effects of GVHD, resulting in a statistically significant superior survival rate (log-rank; p=0.003) at day 40 when compared to the PBMC-only group. [Figure 4A]1 shows a series of graphs and plots showing that expanded activated Treg cells remain suppressive, do not express RORγt, and show a reciprocal increase in IL-10 expression in response to stress. [Figure 4B] 1 shows a series of graphs and plots showing that expanded activated Treg cells remain suppressive, do not express RORγt, and show a reciprocal increase in IL-10 expression in response to stress. [Figure 4C] 1 shows a series of graphs and plots showing that expanded activated Treg cells remain suppressive, do not express RORγt, and show a reciprocal increase in IL-10 expression in response to stress. [Figure 4D] 1 shows a series of graphs and plots showing that expanded activated Treg cells remain suppressive, do not express RORγt, and show a reciprocal increase in IL-10 expression in response to stress. Cord blood Tregs were expanded in culture in the presence of IL-2 and CD3 / CD28 co-expressing beads. Cells were also treated with 0 ng / ml, 40 ng / ml, or 200 ng / ml IL-6. Cells were fed every 48 hours and flow cytometry-based analysis was performed for intracellular staining of RORγγt and cytokine release assays of IL-10 and IL-17. [Figure 5A] FIG. 1 shows a graph demonstrating that cryopreserved cord blood (CB) Treg cells have equivalent suppressive function compared to fresh CB Treg cells. [Figure 5B] FIG. 1 shows a graph demonstrating that cryopreserved cord blood (CB) Treg cells have equivalent suppressive function compared to fresh CB Treg cells. [Figure 5C] FIG. 1 shows a graph demonstrating that cryopreserved cord blood (CB) Treg cells have equivalent suppressive function compared to fresh CB Treg cells. [Figure 5D]Graphs showing that cryopreserved cord blood (CB) Treg cells have comparable suppressive function compared to fresh CB Treg cells. Figure 5A: Positive control includes Tcon cells in the presence of CD3 / 28 beads. Figure 5B: Negative control - Tcon cells in the absence of CD3 / 28 beads. Figure 5C: Co-culture of fresh CB Treg cells suppresses proliferation of Tcon cells. Figure 5D: Co-culture of cryopreserved CB Treg cells suppresses proliferation of Tcon cells. [Figure 6] Figure 1 is a series of graphs showing that expanded cord blood Tregs display a Gaussian (polyclonal) distribution of T cell receptor Vγ repertoires. Total RNA was extracted from Tregs using a commercially available kit (Tel-Test, Friendswood, TX) and cDNA was prepared using reverse transcription (Applied Biosystems, Foster City, CA). CDR3 regions were then amplified for 23 TCR Vβ subsets by polymerase chain reaction (PCR). The resulting PCR products were subjected to capillary electrophoresis and quantitative densitometry to assess fragment length diversity within each TCR Vβ family. [Figure 7A] Expanded cord blood Tregs have been shown to remain suppressive in the presence of dexamethasone (also called "Dex" or a "steroid"). [Figure 7B] Expanded cord blood Tregs are shown to remain suppressive in the presence of dexamethasone (referred to as "Dex" or "steroids"). "Tcon" refers to conventional T cells. "Treg" refers to regulatory T cells. Top and bottom left panels are steroids (-). Top and bottom right panels contain 100 μg / mL steroids. [Figure 8A] Cryopreserved activated Treg cells display a consistent phenotype and are capable of immunosuppression similar to freshly activated Treg cells. [Figure 8B] Cryopreserved activated Treg cells display a consistent phenotype and are capable of immunosuppression similar to freshly activated Treg cells. [Figure 8C]Cryopreserved activated Treg cells display a consistent phenotype, demonstrating that they are capable of immune suppression similar to freshly activated Treg cells. Figure 8A shows the expression of CD25, CD8 and CD127 in cryopreserved Tregs upon thawing. Figure 8B shows that cryopreserved Tregs display high expression of Helios and FoxP3. Figure 8C shows that cryopreserved Tregs suppress proliferating conventional T cells using a CellTrace™ Violet dye-based suppression assay. [Figure 9A] 1 shows the results of a study using a xenogeneic mouse graft-versus-host disease (GVHD) model. [Figure 9B] The results of a study using a xenogeneic mouse graft-versus-host disease (GVHD) model are shown. Using a xenogeneic graft-versus-host disease (GVHD) model (Parmar et al., Cytotherapy 16(10:90-100(2013)), fresh activated Treg cells or cryopreserved (frozen) activated Treg cells were administered at a dose of 1×107 cells one day before donor peripheral blood mononuclear cells at a dose of 1×107 cells for GVHD prophylaxis. FIG. 9A is a graph showing the effect of fresh activated Treg cells or cryopreserved (frozen) activated Treg cells on GVHD scores. FIG. 9B is a graph showing the effect of fresh activated Treg cells or cryopreserved (frozen) activated Treg cells on mouse body weight. "CB" refers to umbilical cord blood. "PBMC" refers to peripheral blood mononuclear cells. [Figure 10A] 1 shows the design of a study using a xenogeneic mouse graft-versus-host disease (GVHD) model. [Figure 10B]The design of the study using a xenogeneic mouse graft-versus-host disease (GVHD) model is shown. FIG. 10A shows the GVHD prevention study design, where NSG mice are sublethally irradiated on day -1, followed by injection of cord blood (CB) Treg-1×107 cells, and PBMC-1×107 cells on day 0. Mice are then followed every other day for measurement of body weight and GVHD score. Peripheral blood and serum are collected at baseline and at weekly intervals thereafter from day +7. FIG. 10B shows the GVHD treatment study design, where NSG mice are sublethally irradiated on day -1, followed by injection of PBMC-1×107 cells on day 0. Injections of CB Treg-1×107 cells are administered on days +4, +11, +18, and +25. Mice are then followed every other day for measurement of body weight, GVHD score, and survival rate. Peripheral blood and serum are collected at baseline and then at weekly intervals from day +7. "PBMC" refers to peripheral blood mononuclear cells. "Frozen Treg" refers to cryopreserved Tregs. "NSG" refers to non-SCID gamma null mice. [Figure 11A] The effect of administration of cryopreserved activated Tregs on body weight variation (FIG. 11A) and survival (FIG. 11B) in a xenogeneic mouse graft-versus-host disease (GVHD) model is shown. [Figure 11B] Figure 11 shows the effect of administration of cryopreserved activated Tregs on body weight variation (Figure 11A) and survival (Figure 11B) in a xenogeneic mouse graft-versus-host disease (GVHD) model. "Prevention" refers to the study design shown in Figure 10A. "Treatment" refers to the study design shown in Figure 10B. "Control" refers to a negative control where no Treg cells were administered. [Figure 12A] 1 shows peripheral blood cytokine analysis results at baseline, day +7 and day +14 post PBMC injection in a xenogeneic mouse graft-versus-host disease (GVHD) model for control, prevention and treatment groups. [Figure 12B] 1 shows peripheral blood cytokine analysis results at baseline, day +7 and day +14 post PBMC injection in a xenogeneic mouse graft-versus-host disease (GVHD) model for control, prevention and treatment groups. [Figure 12C]1 shows peripheral blood cytokine analysis results at baseline, day +7 and day +14 post PBMC injection in a xenogeneic mouse graft-versus-host disease (GVHD) model for control, prevention and treatment groups. [Figure 12D] 1 shows peripheral blood cytokine analysis results at baseline, day +7 and day +14 post PBMC injection in a xenogeneic mouse graft-versus-host disease (GVHD) model for control, prevention and treatment groups. [Figure 12E] 1 shows peripheral blood cytokine analysis results at baseline, day +7 and day +14 post PBMC injection in a xenogeneic mouse graft-versus-host disease (GVHD) model for control, prevention and treatment groups. [Figure 12F] The results of peripheral blood cytokine analysis at baseline, +7 days and +14 days after PBMC injection in a xenogeneic mouse graft-versus-host disease (GVHD) model for control, prevention and treatment groups are shown in Figure 12A: IP-10, Figure 12B: TNFγ, Figure 12C: GM-CSF, Figure 12D: MIP-1, Figure 12E: FLT-3, Figure 12F: IFN-γ. [Figure 13] Images of mice treated with activated Tregs (cord blood (CB) Tregs only) or activated Tregs and PBMCs (CB Treg+PBMCs) are shown. Bioluminescence scans after injection of firefly luciferase-labeled CB Tregs showed that by day +1 post-injection, CB Tregs were detected in the lungs, liver, and spleen of all mice, regardless of PBMC injection. By day +3, CB Tregs were no longer detectable in mice in the continued absence of PBMCs (CB Tregs only), but continued to be detected in PBMC recipient mice (CB Treg+PBMCs). In mice with expanding PBMCs, scans suggest persistence and even proliferation of GVHD target organs. [Figure 14]Images of mice treated with activated Tregs are shown. GFP-labeled HL-60 acute myeloid leukemia (AML) cell line was injected at a dose of 3×106 cells into all 4 groups of NSG mice: 1) control mice (PBS&AML): received HL60+PBS, 2) Treg mice (AML+Treg): received HL60+Treg (1×107 cells), 3) Tcon mice (AML+Tcon): received HL60+Tcon (1×107 cells), 4) Tcon+Treg mice (AML+Tcon+Treg): received HL60+Tcon (1×107 cells)+Treg (1×107 cells). Mice were photographed at weekly intervals to understand the effect of injected Tcon and Treg on tumor volume burden. Mice succumbed to tumors in control (PBS treated) and mice treated with CB Treg only. Recipients of Tcon were able to eliminate tumors but died of GVHD. Recipients of Tcon and Treg were able to enjoy prolonged survival due to tumor control and lack of GVHD. [Figure 15] Figure 1 shows a line graph demonstrating that a single injection of activated Treg cells reduced levels of CD45+ effector T cells 9 weeks after engraftment of SLE-PBMCs in a xenogeneic mouse model of systemic lupus erythematosus (SLE), where SLE-PBMCs (3x106 cells) are injected into NSG mice and CB Tregs (1x107 cells) are injected 1 week after SLE-PBMC injection. "PBMC" refers to peripheral blood mononuclear cells. [Figure 16A] Graph showing that injection of activated Treg cells (1×107 cells) four times a week starting 4 weeks after injection of SLE-PBMCs (3×106 cells) improved survival in a xenogeneic mouse model of systemic lupus erythematosus (SLE). [Figure 16B] 1 shows a bar graph illustrating that four weekly injections of activated Treg cells reduced levels of anti-double stranded DNA antibodies (ds DNA Ig) in a xenogeneic mouse model of systemic lupus erythematosus (SLE). [Figure 17A]17A-B show plots demonstrating that injections of activated Treg cells four times per week reduced urinary albumin levels (FIG. 17A) and reduced urinary creatinine leakage (FIG. 17B) in a xenogeneic mouse model of systemic lupus erythematosus (SLE). [Figure 17B] 17A-B show plots demonstrating that injections of activated Treg cells four times per week reduced urinary albumin levels (FIG. 17A) and reduced urinary creatinine leakage (FIG. 17B) in a xenogeneic mouse model of systemic lupus erythematosus (SLE). [Figure 18] 1 shows a series of images demonstrating that four weekly injections of activated Treg cells improved kidney tissue in a xenogeneic mouse model of systemic lupus erythematosus (SLE). [Figure 19] 1 shows graphs and results of statistical analysis demonstrating that administration of activated Tregs reduces serum concentrations of human sCD40L in a xenogeneic mouse model of systemic lupus erythematosus (SLE). [Figure 20A] Graphs showing that weekly injections of activated cryopreserved Tregs resulted in a sustained reduction in circulating CD8+ effector T cells (FIG. 20A), as well as reduced infiltration of CD8+ effector T cells in the spleen, bone marrow, lungs, and liver in a xenogeneic mouse model of systemic lupus erythematosus (SLE) (FIG. 20B). [Figure 20B] 20A and 20B show graphs showing that weekly injections of activated cryopreserved Tregs resulted in a sustained reduction in circulating CD8+ effector T cells (FIG. 20A), as well as reduced infiltration of CD8+ effector T cells in the spleen, bone marrow, lungs, and liver in a xenogeneic mouse model of systemic lupus erythematosus (SLE). "PBMC" refers to peripheral blood mononuclear cells. [Figure 21A] 1 shows a series of graphs and images showing the effect of administration of Tregs in a xenogeneic mouse model of multiple myeloma. [Figure 21B] 1 shows a series of graphs and images showing the effect of administration of Tregs in a xenogeneic mouse model of multiple myeloma. [Figure 21C] 1 shows a series of graphs and images showing the effect of administration of Tregs in a xenogeneic mouse model of multiple myeloma. [Figure 21D] A series of graphs and images showing the effect of administration of Tregs in a xenogeneic mouse model of multiple myeloma. FIG. 21A is a line graph showing the effect on mouse body weight over time. CB Treg recipients maintain their body weight, while a decline in the "myeloma alone" group shows that weight begins to decrease around week 4 after tumor inoculation. FIG. 21B is a line graph showing the effect on circulating myeloma cells in peripheral blood over time. Weekly blood samples were taken and isolated cells were analyzed for circulating human CD38+ cells. A significant increase in circulating myeloma cells was evident in the "myeloma alone" group compared to Treg recipients (p=0.002). FIG. 21C is a series of images showing visualization of tumor burden. As monitored by weekly bioluminescence imaging, minimal evidence of MM1S cells was visualized in CB Treg recipients compared to the extensive tumors of "myeloma alone" mice. FIG. 21D is a line graph showing quantification of tumor burden over time. Bioluminescence imaging qualification showed significantly higher signals on days 17, 24, and 31. Triangles indicate CB Treg iv injection, and arrows indicate MM1S cell iv injection. [Figure 22] 1 shows a graph showing that administration of activated Tregs improves survival in a xenogeneic mouse model of multiple myeloma. In the xenogeneic myeloma model, injection of cord blood (CB) Tregs prior to myeloma cell injection improved overall survival compared to the "myeloma alone" group. P=0.039 was determined by log-rank test. [Figure 23]Figure 1 shows a bar graph showing that administration of activated Tregs reduces plasma IL-6 levels in a xenogeneic mouse model of multiple myeloma. In a heterogeneous myeloma mouse model, injection of cord blood (CB) Tregs one day prior to injection of myeloma cells prevented myeloma engraftment and led to improved overall survival that correlated with reduced levels of the serum inflammatory cytokine IL-6. Measurement of circulating plasma mouse IL-6 levels showed lower levels compared to "myeloma alone" mice at days 28 and 35. Mean ± SEM. *P<0.0001, **P<0.001, ***P<0.01 determined by unpaired Student's t-test at each time point. [Figure 24A] 24A-24B show bar graphs demonstrating that administration of activated Treg cells reduced myeloma burden in the bone marrow (FIG. 24A) and spleen (FIG. 24B) in a xenogeneic mouse model of multiple myeloma. [Figure 24B] Bar graphs show that administration of activated Treg cells reduced myeloma burden in bone marrow (FIG. 24A) and spleen (FIG. 24B) in a xenogeneic mouse model of multiple myeloma. Three mice from each group were euthanized and organs were harvested on day 25. Bone marrow and spleen cells were stained with CD38 antibody and MM.1S cell populations were analyzed by flow cytometry. [Diagram 25] FIG. 1 shows secretion of the cytokine Granzyme B by activated Treg cells isolated from umbilical cord blood when the cells are exposed to IL-6. [Figure 26] 1 shows a timeline of a clinical trial to evaluate the safety and efficacy of administration of cord blood-derived regulatory T cells in the treatment of amyotrophic lateral sclerosis as described in Example 9. [Figure 27] FIG. 1 shows a diagram of the protocol for a clinical trial to evaluate the safety and efficacy of administration of cord blood-derived regulatory T cells in the treatment of COVID-19 (Coronavirus Disease) mediated acute respiratory distress syndrome (CoV-ARDS) as described in Example 10. [Figure 28] 1 shows a summary of early results from a Phase 1 clinical trial to evaluate the safety and efficacy of administration of cord blood-derived regulatory T cells in the treatment of subjects with bone marrow failure. [Figure 29] 1 is a table providing cord blood selection criteria for various products, including populations of activated human Treg cells. "AABB" refers to the American Association of Blood Banks. "FACT" refers to the Cellular Therapy Accreditation Agency. "CLIA" refers to the Clinical Laboratory Improvement Act. [Diagram 30] 1 is a table providing cord blood selection criteria for various products including populations of activated human Treg cells. [Diagram 31] 1 is a line graph showing the suppression rate by activated Treg cells in the absence or presence of 0.05 μM ruxolitinib 96 hours after the start of co-culture of Treg cells, Tcon cells and ruxolitinib. The x-axis shows the ratio of Treg cells to Tcon cells. Ruxo=ruxolitinib. [Diagram 32] 1 is a bar graph showing the amount of interferon (IFN)-gamma released by pathogenic lupus cells in the presence or absence of a combination of (1) activated Treg cells, (2) ruxolitinib, and / or (3) camptothecin. Rux = ruxolitinib. SLE-PBMC = peripheral blood mononuclear cells from subjects with systemic lupus erythematosus. D6 = day 6. [Diagram 33] Schematic diagram of treatment of a xenogeneic mouse graft-versus-host disease (GVHD) model with ruxolitinib and activated Treg cell regimen. PBMC = peripheral blood mononuclear cells. [Figure 34A] 34A-34B show graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on GVHD score (FIG. 34A) or survival rate (FIG. 34B) in a xenogeneic mouse GVHD model. [Figure 34B] Graphs showing the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on GVHD score (FIG. 34A) or survival rate (FIG. 34B) in a xenogeneic mouse GVHD model. Rux or R=ruxolitinib. PBMC=peripheral blood mononuclear cells. [Figure 35A]1 shows a series of bar graphs depicting (1) activated Treg cells, (2) ruxolitinib, and (3) the effect of treatment with ruxolitinib on activated Treg cells and activated Treg cell persistence in a xenogeneic mouse GVHD model. [Figure 35B] 1 shows a series of bar graphs depicting (1) activated Treg cells, (2) ruxolitinib, and (3) the effect of treatment with ruxolitinib on activated Treg cells and activated Treg cell persistence in a xenogeneic mouse GVHD model. [Figure 35C] Figure 35 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, (3) ruxolitinib on activated Treg cells and activated Treg cell persistence in a xenogeneic mouse GVHD model. Figure 35A shows the percentage of human CD45 cells. Figure 35B shows the percentage of human CD45 cells co-expressing CD4 and CD45. Figure 35C shows the percentage of human CD45 cells labeled as CB Treg cells. Rux or R = ruxolitinib. [Figure 36A] 1 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. [Figure 36B] 1 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. [Figure 36C] Figure 36 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. Figure 36A shows normalized levels of plasma IL-7. Figure 36B shows normalized levels of plasma IL-15. Figure 36C shows normalized levels of plasma IL-4. Ruxo = ruxolitinib. [Figure 37A]1 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. [Figure 37B] 1 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. [Figure 37C] 1 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. [Figure 37D] 1 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. [Figure 37E] Figure 37 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. Figure 37A shows normalized levels of plasma IL-1a. Figure 37B shows normalized levels of plasma IL-17. Figure 37C shows normalized levels of plasma IFNa2. Figure 37D shows normalized levels of plasma FGF-12. Figure 37E shows normalized levels of plasma macrophage-derived chemokine (MDC). Ruxo = ruxolitinib. [Figure 38A] 1 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. [Figure 38B] 1 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. [Figure 38C] Figure 38 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on cytokine secretion in a xenogeneic mouse GVHD model. Figure 38A shows normalized levels of plasma IL-1RA. Figure 38B shows normalized levels of plasma IL-1a3. Figure 38C shows normalized levels of plasma IL-12p70. Ruxo = ruxolitinib. [Figure 39A] 1 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on hematological parameters in a xenogeneic mouse GVHD model. [Figure 39B] Figure 39 shows a series of bar graphs depicting the effect of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on hematological parameters in a xenogeneic mouse GVHD model. Figure 39A shows hemoglobin levels. Figure 39B shows platelet levels. Rux or R = ruxolitinib. [Figure 40A] Schematic diagram of the transwell migration assay: target cells are myeloma cells or leukemia cells (negative control); actor cells are CB Treg cells or Teff cells. [Figure 40B] 1 shows a series of bar graphs depicting the effect of CB Treg cells on myeloma and leukemia target cell migration. [Figure 40C] 1 shows a series of bar graphs depicting the effect of CB Treg cells on myeloma and leukemia target cell migration. [Figure 40D] 1 shows a series of bar graphs depicting the effect of CB Treg cells on myeloma and leukemia target cell migration. [Figure 40E] 1 shows a series of bar graphs depicting the effect of CB Treg cells on myeloma and leukemia target cell migration. [Diagram 40F]A series of bar graphs showing the effect of CB Treg cells on the migration of myeloma and leukemia target cells are shown. Figure 40B shows that CB Tregs are reduced and Teff cells completely block the migration of MM1S (myeloma cell line) (p<0.001). Figure 40C shows that CB Tregs are reduced and Teff cells completely block the migration of RPMI8226 (myeloma cell line) (p=0.04). Figure 40D shows that CB Tregs reduce but not significantly the migration of U266 (myeloma cell line). Teff cells block U266 migration. Figure 40E shows that CB Tregs and Teff cells have no effect on the migration of HL-60 (acute myeloid leukemia cell line). Figure 40F shows that CB Tregs and Teff cells have no effect on the migration of Nalm6 (pre-B cell leukemia cell line). **P<0.05 was determined by unpaired Student's t-test at each time point. The y-axis in Figures 40B-40D shows cell number x 103 / μL. [Diagram 41] FIG. 1 shows a schematic diagram of the design of a phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with bone marrow failure (BMF). [Diagram 42] A diagram summarizing clinical data from a phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF is shown. [Diagram 43] A table summarizing clinical data from a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF is shown. [Diagram 44] 1 is a graph summarizing durability of response data from a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Diagram 45] A diagram summarizing the treatment history of patient 1 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF is shown. [Figure 46A] 1 shows clinical data for patient 1 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF at baseline and 1 and 4 months after Treg cell administration. [Figure 46B]1 shows clinical data for patient 1 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF at baseline and 1 and 4 months after Treg cell administration. [Figure 47]

[0023] Figure 1 is a series of graphs showing inflammatory cytokine levels for patient 1 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. The x-axis indicates days after Treg cell administration. Top left panel: CXCL-5. Top right panel: IL-17. Bottom left panel: IL-15. Bottom right panel: MCP. [Figure 48]

[0036] Figure 1 shows a series of graphs depicting inflammatory cytokine levels for patient 1 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. The x-axis indicates days after Treg cell administration. Top left panel: IL-8. Top right panel: sCD40L. Bottom left panel: MIP-1. Bottom right panel: SDF-1α+1β. [Figure 49] 1 shows a bar graph depicting splenomegaly measurements for patient 1 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF at baseline and 1 and 4 months after Treg cell administration. [Figure 50] A diagram summarizing the treatment history of patient 2 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF is shown. [Figure 51]

[0023] Figure 1 is a series of graphs showing inflammatory cytokine levels for patient 2 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. The x-axis indicates days after Treg cell administration. [Figure 52] 1 shows a graph depicting TPO levels over time for patient 3 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 53] FIG. 1 shows platelet (PLT) transfusion requirements over time for patient 3 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 54] 1 shows packed red blood cell (PRBC) transfusion requirements over time for patient 3 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 55] FIG. 1 shows platelet (PLT) transfusion requirements over time for patient 4 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 56] 1 shows packed red blood cell (PRBC) transfusion requirements over time for patient 4 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 57] FIG. 1 shows platelet (PLT) transfusion requirements over time for patient 6 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 58] 1 shows packed red blood cell (PRBC) transfusion requirements over time for patient 6 in a Phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 59A] Data are presented from studies of xenogeneic lymphoma mouse models treated with i) mimic chimeric antigen receptor (CAR) T cells, ii) cord blood-derived Treg cells, iii) CD19-CAR T cells, or (iv) cord blood-derived Treg cells + CD19-CAR T cells. [Figure 59B] Data are presented from studies of xenogeneic lymphoma mouse models treated with i) mimic chimeric antigen receptor (CAR) T cells, ii) cord blood-derived Treg cells, iii) CD19-CAR T cells, or (iv) cord blood-derived Treg cells + CD19-CAR T cells. [Figure 59C] Data are presented from studies of xenogeneic lymphoma mouse models treated with i) mimic chimeric antigen receptor (CAR) T cells, ii) cord blood-derived Treg cells, iii) CD19-CAR T cells, or (iv) cord blood-derived Treg cells + CD19-CAR T cells. [Fig. 59D] Data are presented from studies of xenogeneic lymphoma mouse models treated with i) mimic chimeric antigen receptor (CAR) T cells, ii) cord blood-derived Treg cells, iii) CD19-CAR T cells, or (iv) cord blood-derived Treg cells + CD19-CAR T cells. [Figure 60A]A table summarizing data from studies of xenogeneic lymphoma mouse models treated with i) mimic chimeric antigen receptor (CAR) T cells, ii) cord blood-derived Treg cells, iii) CD19-CAR T cells, or (iv) cord blood-derived Treg cells + CD19-CAR T cells is shown. [Figure 60B] Figure 60A shows a table summarizing data from a study of xenogeneic lymphoma mouse models treated with i) mock chimeric antigen receptor (CAR) T cells, ii) cord blood-derived Treg cells, iii) CD19-CAR T cells, or (iv) cord blood-derived Treg cells + CD19-CAR T cells. Figure 60A shows a comparison of survival times of the various groups. Figure 60B shows CD19-CAR T cells / μL in various organs. [Figure 61A] 1 shows a series of graphs and images showing the effect of administration of multiple doses of Tregs in a xenogeneic mouse model of multiple myeloma. [Figure 61B] 1 shows a series of graphs and images showing the effect of administration of multiple doses of Tregs in a xenogeneic mouse model of multiple myeloma. [Figure 61C] 1 shows a series of graphs and images showing the effect of administration of multiple doses of Tregs in a xenogeneic mouse model of multiple myeloma. [Figure 61D] 1 shows a series of graphs and images showing the effect of administration of multiple doses of Tregs in a xenogeneic mouse model of multiple myeloma. [Figure 61E] 1 shows a series of graphs and images showing the effect of administration of multiple doses of Tregs in a xenogeneic mouse model of multiple myeloma. [Fig.61F] 1 shows a series of graphs and images showing the effect of administration of multiple doses of Tregs in a xenogeneic mouse model of multiple myeloma. [Figure 61G] 1 shows a series of graphs and images showing the effect of administration of multiple doses of Tregs in a xenogeneic mouse model of multiple myeloma. [Fig.61H]

[0046] Figure 61A shows a series of graphs and images showing the effect of administration of multiple doses of Tregs in a xenogeneic mouse model of multiple myeloma. Figure 61A is a line graph showing the effect on mouse weight over time for mice administered (1) MM.1S myeloma cells alone, (2) myeloma cells and CD3+ T conventional cells (Tcon), (3) myeloma cells and cord blood-derived Treg cells (Treg), or (4) myeloma cells, Tcon cells and Treg cells (Tcon Treg). Figure 61B shows a series of images generated by non-invasive bioluminescence imaging (BLI) of mice treated with CD3+ T conventional cells (Tcon) or a combination of Tcon and Treg cells (Tcon w Treg). Figure 61C is a line graph showing quantification of tumor burden by BLI. Figure 61D is an image showing an example of extramedullary recurrence in a mouse treated with Tcon cells alone. Figure 61E shows the experimental design for administration of bispecific T cell engager against CD3 and BCMA (BiTE®) with Treg cells. Figure 61F shows a series of images generated by non-invasive BLI of mice treated with BiTE® and PanT cells, or a combination of BiTE®, PanT and Treg cells. Figure 61G is a line graph showing the effect of Treg administration on BiTE®-mediated weight loss. Figure 61H is a bar graph showing the effect of Treg administration on GVHD (graft versus host disease) scores. [Figure 62] FIG. 1 is a flow chart showing the "Day 0 (double) enrichment" method for selecting CXCR4-expressing cord blood-derived Treg cells to produce enriched CXCR4+ Treg cell cultures. [Figure 63] FIG. 1 is a flow chart showing the "Day 3 (sequential) enrichment" method for selecting CXCR4-expressing cord blood-derived Treg cells to produce enriched CXCR4+ Treg cell cultures. [Fig. 64A] Data from flow cytometry analysis of CXCR4 unseparated and positive fraction Treg cells generated by the "day 3 (sequential) enrichment" method are shown. [Fig. 64B]Data from flow cytometry analysis of CXCR4 unseparated and positive fraction Treg cells generated by the "day 3 (sequential) enrichment" method are shown. [Fig. 64C] Figure 64 shows data from flow cytometry analysis of CXCR4 unseparated and positive fraction Treg cells produced by the "Day 3 (sequential) enrichment" method. Figure 64A shows the percentage of CXCR4hi cells in the fractions. Figure 64B shows the MFI (mean fluorescence intensity) in the fractions. Figure 64C shows the MFI fold change in the fractions. [Figure 65A] Histograms and contour plots of CXCR4 expression on different isolated populations of D3 (day 3) CB Treg cells are shown. [Figure 65B] Histograms and contour plots of CXCR4 expression on different isolated populations of D3 (day 3) CB Treg cells are shown. [Figure 65C] Histograms and contour plots of CXCR4 expression on different isolated populations of D3 (day 3) CB Treg cells are shown. [Figure 66] Contour plots for CXCR4 expression in D3 (day 3) CB Treg cells are shown. Figure 66A shows data for culture 1. Figure 66B shows data for culture 2. Figure 66C shows data for culture 3. [Figure 67] A line graph showing the number of cells in the categories "CXCR4 enriched", "CXCR4 negative" and "Treg control" at various culture days is shown. The graph shows the combined data from all three cultures (culture 1, culture 2 and culture 3). [Figure 68A] Flow cytometry analysis of cell populations on day 12 is shown. [Figure 68B] Flow cytometry analysis of cell populations on day 12. Figure 68A shows CXCR4 expression in CXCR4 enriched cells vs. Treg control cells. Figure 68B shows intracellular expression of FOXP3 and Helios in D12 (day 12) CXCR4 enriched CBTreg cells. [Figure 69]1 shows a graph depicting the results from a cell suppression assay from various fractions of a representative CXCR4 enriched cell culture. [Figure 70A] D12 (day 12) Transwell migration of CXCR4 enriched CBTreg cell populations is shown. [Figure 70B] Figure 70 shows transwell migration of CXCR4 enriched CBTreg cell populations at D12 (day 12). Figure 70A shows a schematic of the transwell migration assay experiment. Figure 70B shows micrographs of different cell concentrations of Treg control, CXCR4 negative and CXCR4 enriched CB Treg cells in the lower chamber of the transwell migration assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Healthy regulatory T cells (Tregs) protect the body from autoreactive cytotoxic T cells by avoiding thymic deletion or preventing the activation and proliferation of these cells that recognize extrathymic antigens. Thus, Tregs are important for homeostasis and immune control, and for protecting the host against the development of autoimmunity. Furthermore, both infused and innate Tregs are subject to i) proliferation of effector T cells that produce excess IL-2, which is essential for Treg survival, and ii) inflammation areas due to homing signals released by damaged antigen-presenting cells / dendritic cells present in the tissue.

[0027] Several types of Tregs have been described, but the best-characterized and most potent subset expresses CD4 and high levels of CD25 (IL-2Rα) and FoxP3, the forkhead box P3 gene product, and CD127. lo These CD4 + CD25 + FoxP3 + CD127 loTregs can be further subdivided into natural Tregs (nTregs), which arise in the thymus and undergo thymic selection, and endogenous Tregs (iTregs), which arise in the periphery under the influence of cytokines such as transforming growth factor β (TGFβ). (See Ohkura et al., Immunity 38(3):414-23(2013)).

[0028] In their endogenous state, Treg cells play a key role in maintaining immune homeostasis and limiting autoimmune responses by regulating both innate and adaptive immunity. Treg cells are essential for immune homeostasis by maintaining peripheral immune tolerance and inhibiting autoimmune responses and pathogenic tissue damage. (See Burrell et al., J. Immunol 189(10):4705-11(2012); Schneidawind et al., Blood 122(18):3116-21(2013); and Tang et al., Col Spring Harb Perspect Biol 5(11):a015552(2013)). However, in autoimmune diseases, defective endogenous Tregs are unable to effectively protect the body against the onslaught of autoreactive cytotoxic / effector T cells.

[0029] One hurdle to the development of Treg therapy is the instability of regulatory T cells, which often "flip" to a proinflammatory effector T cell phenotype. For example, Treg cells can downregulate FOXP3 expression and thereby acquire effector T cell-like functions through the activity of the E3 ubiquitin ligase Stub1 and in an Hsp70-dependent manner (Chen et al., Immunity. 2013 Aug 22;39(2):272-85).

[0030] To address this challenge, the present disclosure uses Tregs derived from umbilical cord blood. Umbilical cord blood is less immunogenic and is available in excess in public and private cord blood banks. Umbilical cord blood (CB) differs from peripheral blood (PB) in that it has different epigenetic characteristics and different ratios of blood cells due to its high suppressive properties. Furthermore, cord blood cells are primitive, less immunoreactive, naive, exhibit higher proliferation index, and can function across human leukocyte antigen (HLA) boundaries. The cord blood source is unique because Tregs derived from umbilical cord blood are naive, more suppressive and less plastic compared to other Treg sources. Similarly, because cord blood cells are constantly stimulated by many cytokines during the stress of childbirth, they are less sensitive to possible toxic environmental substances.

[0031] Another hurdle to the development of Treg therapy is a clinically relevant number of cells that can be repeatedly infused over a period of time to quell ongoing inflammation. Disclosed herein is a method for producing an expanded population of human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit, where the population is enriched for Treg cells expressing CXCR4 (CXC motif chemokine receptor 4). Treg cells enriched for CXCR4 expression preferentially home to the bone marrow, shortening the transit time from the point of injection to the site of bone marrow inflammation. Splenomegaly is one of the major clinical symptoms of primary myelofibrosis and is directly related to splenic extramedullary hematopoiesis (EMH). Alterations in the CXCL12 / CXCR4 pathway can also result in splenic EMH due to migration of clonal hematopoietic cells from the bone marrow to the spleen (Song et al., Int J Mol Sci, 2018.19:(3)). Uniform delivery of healthy allogeneic CXCR4-expressing Treg cells can result in the resolution of inflammation and restoration of normal hematopoiesis.

[0032] Also disclosed herein is a population of activated human CXCR4-expressing enriched Treg cells produced by the methods described herein. Further disclosed herein is a method for treating a disease or disorder by administering to a subject an effective amount of a population of activated human Treg cells. Further disclosed herein is a method for cryopreserving an expanded population of activated human Treg cells produced from at least one cryopreserved human umbilical cord blood unit. Further disclosed herein is a population of immunosuppressive Treg cells.

[0033] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, shall control. Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" are understood to mean the inclusion of an entirety or group of entireties, but not the exclusion of any other entirety or group of entireties. Unless otherwise required by context, the singular includes the plural and the plural includes the singular. Any examples following the term "eg" or "for example" are not meant to be exhaustive or limiting.

[0034] As used herein, the terms "a," "an," and "the" are understood to be in the singular or plural, unless specifically stated otherwise or clear from the context.

[0035] As used herein, the term "or" is understood to be inclusive unless specifically stated otherwise or clear from the context.

[0036] The term "about" immediately preceding a numerical value means ±0% to 10% of the numerical value, ±0% to 10%, ±0% to 9%, ±0% to 8%, ±0% to 7%, ±0% to 6%, ±0% to 5%, ±0% to 4%, ±0% to 3%, ±0% to 2%, ±0% to 1%, ±0% to less than 1%, or any other value or range of values ​​therein. For example, "about 40" means ±0% to 10% of 40 (i.e., 36 to 44).

[0037] A population of "activated" Treg cells may be defined as a homogenous cell population generated as a result of continuous exposure to high concentrations of interleukin-2 (IL-2) under culture conditions and at cell densities specified herein, in the presence of T cell receptor (TCR) stimulation with CD3 / 28 beads that allows stimulated Treg cells to provide consistent suppression of inflammation.

[0038] As used herein, "antibody fragment" or "antigen-binding fragment" refers to a molecule other than a conventional or intact antibody that contains a portion of a conventional or intact antibody that contains at least the variable region that binds to an antigen. Examples of antibody fragments include Fv, single chain Fv (scFv), Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, and V. H These include, but are not limited to, single domain antibodies (VHH) which contain only the domain.

[0039] As used herein, the term "patient" or "subject" is used interchangeably herein to refer to any mammal, including humans, farm animals and livestock, as well as zoo animals, sports animals, and pet animals such as dogs, horses, cats, and agricultural animals such as cows, sheep, pigs, and goats. One preferred mammal is a human, including adults, children, and the elderly. The subject may also be a pet animal such as a dog, cat, and horse. Examples of agricultural animals include pigs, cows, and goats.

[0040] As used herein, unless otherwise indicated, the terms "treat," "treating," "treatment," and the like refer to reversing, alleviating, inhibiting, or preventing the process of the disease, disorder, or condition to which such term applies, or one or more symptoms of such disease, disorder, or condition, and include administration of any of the compositions, pharmaceutical compositions, or dosage forms described herein to prevent the onset of symptoms or complications, or to alleviate symptoms or complications, or to eliminate the disease, condition, or disorder. In some cases, treatment is curative or ameliorative.

[0041] As used herein, "prevent" means to prevent, in whole or in part, or to ameliorate or control, or to reduce or stop the producing or occurrence of the thing or event to be prevented, e.g., a disease, disorder or condition.

[0042] As used herein, phrases such as "therapeutically effective amount" and "effective amount" refer to the amount required to be administered to a patient, or to a patient's cells, tissues, or organs to achieve a therapeutic effect, such as to ameliorate or replace a therapeutic effect. An effective amount is sufficient to elicit the biological or medical response in a cell, tissue, system, animal, or human that is desired by a researcher, veterinarian, physician, or clinician. Determination of an appropriate effective amount or therapeutically effective amount is within the routine level of skill in the art.

[0043] As used herein, the terms "administering," "administer," "administration," and the like refer to any mechanism of movement, delivery, introduction, or transport of a therapeutic agent to a subject in need of treatment with the therapeutic agent. Such mechanisms include, but are not limited to, intraocular, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

[0044] The term "CXCR4 enrichment," as used herein, refers to the increase in CXCR4 expression (or CXCR4 expression) in a population compared to a population not subjected to the method.+ ) Refers to a population of Treg cells that has been subjected to a method to increase the number of Treg cells.

[0045] Methods for generating expanded populations of regulatory T cells Because Treg cells are present at low frequencies in circulating blood or umbilical cord blood, CD4 + CD25 + Ex vivo enrichment and expansion of Treg cells with this phenotype is required.

[0046] In any of the methods described herein, the cord blood bank and donor may be qualified prior to using the human cord blood in the methods described herein. In some embodiments, the units of human cord blood are provided by public cord blood banks in the United States, the European Union, or other regions that meet the donor qualification criteria. Qualification of the cord blood unit may include verification that the donor has no evidence of relevant infectious diseases based on screening and testing. Additional selection criteria may be applied, including one or more of maternal age, gestational age, total nucleated cell (TNC) count, cell viability before freezing, cryopreserved volume, collection date, storage conditions, race, ethnicity, maternal donor history (e.g., infectious disease history, travel history), family medical history, cytomegalovirus seropositivity, gestational diabetes, hypertension, and the like. Selection criteria may be relevant to ensure the consistency of the cord blood unit prior to use. Cord blood selection criteria for various products, including populations of activated human Treg cells, are provided in Figures 29 and 30.

[0047] In some embodiments, the cell starting material (CBU) is thawed, washed, and isolated using immunomagnetic selection to identify CD25 + Enriched for mononuclear cells (MNC). CD25 +The MNCs are placed into a gas-permeable culture device containing interleukin-2 (IL-2) and anti-CD3 / anti-CD28 beads. The cells are expanded in culture for up to 10 days, up to 12 days, or up to 14 days. In some embodiments, the cells are expanded in culture for 8-10 days or 10-12 days. On day 8, 9, 10, 11, 12, or 14, the expanded cells are harvested and washed, and the CD3 / CD28 beads are immunomagnetically removed. The bead-free cells are then formulated and packaged.

[0048] In some embodiments, a method for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human cord blood unit, the method comprising the steps of: a) thawing the cryopreserved human cord blood unit; b) diluting and washing the thawed cord blood unit in a functionally closed system or in a closed system; and c) detecting CD25 + Isolating endogenous Treg cells using a double selection method based on cell surface expression; and d) isolating the isolated CD25 + and (b) expanding the Treg cells ex vivo in a gas-permeable culture vessel in medium in the presence of an effective amount of interleukin-2 (IL-2) and in the presence of a reagent that specifically binds CD3 and CD28 for up to 14 days, changing the medium about every 48 hours to produce a population of activated CD25+ Treg cells; and + and recovering the cells from the culture medium to produce an expanded population of activated human Treg cells. In some embodiments, the activated human Treg cells have a specific phenotype. In some embodiments, the method further comprises using an algorithm to select optimal cryopreserved cord blood units prior to the thawing step (i.e., step a)). In some embodiments, the method further comprises releasing an expanded population of activated human Treg cells having a characteristic phenotype for clinical use based on defined criteria after the harvesting step (i.e., step f)).

[0049] In some embodiments, a single cord blood unit (CBU) is used. In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) pooled CBUs are used. In some embodiments, two to four pooled CBUs are used. In some embodiments, the CBUs are collected from healthy donors and frozen prior to use.

[0050] In some embodiments, the cryopreserved human cord blood unit is thawed in a single step in a water bath (e.g., at 37°C + / - 1 degree). In some embodiments, thawing the cryopreserved cord blood unit involves gently massaging the bag while it is submerged in a 37°C (+ / - 1 degree) water bath until the bag feels semi-thawed. The cells are then immediately transferred for a washing process.

[0051] In some embodiments, the thawed cord blood unit is subjected to an automated washing using an automated cell processing system (e.g., a functionally closed system or a closed system). In some embodiments, the automated cell processing system is the Sepax system (Biosafe). The Sepax system is a centrifugation and pump device intended for use in cell therapy where specific blood components need to be isolated. Its principle is based on centrifugation and allows separation according to the density and size of blood particles. The blood components are collected in individual bags and are immediately available for transfusion. The automated cell processing system may allow a starting volume of up to 100 ml up to a final volume of 50-150 ml. The dilution ratio between the initial volume and the dilution volume is adjustable in the range of 0.5-2.0 times. The washing cycles may include one cycle of standard washing or, in certain circumstances, two cycles of high washing. The automated cell processing system is programmed to automatically perform the initial product dilution, osmolarity recovery, washing, centrifugation, supernatant extraction, and cell resuspension. Usually, the starting volume is set to 25 mL. The final volume is set to 100 mL and the dilution factor is set to 1.0. The wash reagent contains 5% human serum albumin (HSA) (CSL Behring) and 10% dextran-40 (D-40) (Hospira). After washing, the cord blood cells are collected in the cord blood wash bag.

[0052] In some embodiments, the basic wash medium comprises about 20 ml of 25% HSA and about 1000 ml of PBS / EDTA buffer. In some embodiments, the working wash medium comprises about 300 ml of basic wash buffer and about 50 mg of magnesium chloride (MgCl2) and about 2500 units of DNase. In some embodiments, the modified medium comprises X-Vivo15 medium (Lonza) and about 10 ml of GlutaMAX-1 and about 100 ml of thawed human AB serum. In some embodiments, the wash medium comprises PBS, EDTA, and 0.5% HSA.

[0053] In some embodiments, the washing step does not include manual washing.

[0054] In some embodiments, the automated washed cord blood cells undergo an additional manual wash using working wash medium, where a final volume of 200 mL is constructed and the reconstituted cells are centrifuged at 300 g for 10 minutes at room temperature. Finally, the washed cells are diluted to 100 x 10 in 0.09 ml. 6 Resuspend at a concentration of 1 cell.

[0055] In some embodiments, the reagent that specifically binds to CD25 is an anti-CD25 antibody or an antigen-binding fragment thereof. In some embodiments, the reagent that specifically binds to CD25 is conjugated to a solid support. In some embodiments, the solid support is a bead, a column, or a plate. In some embodiments, the solid support is a magnetic microbead. In some embodiments, the bead comprises cellulose, cellulose derivatives, acrylic resins, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene, polyacrylamide, latex gel, polystyrene, dextran, rubber, silicone, plastic, nitrocellulose, natural sponge, controlled pore glass, metal, cross-linked dextran, or agarose gel.

[0056] In some embodiments, the CD25 microbeads are 100×10 6 CD25 microbeads are added to the washed cord blood cells at a ratio of 0.02 ml per cell. The cells and microbeads are incubated together for 30 minutes at 4° C. In some embodiments, an LS column (Miltenyi) made of ferromagnetic spheres is used in combination with an external magnetic field, allowing unlabeled cells to pass freely while magnetically labeled CD25 + The cells are kept in suspension within the column and do not actually "bind" the column matrix. This suspension allows for efficient sterile washing by minimizing stress on the cells and avoiding cell clumping. The LS column is primed with working wash medium and loaded with CD25 labeled microbeads. +The cells are allowed to pass through the LS column attached to the magnetic field. The LS column is then removed from the magnetic field and a plunger is used to push out the loosely held cells that are bound to the CD25 microbeads and labeled as positive fraction 1. In the double selection method, positive fraction 1 now behaves as the starting solution that can be passed through the primed LS column, and the steps are repeated where positive fraction 2 is collected, and finally, the two positive fractions are mixed to produce the CD25 microbeads for the final selection. + In some embodiments, CD25 cells are isolated using a double ferromagnetic column (e.g., LS column) method. + Isolate the cells.

[0057] In some embodiments, the reagent that specifically binds to CD3 and CD28 comprises an anti-CD3 antibody or antigen-binding fragment thereof, and an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the reagent that specifically binds to CD3 and CD28 comprises an anti-CD3 coated bead and an anti-CD28 coated bead (i.e., "anti-CD3 / anti-CD28 coated beads"). In some embodiments, the anti-CD3 coated beads and the anti-CD28 coated beads are in a 1:1 ratio in the reagent that specifically binds to CD3 and CD28. In some embodiments, the anti-CD25 coated beads are in a 1:1 ratio in the reagent that specifically binds to CD3 and CD28. + When cells are cultured in the presence of a reagent that specifically binds to CD3 and CD28, CD25 + The cells and anti-CD3 / anti-CD28 coated beads are in a 1:1 ratio.

[0058] In some embodiments, the effective amount of IL-2 used in the method for producing an expanded population of activated human Treg cells is up to about 1000 IU / ml. In some embodiments, the effective amount of IL-2 is about 1000 IU / ml. In some embodiments, the IL-2 is human IL-2. In some embodiments, the isolated CD25 + Treg cells are suspended in medium containing IL-2 immediately prior to the culture step of the methods described herein.

[0059] In some embodiments, during the culturing step, the medium is changed about every 48 hours without disturbing the cells, hi some embodiments, the culture is not mixed and resuspended during the culturing step of the methods described herein.

[0060] In some embodiments, about 1×10 6 CD25 + The cells / ml are cultured in the presence of a reagent that specifically binds CD3 and CD28 in a method to produce an expanded population of activated human Treg cells. In some embodiments, CD25 + The cells were initially cultured at 10 cm 2 In some embodiments, the culture is then cultured in a gas permeable incubator having a membrane surface area of ​​100 cm. 2 The cells are transferred to a gas permeable incubator having a membrane surface area of ​​1000 μm.

[0061] In some embodiments, about 0.5×10 9 ~Approx. 12×10 9 , or about 1 × 10 9 ~Approx. 2×10 9 Activated CD25 + The cells are harvested after 14 days of culture in the presence of a reagent that specifically binds to CD3 and CD28. In some embodiments, the manufacturing process described herein comprises the steps of: + CD25 + This results in a 50-fold or greater expansion of the Treg population. In some embodiments, the expanded population of activated human Treg cells is cryopreserved after the harvesting step. In some embodiments, the expanded population of activated human Treg cells is not cryopreserved after the harvesting step and is rapidly released for administration.

[0062] Additionally, a method for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human cord blood unit is provided, the method comprising: a) thawing the cryopreserved human cord blood unit in a single step in a water bath; b) diluting and washing the thawed cord blood unit in a solution comprising PBS, EDTA, and about 0.5% human serum albumin in a functionally closed system with no manual washing; and c) isolating and isolating CD25 Treg cells from at least one cryopreserved human cord blood unit using a dual ferromagnetic column method. + isolating endogenous Treg cells using a double selection method based on cell surface expression; and d) culturing the isolated CD25+ Treg cells in the presence of about 1000 IU / ml interleukin-2 (IL-2) in medium in a gas-permeable incubator and in the presence of anti-CD3 and anti-CD28 coated beads for up to 10 days, up to 12 days, or up to 14 days. + and ex vivo expansion of Treg cells, said medium being replaced about every 48 hours to induce activation of CD25 + To generate a population of Treg cells, + Treg cells and the anti-CD3 and anti-CD28 coated beads are in a 1:1 ratio, the cultures are not mixed or resuspended, and e) activated CD25 + and harvesting the cells to produce an expanded population of activated human Treg cells.

[0063] After harvesting, the Treg cells may be tested for contamination, viability, purity, cell number counted, and / or examined using flow cytometry.

[0064] In some embodiments, the active ingredient (DS) is a T-regulatory T cell phenotype (CD4 + CD25 + In some embodiments, the DS is a liquid cell suspension that comprises or consists of nucleated cord blood cells, about 60% or more of which have a regulatory T cell phenotype (CD4 + CD25 +), and approximately less than 10% have a T cytotoxic / suppressor cell phenotype (CD4 - CD8 + In some embodiments, the Final Product (DP) is a liquid cell suspension comprising or consisting of the active ingredient suspended in an excipient solution comprising or consisting of Plasma-Lyte A with 0.5% human serum albumin (HSA) in a final volume of 50 mL.

[0065] In some embodiments, the conditional CD8 + A cell depletion step can be optionally performed to determine the CD4 T cell population in the activated Treg cell population before the final preparation. - CD8 + Used to reduce the content of cytotoxic / suppressor T cells. Prior to harvest, CD8 cells are depleted from the medium using a reagent that specifically binds to CD8 (i.e., an anti-CD8 antibody or an antigen-binding fragment thereof). + Cells can be depleted to remove any cells that bind to the reagent. In some embodiments, the reagent can be conjugated to a solid support, such as beads, columns, and plates. For example, the beads can be magnetic microbeads coated with anti-CD8 antibodies. The beads can be made from any material commonly used in the art, including, but not limited to, cellulose, cellulose derivatives, acrylic resins, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, vinyl and acrylamide copolymers, polystyrene cross-linked with divinylbenzene, polyacrylamide, latex gel, polystyrene, dextran, rubber, silicon, plastic, nitrocellulose, natural sponge, silica gel, controlled pore glass, metal, cross-linked dextran, and agarose gel.

[0066] CD8 + Following cell depletion, the methods described herein include the step of isolating CD4 - CD8 + The method may further include analyzing the cells remaining in the medium for the presence of CD4 - CD8 +The method may include determining the number of cells remaining in the medium that are CD4+. - CD8 + If the cells are CD8 + Cell depletion can be performed.

[0067] At the end of cell culture, the concentration was 3 × 10 6 If there are more than 100 per cell, an additional step can be performed to remove the anti-CD3 / anti-CD28 coated beads.

[0068] Criteria for releasing expanded populations of activated human Treg cells with a distinctive phenotype for clinical use include 7-amino-actinomycin-D (7-AAD) viability of 70% or more, CD4 + CD25 + These may include purity, "organism-free" Gram stain, and endotoxin less than 5EU / kg.

[0069] In some embodiments, large volumes of product can be generated with extensive expansion of up to 1000-fold, with final populations of cells ranging from approximately 0.5×10 cells harvested after up to 14 days of culture. 9 ~12×10 9 The final product is homogenous and well-defined Treg cells ranging from 10 to 100 Treg cells. In some embodiments, the final product may remain stable for up to 8 hours when stored at room temperature and 96 hours when stored at 4°C.

[0070] A. Methods for Producing Expanded Populations of CXCR4-Expressing Regulatory T Cells Exploiting CXCR4 expression on the Treg cell surface allows homing of Treg cells to areas of dysregulation of the CXCR4 / CXCL12 axis. In the case of primary myelofibrosis, these areas would be the bone marrow and potentially ongoing extramedullary hematopoiesis from clonal hematopoietic cells in the spleen. Targeting the CXCR4 / CXCL12 axis allows repeated infusion of CB Treg cells to capture any residual disease or escape cells.

[0071] CK0804 is a product that has undergone additional enrichment to ensure that there is consistent cell surface expression of CXCR4 in the cultured CB Treg cells, and that the cell surface expression of CXCR4 is retained during the freeze / thaw process. The starting material for CK0804 remains HLA-independent but meets predefined eligibility tests for manufacturing purposes. The one-batch manufacturing process results in multiple doses that are cryopreserved at a single dose of 100 million cells each. The product generated from a single expansion can be cryopreserved and administered to different patients. The same patient can receive multiple doses from different batches.

[0072] A population of CB-derived CXCR4-expressing Treg cells (e.g., CK0804 product) was isolated by thawing and washing the CBU and transfecting the CBU with CD25 + Starting with immunomagnetic enrichment of mononuclear cells (MNC), the process can be completed in 10-12 days. + The enriched MNCs are placed in culture with X-Vivo 15 supplemented with GlutaMAX (2 mM) and IL-2 (1000 IU / mL), and anti-CD3 / CD8 beads, and incubated at 37°C, 5% CO2. The cultured cells are enriched for CXCR4 at the third feed of culture expansion. Enrichment can be done by using magnetic microbeads coated with anti-CXCR4 antibodies to isolate CXCR4 expressing Treg cells from the medium. Final formulation and loading of the CK0804 end product (DP) is done on the last day of the expansion process on day 4 or 5. If necessary, the cells can be enriched for CD4 - CD8 + To ensure that the T cytotoxic / suppressive cell content is less than 10%, cells undergo CD8 depletion prior to harvesting and final formulation.

[0073] Ex-vivo expanded Treg cells are harvested, washed, depleted of residual CD3 / CD28 beads, and formulated in an insoluble cryopreservation medium containing 10% dimethylsulfoxide (DMSO). This product can then be cryopreserved in cryobags utilizing a controlled rate freezer and transferred to vapor phase LN2 (<-150°C).

[0074] CXCR4 from cryopreserved human umbilical cord blood units + 1. A method for producing an expanded population of human Treg cells enriched for Treg cells, the method comprising: (a) thawing a cryopreserved human umbilical cord blood unit; (b) diluting and washing the thawed cord blood unit in a functionally closed system; and (c) detecting CD25 + (d) ex vivo expansion of the isolated CD25+ Treg cells in a gas-permeable culture vessel in medium in the presence of (1) an effective amount of interleukin-2 (IL-2), (2) a reagent that specifically binds CD3 and CD28, and (3) anti-CXCR4 magnetic microbeads for up to 10 days or up to 12 days, with the medium being changed about every 48 hours to detect CD25+ Treg cells. + (e) generating a CXCR4-enriched culture of Treg cells; and + CXCR4 + Cells were harvested from the culture and stained for CXCR4 + producing an expanded population of human Treg cells enriched for Treg cells.

[0075] In some embodiments, in step (d), (1) ex vivo expansion is initiated on day 0. (2) an effective amount of IL-2 is administered to isolated CD25 + (3) adding a reagent that specifically binds CD3 and CD28 to the isolated CD25 + The Treg cells are added on day 0. In some embodiments, in step (d), an effective amount of IL-2 is administered to the isolated CD25 +The medium containing the Treg cells is added approximately every 48 hours.

[0076] In some embodiments, the reagents that specifically bind CD3 and CD28 are added to the medium twice: once with IL-2 at the beginning of the expansion step, and a second time after the anti-CXCR4 magnetic microbeads are removed. In some embodiments, the reagents that specifically bind CD3 and CD28 are removed from the medium before the anti-CXCR4 magnetic microbeads are added to the medium. In some embodiments, the reagents that specifically bind CD3 and CD28 are added back to the medium after the CXCR4 enrichment step is completed. In some embodiments, the reagents that specifically bind CD3 and CD28 include anti-CD3-coated beads and anti-CD28-coated beads (e.g., magnetic microbeads). In some embodiments, the anti-CD3-coated beads and anti-CD28-coated beads are removed by immunomagnetic methods.

[0077] In some embodiments, in step (d), the anti-CXCR4 magnetic microbeads are added to the medium 3 or 4 days after ex vivo expansion has commenced. In some embodiments, in step (d), the anti-CXCR4 magnetic microbeads are added to the medium 3 or 4 days after ex vivo expansion has commenced. + It is added to the culture medium for approximately 30 minutes before being used to isolate Treg cells.

[0078] In some embodiments, the effective amount of IL-2 is about 1000 IU / ml.

[0079] In some embodiments, the isolated CD25 + Treg cells are enriched for CXCR4 at the third feed of ex vivo expansion.

[0080] In some embodiments, step (d) is carried out for 4 or 5 days.

[0081] CXCR4 from cryopreserved human umbilical cord blood units +1. A method for producing an expanded population of human Treg cells enriched for Treg cells, the method comprising: (a) thawing a cryopreserved human umbilical cord blood unit; (b) diluting and washing the thawed cord blood unit in a functionally closed system; and (c) detecting CD25 + (d) ex vivo expansion of the isolated CD25+ Treg cells in a gas-permeable culture vessel in a medium, the ex vivo expansion step comprising: (1) on day 0, injecting anti-CD3 and anti-CD28 coated beads into the CD25+ Treg cells in the medium; + (1) on day 2, adding about 1000 IU / mL of IL-2 to the medium; (2) on day 2, removing the anti-CD3 and anti-CD28 coated beads from the medium and adding anti-CXCR4 magnetic microbeads to the medium; and (3) on day 3 or 4, removing the anti-CD3 and anti-CD28 coated beads from the medium and adding anti-CXCR4 magnetic microbeads to the medium. + The anti-CXCR4 magnetic microbeads attached to the Treg cells were removed from the medium, and fresh anti-CD3 and anti-CD28 coated beads were added to the CXCR4 magnetic microbeads. + adding to the Treg cells, ex vivo expansion for up to 10 days or up to 12 days, changing the medium about every 48 hours, and + (e) producing a CXCR4 enriched culture of Treg cells; and + CXCR4 + Cells were harvested from the culture medium and then subjected to immunofluorescence assay using CXCR4 + and producing an expanded population of human Treg cells enriched for Treg cells.

[0082] In some embodiments, in step (d), IL-2 is administered to isolated CD25 + The medium containing the Treg cells is added approximately every 48 hours.

[0083] In some embodiments, the anti-CD3 coated beads and the anti-CD28 coated beads are in a 1:1 ratio. +The cells and anti-CD3 and anti-CD28 coated beads are in a 1:1 ratio.

[0084] In some embodiments, in step (e), about 1×10 6 CD25 + Cells / mL are cultured.

[0085] In some embodiments, in step (e), the cells are initially cultured at 10 cm 2 In some embodiments, the culture is then cultured in a gas permeable incubator having a membrane surface area of ​​100 cm. 2 In some embodiments, the culture is not rocked or agitated when IL-2 is added in step (d).

[0086] In some embodiments, in step (a), the cryopreserved human umbilical cord blood unit is thawed in a single step in a water bath.

[0087] In some embodiments, step (b) does not include manual washing. In some embodiments, step (b) is performed in a solution comprising PBS, EDTA, and about 0.5% human serum albumin.

[0088] In some embodiments, in step (c), a dual ferromagnetic column method is used to detect CD25 + Isolate Treg cells.

[0089] In some embodiments, the method comprises administering to a subject a subject comprising administering to said subject a CXCR4 + The method further comprises cryopreserving the expanded population of human Treg cells enriched for Treg cells.

[0090] An exemplary manufacturing process for producing an enriched population of Treg cells expressing CXCR4 (e.g., CK0804 product) is provided below. + The cell depletion step is optional and %CD3 + CD4 - CD8+ This is only performed if the cell population represents >10% of culture-grown cells.

[0091] Step 1: Thaw the CBU (Day 0) Input: CBU Output: CBU after decompression Frozen CBUs are removed from LN2 vapor storage and placed in a plastic overwrap bag to prevent contamination of the port during thawing. The wrapped cryobag is rapidly thawed by immediately placing it in a 37°C water bath and gently kneading the bag to ensure uniform thawing. The output, thawed CBUs, are sampled for: Total nucleated cell (TNC) count (cellometer) % NC (nucleated cell) viability (cellometer) The test results are used to monitor the process.

[0092] Step 2: Dilute and wash CBU (Day 0) Input: Thawed CBU Output: CBU after cleaning Immediately after rapid thawing, the contents of the post-thaw CBU bag are attached to the input line of the Sepax single-use disposable kit. The cells are diluted and washed in the Sepax system with 10% LMD in 0.9% NaCl. The output of the Sepax wash (Post-Wash CBU) is approximately 100 mL and is sampled for: Total nucleated cell (TNC) count (cellometer) % NC (nucleated cell) viability (cellometer) %NC Viability (Flow Cytometry-7-AAD) %CD3+CD4-CD8+(flow cytometry) %CD3+CD4+CD25+(flow cytometry) The test results are used to monitor the process.

[0093] Step 3: Pre-selection wash (day 0) Input: CBU after cleaning Output: CB mononuclear cells (MNC) The washed CBU cells are centrifuged at 300 x g for 10 min at room temperature. After removing the supernatant by gentle aspiration, the cells (CB MNCs) are resuspended in a volume of approximately 8-10 mL in Miltenyi PBS / EDTA buffer supplemented with Pulmozyme and MgCl2. The output, CB MNCs, are not sampled.

[0094] Step 4: CD25 antibody incubation (day 0) Input:CB MNC Output: CB MNC after incubation CB mononuclear cells are incubated with Miltenyi anti-CD25 microbeads for 30 minutes at 2-8°C with automated mixing. After incubation, the cell and anti-CD25 microbead mixture is washed and resuspended in Miltenyi PBS / EDTA buffer supplemented with Pulmozyme and MgCl2 in a volume of approximately 10 mL. The output, post-incubation CB MNCs, is not sampled.

[0095] Step 5: CD25 positive selection (day 0) Input: CB MNC after incubation Output: CD25 + MNC Following the incubation step with the Miltenyi CD25 antibody reagent, the incubated CB MNCs are transferred into a Miltenyi LS column attached to a MidiMACS device, which captures the anti-CD25 labeled cells by using a magnet. After immunomagnetic selection, the cells are released from the magnetic field. This selection is performed on each of the two separate fractions of incubated CB MNCs, and the output is combined. Output, CD25 + The MNCs are sampled for: ·TNC number (cellometer) %NC Viability (Cellometer) % Viability NC (Flow Cytometry - 7-AAD Flow Cytometry) %CD3+CD4-CD8+(flow cytometry) %CD3+CD4+CD25+(flow cytometry) ·Sterility (negative fraction) The test results are used to monitor the process.

[0096] Step 6: Initiating culture growth (day 0) Input: CD25 + MNC Output: Day 0 of culture CD25 + The MNCs selected in are washed and suspended in X-Vivo15 with 1% glutamine and 10% human AB serum with interleukin-2 (IL-2, 1000 IU / mL), then mixed with CD3 / CD28 beads at a 1:1 ratio of cells to beads. The cell+bead mixture is transferred to the G-Rex 10M system and incubated at 37°C, 5% CO2. There is no rocking or agitation of the cell suspension. No sampling is performed at this step.

[0097] The Bioreactor 10M system consists of a cylindrical, sterile, single-use, disposable plastic device. After the cells and media are transferred to the bioreactor, the cells reside at the bottom of the vessel and the surface is gas permeable. The gas permeable membrane of the Bioreactor 10M system is 10 cm 2 The system is placed in a conventional incubator but can be removed intermittently as needed for sampling, media removal, media addition, or cell harvesting.

[0098] Step 7: Addition of IL-2 (Day 2) Input: Day 0 of culture Output: Day 2 culture + IL-2 On day 2, fresh IL-2 is added to the cultured cells in the G-Rex 10M system at 1000 IU / mL to replenish any IL-2 that may have been consumed. No sampling is performed at this step. The cells in the G-Rex 10M system are returned to incubation at 37° C. and 5% CO2. The cell suspension is not rocked or agitated. To monitor the culture-growth process, sampling for lactate in the culture supernatant is obtained at harvesting days 2 or 3, 4, 6, 8, 10, 12, and 14.

[0099] Step 8: CXCR4 Enrichment (Day 3 or Day 4) Input: Day 2 culture + IL-2 Output: Day 3 or Day 4 CXCR4 enriched cells On day 3 or 4, cultured cells are incubated with Miltenyi anti-CXCR4 microbeads for 30 min at 2-8 °C with automated mixing. After incubation, the mixture of cells and anti-CXCR4 microbeads is washed and resuspended in a volume of approximately 10 mL in Miltenyi PBS / EDTA buffer supplemented with Pulmozyme and MgCl2. The post-incubation cell culture, which is the output, is not sampled.

[0100] Step 9: CXCR4 enrichment selection (day 3 or day 4) Input: Cultured cells on day 3 or 4, after incubation Output: Day 3 or Day 4 CXCR4 enriched cells Following the incubation step with the Miltenyi CXCR4 antibody reagent, the post-incubation CB MNCs are transferred into a Miltenyi LS column attached to a MidiMACS device, which captures the anti-CXCR4 labeled cells by using a magnet. The output, CXCR4 enriched cells, are sampled for: ·TNC number (cellometer) %NC Viability (Cellometer) % Viability NC (Flow Cytometry - 7-AAD Flow Cytometry) %CD3+CD4-CD8+(flow cytometry) %CD3+CD4+CD25+(flow cytometry) %CD3+CD4+CD25+CXCR4+(flow cytometry) ·Sterility (negative fraction) The test results are used to monitor the process.

[0101] Step 10: Migrate and Feed (Day 3 or 4) Input: Day 4 CXCR4 enriched cultures Output: Day 4 culture of CXCR4 + IL-2 The CXCR4 enriched cells are returned to culture with fresh medium + IL-2 (1000 IU / mL) in the G-Rex 10M system. No sampling is performed at this step. The cells in the G-Rex 10M system are returned to incubation at 37°C and 5% CO2. There is no rocking or agitation of the cell suspension.

[0102] Step 11: Migrate and Feed (Day 6 or 7) Input: Day 3 or 4 culture + IL-2 Output: Day 6 or 7 culture + fresh medium + IL-2 On day 6 or 7 (less than 66 hours after the final media / IL-2 exchange), an aliquot of cells cultured in the G-Rex 10M system is removed and sampled for: ·TNC number (cellometer) %NC Viability (Cellometer)

[0103] TNC count and % viability are used to monitor the process of culture growth. The remaining cultured cells in the G-Rex10M system are transferred to the G-Rex100M system and fresh medium is added to 2x10 6 A concentration of 1000 cells / mL is obtained (X-Vivo15 with 1% glutamine and 10% human AB serum, IL-2 1000 IU / mL). The cells in the G-Rex 100M system are returned to incubation at 37° C. and 5% CO2. There is no shaking or agitation of the cell suspension.

[0104] The Bioreactor 100M system consists of a cylindrical, sterile, single-use, disposable plastic device. After transferring the cells and media to the G-Rex, the cells reside at the bottom of the vessel and the surface is gas permeable. The gas permeable membrane of the Bioreactor 100M system is 100 cm2 The system is placed in a conventional incubator but can be removed intermittently as needed for sampling, media removal, media addition, or cell harvesting.

[0105] Step 12: Addition of IL-2 (Day 8 or Day 9) Input: Day 6 or 7 culture + fresh medium + IL-2 Output: Day 8 or 9 culture + IL-2 On day 8 or 9 (<66 hours after the last media / IL-2 exchange), fresh IL-2 is added to the cultured cells in the G-Rex 100M system to replenish the IL-2 presumably consumed. The cell suspension is sampled for TNC and % viability (Cellometer) and used for process monitoring. The cells in the G-Rex 100M system are returned to incubation at 37° C. and 5% CO2. There is no rocking or agitation of the cell suspension.

[0106] Step 13: (Day 10) Input: Day 10 culture + IL-2 Output: Day 10. If CXCR4 meets criteria, collect (go to step 15). An aliquot of cultured cells in the G-Rex 100M system is removed and sampled for: ·TNC number (cellometer) %NC Viability (Cellometer) % Viability NC (Flow Cytometry - 7-AAD Flow Cytometry) %CD3+CD4-CD8+(flow cytometry) %CD3+CD4+CD25+(flow cytometry) %CD3+CD4+CD25+CXCR4+(flow cytometry) or Day 10 culture (if CXCR4 criteria are not met) On day 10, return the cells in the G-Rex 100M system to incubation at 37° C. with 5% CO2. Do not rock or agitate the cell suspension. Proceed to step 14.

[0107] Step 14: Addition of IL-2 (Day 10) Input: Day 8 or 9 culture + IL-2 Output: Day 10 culture + IL-2 On day 10 (<66 hours after the last media / IL-2 change), fresh IL-2 is added to the cultured cells in the G-Rex 100M system to replenish the IL-2 presumably consumed. The cell suspension is sampled for TNC and % viability (Cellometer) and used for process monitoring. The cells in the G-Rex 100M system are returned to incubation at 37° C. and 5% CO2. There is no rocking or agitation of the cell suspension.

[0108] Step 15: Pre-harvest sample (day 10 or 12) Input: Day 10 or 12 culture + IL-2 Output: 10 or 12 days before collection, sampling On days 10 or 12, prior to harvesting the culture-expanded T-Reg cells, the cell suspension is sampled for: Mycoplasma Mycoplasma test results will be included in the final release criteria.

[0109] After mycoplasma sampling, 60% of the total culture volume is removed from the cell suspension volume in the G-Rex100M system and the remaining culture medium is sampled as follows: ·TNC number (cellometer) %NC Viability (Cellometer) % Viability NC (Flow Cytometry - 7-AAD Flow Cytometry) %CD3+CD4-CD8+(flow cytometry) %CD3+CD4+CD25+(flow cytometry) %CD3+CD4+CD25+CXCR4+(flow cytometry)

[0110] TNC count and % viability are used to monitor the process. + CD4 - CD8 + is %CD3 + CD4 - CD8 + If the cell population represents >10% of culture-expanded cells, CD8 + Immunomagnetic depletion of cells is used to determine the need for CD8 depletion. If CD8 depletion is required, cells are depleted prior to harvest on day 10 or day 12.

[0111] Step 17: Harvest (Day 10 or 12) Input: 10 or 12 days before collection, sampling Output: T-Reg harvest Following sampling, transfer the remaining volume in the G-Rex 100M system to one or more 500 mL conical tubes in 150 mL increments while rinsing the G-Rex to optimize cell recovery. Bring the volume in each tube to 400 mL with injection buffer (Plasma-Lyte A with 0.5% HSA). Centrifuge the 500 mL conical tubes twice at 400 x g for 10 min at room temperature to wash the cells with Plasma-Lyte A with 0.5% HSA, and bring the cell suspension to a volume of 10 mL with Plasma-Lyte A with 0.5% HSA in a 15 mL conical tube for bead removal (step 15).

[0112] Conditional step: CD8 depletion (day 10 or 12) Input: T-Reg collection on day 10 or 12 Output: After CD8 depletion If the %CD3+CD4-CD8+ flow cytometry results from sampling in step 14 indicate that the CD3+CD4-CD8+ cell population represents >10% of culture-expanded cells, perform CD8 depletion. To deplete CD8, the T-Reg harvest is incubated with Miltenyi CD8 microbeads for 15 min at 4-8 °C with gentle agitation, then transferred to a Miltenyi LS column and then immunomagnetically selected using a MidiMACS device. The output after CD8 depletion is sampled for: ·TNC number (cellometer) %NC Viability (Cellometer) %CD3+CD4-CD8+(flow cytometry) NC counts and % viability are used to monitor the process. If %CD3+CD4-CD8+ is ≦10% after CD8 depletion, the process is restarted with the following step (wash and removal of CD3 / CD28 beads, step 15). If %CD3+CD4-CD8+ is >10%, a second CD8 depletion is performed.

[0113] Step 18: Washing and Removal of CD3 / CD28 Beads (Day 10 or 12) Input: 10th or 12th day of collection Output: T-Reg harvested material, debeaded material The 15 mL conical tube containing the harvested T-Reg cell suspension is placed in a Dynal MPC-1 magnet for 2 minutes. The supernatant (containing cells and no CD3 / CD28 beads) is collected in another 15 mL conical tube ("De-beaded #1") before releasing the magnet. Once the magnet is released, the remaining beads and cells are resuspended in 2 mL of Plasma-Lyte A with 0.5% HSA and placed in a Dynal MPC-1 magnet for 2 minutes, the supernatant is collected and transferred to the "De-beaded #1 tube". The "De-beaded #1" tube is then placed in a Dynal MPC-1 magnet for 2 minutes, and the supernatant is collected in another 15 mL conical tube ("De-beaded #2") before releasing the magnet. This process can be done up to three times depending on the harvest volume. The cell suspension in the "De-beaded #2" tube is now approximately 17 mL in volume and is pooled with the additional De-beaded #2 tube and sampled for: ·TNC number (cellometer) %NC Viability (Cellometer) %CD3+CD4-CD8+(flow cytometry) %CD3+CD4+CD25+(flow cytometry) %CD3+CD4+CD25+CXCR4+(flow cytometry) %NC Viability (Flow Cytometry-7-AAD) Residual beads The output of this step, the T-Reg harvest, de-beaded material, is the active ingredient (pharmaceutical substance). TNC counts and % viability (Celometer) are used to monitor the process. TNC is also used to calculate cell dose for lot release. %CD3+CD4-CD8+, %CD3+CD4+CD25+, %viability (7-AAD), and residual bead assays are used for final product release.

[0114] Step 19: Wash and enrich T-Regs (days 10–12). Input: T-Reg harvest, debeaded material Output: Washed / enriched T-Regs The T-Reg harvest, de-beaded, is transferred from the 15 mL conical tube to a 500 mL conical tube. The conical tube is rinsed with 10 mL Plasma-Lyte A + 0.5% HSA and this rinse is added to the 500 mL conical tube. The cell suspension in the conical tube is brought to a volume of 150 mL with Plasma-Lyte A + 0.5% HSA and centrifuged at 400 x g for 10 minutes at room temperature. The supernatant is removed, leaving the concentrated cell pellet in the tube.

[0115] Step 20: Add cryoprotectant and fill bags (day 10 or 12). Input: Washed / enriched T-Regs Output: CK0804 final product, before freezing. Add CryoStor® Freeze Medium CS10 to the concentrated cell pellet at a volume based on the total number of cells and a target final cell concentration of 10-12 Treg cells / mL. Add 10 mL (100 x 10) of the cell suspension containing CryoStor® CS10 to OriGen CryoStor® CS50N freezing bags. 6 Treg) or 30 mL (300 × 10 6 The cells are filled with the filling volume of Tregs. The remaining cell suspension is sampled for: Endotoxin ·Sterility Endotoxin and sterility results are used for release of the final product.

[0116] Step 21: Controlled rate freezing (day 10 or 12). Input: CK0804 final product, before freezing. Output: CK0804 final product frozen in bags. The bags containing the CK0804 final product are placed in aluminum cassettes, transferred to a controlled rate freezer, frozen according to a pre-set program, and then overwrapped in plastic bags, which are sealed and transferred to an LN2 vapor freezer for storage.

[0117] Process Control Process control at step 16 (pre-collection sample): %CD3 + CD4 - CD8 + If the cells (assayed by flow cytometry) are >10% then a conditional step (CD8 depletion) is performed. + CD4 - CD8 + If the post-CD8 depletion value for the cells is ≦10%, the manufacturing process resumes at step 17. + CD4 - CD8 + If the post-CD8 depletion value for cells is still >10%, a second CD8 depletion procedure is performed. This results in a final product of %CD3 + CD4 - CD8 + Ensure that lot release criteria for cells are met.

[0118] An overview of the analytical procedures for CK0804 is provided in Tables 1a and 1b.

[0119] [Table 1-1]

[0120] [Table 1-2]

[0121] B. Methods for characterizing expanded populations of CXCR4-expressing regulatory T cells Expanded populations of CXCR4-expressing regulatory T cells (eg, CK0804 products) can be characterized by the following methods.

[0122] Phenotypic Analysis: Fresh or "in process" samples from each run can be analyzed for phenotypic markers including CD3, CD4, CD25, CD127, CXCR4, FOXP3 and Helios on days 0, 3 and 12. Phenotypic analysis of cryopreserved samples can be performed for CK0804, CD3, CD4, CD25, CD127, CXCR4, FOXP3 and Helios. Samples can be used at predefined time points after cryopreservation. Post-thaw stability of the product can be analyzed for viability and phenotypic markers including CD3, CD4, CD25, CD127, CXCR4, FOXP3 and Helios. Phenotypic analysis of CK0804 can be performed using anti-human specific antibodies including CD4 / APC eFluor780 (Thermo Fischer Scientific, Waltham, MA, USA), CD8 / FITC (Becton Dickinson, Franklin Lakes, NJ, USA), CD25 / PE (Becton Dickinson, Franklin Lakes, NJ, USA), CD127 / Alexa Fluor647 (Becton Dickinson, Franklin Lakes, NJ, USA), CXCR4 (CD184) / PE-Cy™5 (Becton Dickinson, Franklin Lakes, NJ, USA) for surface staining, and FoxP3 / PECy7 (Thermo Fischer Scientific, Waltham, MA, USA) and Helios / APC (Thermo Fischer Scientific, Waltham, MA, USA) for intracellular staining. True-Nuclear Transcription Factor Buffer Set (BioLegend, San Diego, CA, USA) can be used for intracellular staining. As a viability dye, 7-amino-actinomycin-D (7 AAD, TONBO biosciences, Tucson, AZ, USA) for surface staining and Ghost Dye V510 (TONBO biosciences, Tucson, AZ, USA) for intracellular staining can be used, respectively.Cells are acquired using a BD FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA) or a BD LSRFortessa X20 (Becton Dickinson, Franklin Lakes, NJ, USA). Flow cytometry data can be analyzed using FlowJo software version 10 (Becton Dickinson, Franklin Lakes, NJ, USA).

[0123] Cellular suppression assay: Samples during the process can be collected and analyzed for their suppressive function on day 12. The suppressive function analysis can also be performed on samples collected at predetermined time points after cryopreservation.

[0124] Conventional T cells (Tcon) CD4 + twenty five -Phenotypes (ReachBio LLC, Seattle, WA) can be stained using CellTrace™ Violet Cell Proliferation Kit (Invitrogen, Carsbad, CA, USA) to track multiple generations using dye dilution by flow cytometry. 50,000 Tcon are seeded in 96-well U-bottom plates and co-cultured with different numbers of CB Tregs at ratios of 4:1, 2:1, 1:1, 1:2, 1:4, and 1:8. CD3 / 28 T cell activator beads are added at a ratio of 1:1 with Tcon cultured in RPMI medium 1640+Glutamax-I (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, heat inactivated (Thermo Fischer Scientific, Waltham, MA, USA). Cells are incubated at 37°C in a 5% CO2-in-air atmosphere for 4 days. Tcon proliferation can be measured using flow cytometry. Cells are acquired using either a BD FACSCanto or a BD LSRFortessaX20 flow cytometer. Data can be analyzed using FlowJo software. For CellTrace™-based measurements, inhibitory potency is calculated using the following formula: 100×(1−% CellTrace in target wells) 低 Tcon / %CellTrace 低 Tcon alone).

[0125] Migration Assay: The Transwell cell migration assay measures the chemotactic performance of cells towards chemoattractants. The purpose of this experiment is to capture the chemotaxis of an enriched population and compare it to a standard (non-enriched) population. It is expected that CXCR4-expressing enriched cells have the ability to cross the transwell membrane faster in response to SDF1α signaling. Place the desired amount of chemoattractant (SDF-1α=500ng / mL) in the bottom of the lower chamber in a 24-well plate. Place a 0.5 micron sized transwell chamber. Place 1x10 CXCR4 enriched cell population or standard on the top of the transwell. 6Cells are placed at a cell concentration of 1.0 cells / mL. Cells are allowed to migrate from the top chamber to the bottom well. Images and cell counts are captured at 0.5 hours, 1.0 hours, and 1.5 hours.

[0126] In vivo analysis can be performed to examine the biodistribution of injected CK0804 cells and to quantify their persistence in target organs and circulation. To detect the cell of origin and differentiation destination, CK0804 can be mismatched with donor cells on the HLA-A2 locus.

[0127] Bone marrow homing studies: The transit time of CXCR4-enriched or standard Tregs can be analyzed by assessing their concentration in the target organ of interest. 8-10 week old NOD-scid IL2Rγ cells purchased from Jackson Laboratories ヌル (NSG) mice can be used. CXCR4-enriched Tregs or standard Tregs can be added at 1×10 7 NSG mice can be injected intravenously (iv) with 100,000 IU of IL-2 immediately after injection. Femoral bone marrow is harvested 24 hours after injection and human Treg cells are identified based on CD25 positivity by flow cytometry. Data is analyzed with FlowJo software.

[0128] In each experiment, at least 3 mice are assigned to each group. The experiment is repeated at least three times. Organs including lung, liver, bone marrow, spleen, skin, and intestine are harvested for flow analysis and pathological study at euthanasia.

[0129] The above experiment is repeated with two additional groups: i) ruxolitinib alone and ii) ruxolitinib + CK0804. 100 mg of ruxolitinib powder is dissolved in DMSO, PEG-300, and sterile water to a total volume of 10 mL (1 mg / 100 μL) and 100 μL aliquots are stored at −20° C. Mice are given 1 mg (100 μL) of ruxolitinib daily for the duration of the study.

[0130] Methods for cryopreservation of activated regulatory T cells Provided herein are methods for cryopreserving populations of ex vivo expanded human Treg cells (eg, activated human Treg cells).

[0131] In some embodiments, a method for cryopreserving an expanded population of activated human regulatory T (Treg) cells produced from at least one cryopreserved human cord blood unit, the method comprising the steps of: a) thawing a cryopreserved human cord blood unit; b) diluting and washing the thawed cord blood unit in a functionally closed system; and c) detecting CD25 + isolating endogenous Treg cells using a double selection method based on cell surface expression; and d) culturing the isolated CD25+ Treg cells in a gas-permeable culture vessel in medium in the presence of an effective amount of interleukin-2 (IL-2) and in the presence of a reagent that specifically binds to CD3 and CD28 for up to 14 days. + and ex vivo expansion of Treg cells, wherein the medium is changed about every 48 hours to induce activation of CD25 + e) extracting from the culture medium an activated CD25 + harvesting the cells to produce an expanded population of activated human Treg cells; and f) cryopreserving the expanded population of activated human Treg cells.

[0132] In some embodiments, the method further comprises releasing the activated cultured human Treg cells for clinical use based on the criteria defined between steps e) and f).

[0133] Any suitable cryopreservation process known in the art can be used in the methods described herein. For example, expanded populations of human Treg cells can be cryopreserved using a freezing cocktail containing dimethyl sulfoxide (DMSO) followed by placing in a controlled speed freezer using a specifically defined program. The cryopreserved product can be stored at -180°C for at least several months. Upon thawing the cryopreserved product, Treg cells maintain a cell surface and intracellular phenotype with high expression of FOXP3 (forkhead box P3) and Helios and can retain their suppressive function as shown by in vitro cell suppression assays (Figures 8A-8C) as well as in vivo data in different animal models (Figures 9A-9B).

[0134] In some embodiments, about 10×10 per ml 6 At a cell concentration of up to approximately 50 x 10 per 5 ml vial 6 In some embodiments, about 100×10 cells are cryopreserved. 6 ~ approx. 1 x 10 cells 8 Cells can be cryopreserved in a single cryogenic bag with a maximum volume of 10ml to 100ml.

[0135] In some embodiments, for cryopreservation purposes, the harvested expanded population of human Treg cells may be centrifuged at 400 g for 10 minutes at a temperature of 4° C. The total cell number may be calculated using an automated cell counter and the number of cryovials may be calculated based on the total cell number of 50×10 6 Subsequently, frozen stock solutions were prepared using preformulated solutions containing 5% or 10% dimethylsulfoxide (DMSO) (Cryostor®) to obtain a maximum of 50×10 cells per 5 ml cryovial. 6The cells may be cryopreserved. While the cells are being centrifuged, the controlled speed freezer is turned on and once the controlled speed freezer has reached the appropriate starting temperature, the command "Program waiting for user - click here to continue" is displayed. Upon mixing with the freezing stock solution, up to 50 × 10 cells may be cryopreserved. 6 Cryovials of cells are placed into a controlled-rate freezer using a freezing algorithm, allowing the rate of cell freezing to be tuned to avoid cell death and maintain cell function. After the freezing program is complete, the cryovials are removed from the controlled-rate freezer and placed into a liquid nitrogen cryogenic freezer at temperatures as low as -190°C for long-term frozen storage.

[0136] The expanded Treg population can be cryopreserved in several aliquots to generate a clinical dose suitable for therapeutic administration.

[0137] Regulatory T cell populations and pharmaceutical compositions Disclosed herein is a population of human Treg cells produced by the methods described herein. The population is suitable for use in allogeneic cell therapy. In some embodiments, the human Treg cells are immunosuppressive.

[0138] In some embodiments, the population of human Treg cells is positive for CD4 and CD25. In some embodiments, the population of human Treg cells is positive for CD3, CD4 and CD25. In some embodiments, the population of human Treg cells is positive for CD3, CD4, CD25, CD45RO, CD45RA, CD95 and CD28.

[0139] Provided herein are antibodies that are at least about 60% CD4 + CD25 + and approximately 10% or less CD4 - CD8 + In some embodiments, the population of human Treg cells is at least about 60% CD4 + CD25 +and approximately 10% or less CD4 - CD8 + A population of human Treg cells further co-expresses CD45RA and CD45RO.

[0140] In some embodiments, the population of human Treg cells is at least about 90% CXCR4 + In some embodiments, the population of human Treg cells is at least about 95% CXCR4 + , at least about 95% are CD45RA + , and at least about 80% are CD45RO + In some embodiments, the population of human Treg cells is at least about 95% CXCR4 + , at least about 95% are CD45RA + , at least about 80% are CD45RO + , at least about 95% are CD95 + , at least about 95% are HLADR + , at least about 95% are alpha4beta7 + , at least about 15% are CXCR3hi + , at least about 95% are CCR6 + , at least about 95% are CD54 + , at least 95% are CD11A + , at least about 85% are CD45RARO + , at least about 80% are CTLA4 + , at least about 80% are GPR83 + and at least about 80% CD62L + In some embodiments, the expression of such cell surface markers is measured by flow cytometry. In some embodiments, the population of human Treg cells is expanded ex vivo.

[0141] In some embodiments, the population of human Treg cells is CD4 + CD25 + CD127 lo FOXP3 hi phenotype and CD45RA + CD45RO+ In some embodiments, the population of human Treg cells comprises human Treg cells that exhibit additional co-expression of CD4 + CD25 + CD127 - FoxP3 hi and Helios + In some embodiments, the expanded phenotype of activated human Tregs is: γ4β7 hi CCR3 lo CCR4 hi CCR6 hi CCR7 hi CD103 lo CD11a hi CD137 lo CD28 hi CD31 + CD39 lo CD54 hi CD62L hi CD7 h iCD95 hi CXCR3 lo CXCR4 hi HLA-ABC hi HLADR hi PD1 lo PD-LI lo and intracellular CD154 hi FOXP3 hi Helios hi GITR hi RORγt lo Tbet lo In some embodiments, the population of neurotropic human Tregs is CD95 / CXCR4 / CD31 / CD39 hi / CTLA4 / HELIOS / CXCR3 / CD28 phenotype.

[0142] In some embodiments, the population of human Treg cells is greater than or equal to about 60% CD4 + CD25 + Treg cells and less than 10% of CD4 - CD8 + Has a T cytotoxic / suppressor cell flow cytometry phenotype.

[0143] In some embodiments, the population of human Treg cells comprises human Treg cells that exhibit high expression of FOXP3 and low expression of RORγt. In some embodiments, the population of human Treg cells comprises human Treg cells that do not secrete IL-17 or exhibit RORγT under stressful conditions. In some embodiments, the population of human Treg cells comprises human Treg cells that maintain their polyclonal T cell receptor Vγ (TCR Vγ) repertoire. In some embodiments, the population of human Treg cells is cryopreserved prior to use.

[0144] In some embodiments, the population of human Treg cells express intracellular Helios. In some embodiments, the human Treg cells produced by the methods disclosed herein retain their immunosuppressive function and phenotype under stressful conditions. In some embodiments, the human Treg cells produced by the methods disclosed herein retain their viability and suppressive function in the presence of steroids (e.g., dexamethasone, prednisone or prednisolone). In some embodiments, the human Treg cells produced by the methods disclosed herein, due to the nature of their epigenetic signature and the selection / expansion protocols described herein, are resistant to interleukin-17 (IL-17) secretion and are less likely to "flip" into pro-inflammatory TH17 cells compared to peripheral blood Tregs.

[0145] The biological activity of interest of Treg cells in the populations described herein is immunosuppressive function, which can be measured by an in vitro suppression assay using intracellular dyes of CFSE (carboxyfluorescein succinimidyl ester) or CellTrace™ Violet dye. In this assay, Treg cells are co-cultured with normal peripheral blood T responder (Tresp) cells at various ratios and proliferating cells are detected using flow cytometry to detect the uptake of intracellular dyes of CFSE or CellTrace™ Violet, which can track cell proliferation for up to eight cell divisions. The degree of suppression of T responder (Tresp) cells by Treg cells can be quantified in relation to the ratio of Treg cells to Tresp cells and the generation of divided cells. When there is effective suppression by Treg cells, the suppression in first generation divided responder cells is greater at higher ratios of Tresp cells to Treg cells compared to lower ratios of Treg cells to Tresp cells. In some embodiments, Treg cells in the populations described herein are considered to be immunosuppressive if the Treg cells suppress at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of proliferating T conventional (Tcon) cells at a Treg:Tcon ratio of 4:1.

[0146] In some embodiments, a population of human Treg cells exhibits paracrine functions such as increased production of the inhibitory cytokine interleukin-10 (IL-10), but not transforming growth factor beta (TGFβ). In some embodiments, a population of human Treg cells secretes granzyme B in response to IL-6 treatment (see, e.g., FIG. 25).

[0147] Provided herein are at least about 1×10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% CD4 + CD25 + and (ii) ≤10% CD4 - CD8 +Further provided herein is a population of human Treg cells, comprising at least about 1×10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% CD4 + CD25 + , (ii) ≥ 60% CD4 + CD25 + CXCR4 + and (iii) ≤10% CD4 - CD8 + and the human Treg cells are immunosuppressive. Further provided herein is a population of at least about 1×10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% CD4 + CD25 + , (ii) ≥ 60% CD4 + CD25 + α4β7 + and (iii) ≤10% CD4 - CD8 + and the human Treg cells are immunosuppressive. Also provided herein is a population of human Treg cells comprising at least about 1×10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% CD4 + CD25 + , (ii) ≥ 60% CD4 + CD25 + CD11a + and (iii) ≤10% CD4 - CD8 + and the human Treg cells are immunosuppressive. In some embodiments, the population of human Treg cells disclosed herein is at least about 1×10 9 human Treg cells or at least about 1×10 10 In some embodiments, the population of human Treg cells disclosed herein comprises about 1×10 8 ~1×10 10 pieces, about 1×10 8 ~1×10 9Pieces, or about 1 x 10 9 ~1×10 10 Contains human Treg cells.

[0148] In some embodiments, the population of human Treg cells is formulated as a fresh, single-dose product (e.g., CK0801). The CK0801 product is produced from umbilical cord blood that is at least 3 of 6 HLA (human leukocyte antigen) matched (e.g., 3 of 6, 4 of 6, 5 of 6, or 6 of 6 HLA matched) to the subject to whom the product is administered. The CK0801 product is administered to the subject as a single infusion at a dose based on the subject's weight. The product contains immunosuppressive Treg cells.

[0149] In some embodiments, the CK0801 product is CD25 + The CK0801 product is isolated via selection and after a 14 day culture period. In some embodiments, the release criteria for the CK0801 product are: (i) > 60% CD4 + CD25 + (T regulatory phenotype), (ii) ≦10%CD4 - CD8 + (T cytotoxic / suppressive phenotype). In some embodiments, a CK0801 product is administered to a subject to treat an inflammatory bone marrow disorder or Guillain-Barre syndrome.

[0150] In some embodiments, the population of human Treg cells is formulated as a cryopreserved and / or multi-dose product (e.g., CK0804, CK0802, CK0803, or CK0805). In some embodiments, CK0804, CK0802, CK0803, or CK0805 is formulated in an injectable cryopreservation medium containing 10% dimethylsulfoxide (DMSO). CK0804, CK0802, CK0803, and CK0805 are not HLA-matched to the subject to whom the product is administered. In some embodiments, these products are 2 of 6, 1 of 6, or 0 of 6 HLA matches to the subject to whom the product is administered. Each of these products is administered to the subject as a fixed-dose, multi-dose infusion. These products contain immunosuppressive Treg cells.

[0151] In some embodiments, the CK0802 product is CD25 + The CK0802 product is isolated via selection and after a culture period of 14 days. In some embodiments, the release criteria for the CK0802 product is: (i) 100×10 in 10 mL 6 Treg / bag (10 x 10 6 Treg / mL), (ii) ≥ 60% CD4 + CD25 + (Regulatory T phenotype) (iii) ≦10%CD4 - CD8 + (T cell toxic / suppressive phenotype). In some embodiments, the CK0802 product is administered to a subject to treat acute respiratory distress syndrome (ARDS) (e.g., CoV-ARDS) or cytokine release syndrome (CRS) (e.g., CRS due to chimeric antigen receptor T cell therapy). In some embodiments, the CK0802 product is administered to a subject on days 0, 3, and 7.

[0152] In some embodiments, the CK0804 product is CD25 + Through selection and further enrichment for CXCR4, and after a culture period of 10-12 days, the release criteria for CK0804 product are (i) 100×10 in 10 mL.6 Treg / bag (10 x 10 6 Treg / mL), (ii) ≥ 60% CD4 + CD25 + (regulatory T phenotype), (iii) ≥ 60% CD4 + CD25 + CXCR4 + (bone marrow homing subtype), (iv) ≤10% CD4 - CD8 + (T cell toxic / suppressive phenotype). In some embodiments, the CK0804 product is administered to the subject to treat myelofibrosis, aplastic anemia, or immune thrombocytopenia. In some embodiments, the CK0804 product is administered to the subject monthly for up to six months.

[0153] CK0804 is a product containing cord blood (CB) derived Treg cells enriched for CXCR4 expression to enable faster delivery to the bone marrow stroma in patients suffering from inflammatory bone marrow disorders, including primary myelofibrosis. CK0804 is an off-the-shelf cryopreserved product. CK0804 is prepared from a single unit of cord blood (CBU) for each batch. One batch manufacturing process produces multiple products that are frozen in cryobags at a fixed dose of 100 million cells per bag.

[0154] In some embodiments, the CK0805 product is CD25 + Through selection and further enrichment for α4β7, and after a culture period of 8-10 days, the release criteria for the CK0805 product are (i) 100×10 in 10 mL. 6 Treg / bag (10 x 10 6 Treg / mL), (ii) ≥ 60% CD4 + CD25 + (regulatory T phenotype), (iii) ≥ 60% CK0805 (gastrointestinal homing subtype), (iv) ≤ 10% CD4 - CD8 +(T cell toxic / suppressive phenotype). In some embodiments, the CK0805 product is administered to a subject to treat gastrointestinal graft-versus-host disease or inflammatory bowel disease. In some embodiments, the CK0805 product is administered to a subject with the following dosing regimen: (i) induction: weekly for up to 4 weeks, and (ii) maintenance: monthly for up to 6 months.

[0155] In some embodiments, the CK0803 product is CD25 + Through selection and further enrichment on CD11a and after a culture period of 8-10 days, the release criteria for the CK0803 product are (i) 100×10 in 10 mL. 6 Treg / bag (10 x 10 6 Treg / mL), (ii) ≥ 60% CD4 + CD25 + (Treg phenotype) (iii) ≥ 60% CD4 + CD25 + CD11a + (neuron-homing subtype), (iv) ≤10% CD4 - CD8 + (T cytotoxic / suppressive phenotype). In some embodiments, the CK0803 product is administered to a subject to treat amyotrophic lateral sclerosis, multiple sclerosis, or demyelinating neuropathy. In some embodiments, the CK0803 product is administered to a subject with the following dosing regimen: (i) induction: weekly for up to 4 weeks, and (ii) maintenance: monthly for up to 6 months.

[0156] The cord blood unit selection criteria for various populations of human Treg cells are provided in Figures 29 and 30.

[0157] The cellular starting material for CK0802 is a single unit of umbilical cord blood (CBU) from a normal, healthy, unrelated donor. To generate a clinically relevant dose of Treg cells, CD4 + CD25 + In some embodiments, the 14-day manufacturing process includes ex vivo enrichment and expansion of Treg cells having the CD4 + CD25+ This results in a 50-fold or greater expansion of the Treg population. Multiple doses intended for different recipients can be produced from a single expansion process. Treg cells are harvested, cryopreserved, tested, and released for clinical use before being shipped to the clinical site for infusion.

[0158] CK0802 is polyclonal, expresses a broad V-beta repertoire and has high intracellular FOXP3 staining. CK0802 is also associated with consistent hypomethylation of the TSDR (Treg-specific demethylated region), which is common in naturally occurring human Tregs.

[0159] In some embodiments, the CK0802 active drug substance (DS) is a liquid cell suspension consisting of nucleated cord blood cells, 60% or more of which are of regulatory T cell phenotype (CD3 + CD4 + CD25 + ), of which less than 10% have a T cytotoxic / suppressor cell phenotype (CD3 + CD4 - CD8 + In some embodiments, the CK0802 final formulation (DP) is 10×10 in injectable cryopreservation medium containing 10% dimethylsulfoxide (DMSO). 6 It is a suspension of live cells containing the active pharmaceutical ingredient CK0802 suspended at a cell concentration of Treg cells / mL.

[0160] An example of the composition of a CK0802 formulation is provided in Table 2.

[0161] [Table 2]

[0162] Further disclosed herein is a pharmaceutical composition comprising a population of activated human Treg cells and one or more pharma- ceutically or veterinarily acceptable carriers, diluents, excipients, or vehicles.

[0163] The terms "pharmaceutical acceptable" and "veterinarily acceptable" refer to pharma- ceutical or veterinarily acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials. Each component must be "pharmaceutical acceptable" or "veterinarily acceptable" in the sense of being compatible with the other components of a pharmaceutical formulation, and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication commensurate with a reasonable benefit / risk ratio. (See Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th ed., Rowe et al., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd ed., Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Pre-formulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004).

[0164] A pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration (i.e., intraocular, subretinal, parenteral, intravenous, intraarterial, intradermal, subcutaneous, oral, inhalation, transdermal, topical, transmucosal, intraperitoneal or intrapleural and / or rectal administration).

[0165] It will be understood that administration of therapeutic entities according to the present disclosure will be with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, and the like. A number of suitable formulations are described in the following formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 therein by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be suitable in treatments and therapies according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation, and the formulation is physiologically compatible and tolerable to the route of administration. See Baldrick P. "Pharmaceutical excipients development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000); Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2)1-60 (2000); Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci. 89(8):967-78 2000; Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998) and citations therein for additional information regarding formulations, excipients and carriers well known to pharmaceutical chemists.

[0166] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions of cells. In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0167] Sterile injection solution can be prepared by incorporating the required amount of active ingredient into a suitable solvent, with one or combination of the above-listed ingredients as required, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients required from the above-listed ones.For the sterile powder for preparing sterile injection solution, the preparation method is vacuum drying and freeze-drying, which allows the powder of active ingredient plus any additional desired ingredients to be obtained from its previously sterile-filtered solution.

[0168] In some embodiments, the active ingredient may be prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for the preparation of such formulations will be apparent to those skilled in the art. Materials may also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes that target infected cells with monoclonal antibodies against viral antigens) may be used as pharma-ceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0169] As used herein, unit dosage form refers to a physically separate unit suitable as a unitary dose for the subject to be treated.Each unit contains a predetermined amount of active material calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specification of the unit dosage form of the present disclosure is determined and directly depends on the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the art of compounding such active compound for the treatment of an individual.

[0170] An example of the composition of the final product (consisting of active ingredients suspended in excipients) is shown in the table below. In some embodiments, the final dosage form has a volume of about 50 mL to about 100 mL. In some embodiments, the cellular component of the final product is culture-expanded, primarily CD4 IgG1-associated cellular components derived from a single cord blood unit or multiple pooled cord blood units. + CD25 + It consists of mononuclear cells derived from umbilical cord blood that have a regulatory T cell phenotype.

[0171] [Table 3] *Total nucleated cells in in-process and final product samples are counted by conventional manual methods using a hemocytometer and light microscope, the results are expressed as nucleated cells per volume, and calculations are performed using the volume of the product, which represents the total nucleated cell content in the product.

[0172] In some embodiments, the final formulated product is provided for use contained in a sealed 300 mL polyvinyl chloride (PVC) plastic blood bag that has a port that can be accessed with the plastic spike of a conventional intravenous (IV) administration set used for administration to a patient.

[0173] In some embodiments, excipients used to formulate the final product may include:

[0174] [Table 4]

[0175] In some embodiments, the composition comprises a population of activated human Treg cells produced by the methods described herein and one or more other therapeutic agents. Also provided herein are kits for treating one or more autoimmune diseases, disorders, or conditions that include a composition described herein (e.g., a container, pack, or dispenser) together with instructions for use or administration. Also provided are articles of manufacture that include a container containing any of the populations of activated human Treg cells described herein and instructions for use.

[0176] Therapeutic methods and uses Provided herein is a method for treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject an effective amount of a population of human Treg cells (e.g., activated human Treg cells) produced by any of the methods described herein. Further provided herein is a method for treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject an effective amount of a population of human Treg cells disclosed herein. In some embodiments, the disease, disorder, or condition is an autoimmune disease, disorder, or condition. In some embodiments, the disease, disorder, or condition is an inflammatory disease, disorder, or condition. In some embodiments, the disease, disorder, or condition is graft-versus-host disease (GVHD), inflammatory bowel disease, bone marrow failure (e.g., aplastic anemia, primary myelofibrosis, or myelodysplastic syndrome), systemic lupus erythematosus (SLE), inflammatory cancer (e.g., multiple myeloma or inflammatory breast cancer), neuroinflammatory disorder (e.g., Guillain-Barre syndrome, amyotrophic lateral sclerosis (ALS), multiple sclerosis, or demyelinating polyneuropathy), cytokine release syndrome (CRS), or immune deficiency syndrome (e.g., iPEX (immune dysregulation polyendocrinopathy enteropathy X-linked)). In some embodiments, the disease, disorder, or condition is a respiratory disease, disorder, or condition associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In some embodiments, the disease, disorder, or condition is COVID-19 (coronavirus disease)-mediated acute respiratory distress syndrome (CoV-ARDS).

[0177] In some embodiments, the population of human Treg cells is produced from one or more cord blood units that are human leukocyte antigen (HLA) matched to the intended recipient. In some embodiments, the population of human Treg cells is produced from one or more cord blood units that are not HLA matched to the intended recipient. In some embodiments, the population of human Treg cells is prepared from one or more cord blood units of a blood type compatible with the subject.

[0178] In some embodiments, cord blood derived Tregs may exhibit one or more of the following properties to produce an anti-inflammatory effect: (1) direct binding to recipient antigen presenting cells (APCs) and blocking interactions with T effector (Teff) cells (i.e., by suppressing pro-inflammatory immune cells through direct interactions); (2) release of inhibitory cytokines including transforming growth factor beta (TGFβ), interleukin-10 (IL-10), and interleukin-35 (IL-35); (3) depletion of IL-2 supply for Teffs leading to their apoptosis; and / or (4) playing a role in granzyme / perforin production (i.e., by secreting granzyme B or perforin, thereby suppressing natural killer (NK) cells and CD8 cells). + Furthermore, local expansion of infused cord blood-derived Tregs at the site of inflammation may confer a survival advantage and produce the anti-inflammatory effects necessary for disease control.

[0179] The dosage of Treg cells in the final product may be expressed as the number of cells per kg of subject body weight. Determination of the appropriate cell dose for use in any of the methods described herein is within the routine level of skill in the art. In some embodiments, an effective amount of a population of activated human Treg cells is 1×10 6 cells per kg of subject body weight. 5~ Approximately 1×10 8 Treg cells / kg, or approximately 1 × 10 6~ Approximately 1×10 7 In some embodiments, the cell dose for any of the methods described herein may be as follows: Dose level 1: approx. 1×10 6 Treg cells / kg Dose level 2: approx. 3×10 6 Treg cells / kg Dose level 3: approx. 1×10 7 Treg cells / kg

[0180] In some embodiments, a fixed dose may be administered that is not dependent on the subject's weight. In some embodiments, the dose is about 1×10 8 Activated human Treg cells ~ approx. 3 × 10 8 For example, the dose may be about 1×10 Treg cells. 8 , about 3×10 8 , or about 1 × 10 9 The cells may be activated human Treg cells.

[0181] In some embodiments, an effective amount of the population of activated human Treg cells is administered intravenously to the subject.

[0182] In some embodiments, a single dose of an effective amount of the population of human Treg cells is administered to the subject. In some embodiments, multiple doses of an effective amount of the population of activated human Treg cells are administered to the subject. In some embodiments, up to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more repeated doses of Treg cells may be administered. When multiple doses are administered, these doses may be administered periodically (i.e., every 3 days, every 4 days, every 5 days, every 6 days, every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 1-2 weeks, every 1-3 weeks, every 1-4 weeks, every 1-5 weeks, every 1-6 weeks, every 2-3 weeks, every 2-4 weeks, every 2-5 weeks, every 2-6 weeks, every 3-4 weeks, every 3-5 weeks, every 3-6 weeks, every 4-5 weeks, every 4-6 weeks, or every 5-6 weeks). In some embodiments, doses are administered to a subject about every 4-6 weeks, hi some embodiments, Treg cells may be administered weekly for a period of 4 weeks, and then monthly for a period of at least 6-9 months (i.e., 6, 7, 8, or 9 months).

[0183] In some embodiments, following administration of an effective amount of the population of activated human Treg cells, the level of a circulating inflammatory cytokine in the subject is reduced compared to the level of a circulating inflammatory cytokine in the subject prior to administration, in some embodiments, the circulating inflammatory cytokine is interleukin-6 (IL-6), interferon gamma (IFNγ), or tumor necrosis factor-alpha (TNFγ).

[0184] In some embodiments, prior to treatment, the subject's serum biomarkers are examined to determine whether the subject responds to an effective amount of a population of activated human Treg cells. In some embodiments, after treatment, the subject's serum biomarkers are examined to determine correlations with clinical response. In some embodiments, serum biomarkers are examined serially to determine whether subsequent retreatment with Treg cells is required.

[0185] In some embodiments, diphenhydramine is administered to the subject prior to administration of an effective amount of a population of activated human Treg cells. In some embodiments, about 50 mg of diphenhydramine is administered. In some embodiments, diphenhydramine is administered approximately 30 minutes prior to administration of an effective amount of a population of activated human Treg cells.

[0186] Further provided herein is the use of the population of human Treg cells disclosed herein in the preparation of a medicament, which can be used to treat or prevent a disease, disorder or condition.

[0187] Graft-versus-host disease (GVHD) Provided herein is a method for treating or preventing graft-versus-host disease (GVHD) in a subject, the method comprising administering to the subject an effective amount of a population of activated human Treg cells produced by the methods disclosed herein, or an effective amount of a population of human Treg cells disclosed herein.

[0188] In some embodiments, the methods described herein improve, reduce, or prevent one or more symptoms of GVHD in a subject. In some embodiments, the methods described herein prolong the survival of a subject with GVHD. In some embodiments, the methods described herein prevent a subject from developing GVHD after receiving a transplant.

[0189] Further provided herein is a method for treating or preventing GVHD in a subject, the method comprising administering to the subject (i) an effective amount of a population of activated human Treg cells produced by a method disclosed herein or an effective amount of a population of human Treg cells disclosed herein, and (ii) ruxolitinib. In some embodiments, ruxolitinib is administered to the subject continuously and the human Treg cells are administered to the subject every 2, 3, or 4 weeks. In some embodiments, ruxolitinib is administered orally twice daily as a 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg tablet.

[0190] Allogeneic hematopoietic stem cell transplantation (HSCT) is the only treatment option for many hematologic malignancies. However, a major barrier to the more widespread use of this procedure is the development of GVHD, which occurs when T cells from the graft recognize the host's tissues as foreign and is a major cause of morbidity and mortality. (See Warren et al., Tissue Antigens 81(4):183-93 (2013); Sung et al., Stem Cells Transl Med 2(1):25-32 (2013); and Qian et al., J Cell Mol Med 17(8):966:75 (2013).) Acute GVHD (aGVHD) usually occurs within the first 100 days after HSCT and involves a "cytokine storm" from activated T cells that recruits other inflammatory cell types, such as NK cells and macrophages, resulting in inflammatory lesions in tissues such as the skin, gut, and liver. aGVHD causes death in approximately 15% of transplant patients. (See Sung et al., Stem Cells Transl Med 2(1):25-32 (2013), and Qian et al., J Cell Mol Med 17(8):966:75- (2013).) Chronic GVHD (cGVHD) occurs after the first 100 days after transplantation and is characterized by systemic inflammation and tissue destruction affecting multiple organs, particularly the gut, liver, lungs, bone marrow, thymus, and skin. cGVHD occurs in 30-65% of allogeneic HSCT recipients and causes extreme morbidity with a 5-year mortality rate of 30-50%, primarily due to a reduced ability to fight infection. aGVHD is believed to be primarily a Th1 / Th17-driven process, whereas cGVHD is believed to be primarily driven by a Th2-driven response. In some embodiments, the methods described herein improve, reduce, or prevent one or more symptoms of aGVHD in a subject. In some embodiments, the methods described herein improve, reduce, or prevent one or more symptoms of cGVHD in a subject.In some embodiments, the therapeutic methods described herein may be used to suppress graft-versus-leukemia (GVHD) without losing the benefit of GVL activity, a beneficial immune response by allogeneic immune cells that kill leukemic cells (see Edinger et al., Nat Med 9(9):1144-50 (2003)).

[0191] Current strategies to minimize GVHD call for long-term immunosuppressive therapy with drugs such as calcineurin inhibitors (CNIs), cyclosporine, and tacrolimus. However, this long-term immunosuppression leads to delayed immune function and the risk of infectious complications and post-transplant lymphoproliferative disease. In some embodiments, provided herein is a method for treating or preventing GVHD in a subject, comprising administering to the subject an effective amount of a population of activated human Treg cells produced by the methods disclosed herein or an effective amount of a population of human Treg cells disclosed herein, without administering other immunosuppressive therapy.

[0192] Xenogeneic mouse models of GVHD can be used to evaluate the function of cord blood-derived regulatory T cells in the treatment of GVHD. (See Parmar et al., Cytotherapy 16(10:90-100(2013)).

[0193] Bone marrow failure syndrome (BMF) Provided herein are methods for treating or preventing bone marrow failure syndrome (BMF) in a subject, comprising administering to the subject an effective amount of a population of activated human Treg cells generated by a method disclosed herein, or an effective amount of a population of human Treg cells disclosed herein. In some embodiments, an effective amount of a cryopreserved Treg cell product enriched for CXCR4 expression (e.g., CK0804) is administered to treat or prevent BMF.

[0194] In some embodiments, the methods described herein improve, reduce, or prevent one or more symptoms of BMF in a subject. In some embodiments, the methods described herein extend the survival of a subject with BMF.

[0195] BMF refers to a reduction in the production of one or more major hematopoietic systems, leading to a reduction or absence of hematopoietic precursors in the bone marrow (BM). It can be divided into two categories: acquired and inherited. Acquired BMF syndromes include aplastic anemia, myelodysplastic syndromes, and primary myelofibrosis. The pathogenesis of acquired BMF syndromes involves the BM microenvironment and environmental factors. For the majority of these syndromes, the role of immune dysfunction is recognized as important in both the cause and maintenance of BM defects.

[0196] Aplastic anemia (AA) Provided herein is a method for treating or preventing aplastic anemia (AA) in a subject, the method comprising administering to the subject an effective amount of a population of activated human Treg cells generated by the methods disclosed herein, or an effective amount of a population of human Treg cells disclosed herein.

[0197] AA is characterized by pancytopenia in peripheral blood (PB), whereas bone marrow (BM) aplasia is characterized by CD8 + Cytotoxic T cells, CD4 + It is a BMF syndrome characterized by attack by autoreactive cytotoxic T cells, such as Th1 and Th17 cells (see Brodksy et al., Lancet 365(9471):1647-56(2005); Li et al., Crit Rev Oncol Hematol 75(2):79-93(2010); Young et al., Curr Opin Hematol 15(3):162068(2008); and de Latour et al., Blood 116(20):4175:84(2010)).

[0198] Mechanisms of immune-mediated hematopoietic destruction include interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and the suppression of autologous CD34 by T cell populations. + These include Th1 polarized responses that result in direct toxicity to cells and overproduction of inhibitory cytokines, such as Th17 immune responses. (See de Latour et al., Blood 116(20):4175-84(2010); Giannakoulas et al., Br J Haematol 124(1):97-105(2004); Sloand et al., Blood 100(4):1185-91(2002); and Solomou et al., Blood 107(10):3983:91-(2006).) In that sense, AA is a specific autoimmune disease, with BM being the main target organ, due to an excess of cytotoxic autoreactive T cells combined with defective and deficient regulatory T cells that cause abnormal T cell immune homeostasis.

[0199] Also provided herein is a method of treating acquired idiopathic aplastic anemia in a subject, where the subject is ineligible for matched sibling donor hematopoietic stem cell transplantation (MSD-HSCT) or is predicted to be poorly responsive to immunosuppressive therapy (IST).

[0200] The diagnosis of acquired AA can be based on the exclusion of other disorders that can cause pancytopenia and the well-known Camitta criteria (see Camitta et al., Blood 45(3):355-63 (1975)).

[0201] The AA response criteria (see Killicket et al., Br J Haematol 172(2):187-207(2016)) can be used to determine the response of a subject with AA to the treatments described herein, as shown in the table below.

[0202] [Table 5]

[0203] Myelodysplastic syndromes (MDS) Provided herein is a method for treating or preventing myelodysplastic syndrome (MDS) in a subject, the method comprising administering to the subject an effective amount of a population of activated human Treg cells generated by the methods disclosed herein, or an effective amount of a population of human Treg cells disclosed herein.

[0204] MDS is characterized by ineffective hematopoiesis, where impaired blood cell production may be the result of increased apoptosis. Clonal expansion of abnormal progenitor cells that escape apoptosis may lead to evolution into overt acute leukemia. (See Rosenfeld, Leukemia 14(1):2-8(2000) and Barrett et al., Semin Hematol 37(1):15:29(2000)). Dysregulation of immune function is a recognized fact in MDS. (See Fozza et al., Exp Hematol 37(8):947-55(2009)). Among the possible mechanisms, inhibition of T-cell-mediated hematopoiesis has been recognized as a typical feature, especially of low-risk and hypocellular MDS. (See Epperson et al., Leuk Res 25(12):1075-83(2001)). Cytopenias in some types of MDS may be due to either cytokine or cell-mediated autoimmune suppression of normal and abnormal bone marrow (BM) progenitor cells. (See Barrett et al., Semin Hematol 37(1):15-29(2000)). These mechanisms may be operative especially in the hypoplastic form of MDS (HMDS) (See Tuzuner et al., Br J Haematol 91(3):612-17(1995)). It often overlaps clinically with aplastic anemia (AA), a disease with an established autoimmune etiology. (See Young et al., N Engl J Med 336(19):1365-72(1997)).

[0205] Patients with MDS have a decreased CD4:CD8 ratio, multiple activated CD8 + It shows T cell clonal expansion and overproduction of inhibitory cytokines. (See Selleri et al., Cancer 95(9):1911-22(2002)). Immune effector mechanisms in MDS patients may include not only direct killing but also the release of cytokines with inhibitory activity against hematopoietic progenitor cells, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and Fas-ligand (Fas-L). (See Zang et al., Blood 98(10):3058-65(2001)). Consistent with these pathophysiological pathways, increased levels of these cytokines have been described in the blood and bone marrow of MDS patients and may be responsible for the large number of apoptotic bone marrow cells seen in these patients. (See Selleri et al., Cancer 95(9):1911-22(2002)).

[0206] Currently, the diagnosis of MDS (see Gangat et al., Am J Hematol 91(1):76-89(2016)) is defined as (i) persistent (>6 months duration) and significant cytopenias with hemoglobin <10 g / dL, absolute neutrophil count <1.8 × 10 9 / L, platelet count <100×10 109 Establishment of a diagnosis of erythropoietin-positive leukemia is based on (ii) the presence of significant myelodysplasia or excess blasts or typical cytogenetic abnormalities, and (iii) the exclusion of differential diagnoses. (See Barrett et al., Semin Hematol 37(1):15-29 (2000).) Common peripheral blood findings include macrocytic anemia, reticulocytopenia, neutropenia with hypolobulated neutrophils (pseudo-Pelger-Huett), circulating immature myeloid cells including myeloblasts, and thrombocytopenia.

[0207] The International Working Group (IWG) response criteria (see Cheson et al., Blood 108(2):419-25 (2006)) can be used to determine a subject's response to the therapeutic approaches described herein, as shown in the table below.

[0208] [Table 6]

[0209] Primary myelofibrosis (PMF) Provided herein are methods for treating or preventing primary myelofibrosis (PMF) in a subject, the methods comprising administering to the subject an effective amount of a population of activated human Treg cells produced by the methods disclosed herein, or an effective amount of a population of human Treg cells disclosed herein. In some embodiments, the population of human Treg cells administered to the subject to treat or prevent PMF is at least about 90% CXCR4 + In some embodiments, an effective amount of a cryopreserved Treg cell product enriched for CXCR4 expression (e.g., CK0804) is administered to treat or prevent PMF. In some embodiments, the unit dose of cryopreserved Treg cell product enriched for CXCR4 expression administered to a subject is 100 million cells in 10-20 mL. The unit dose may be administered in up to 6 doses (i.e., up to 6 cycles) on day 1 of each 28-day cycle.

[0210] Further provided herein is a method for treating or preventing PMF in a subject, the method comprising administering to the subject (i) an effective amount of a population of activated human Treg cells produced by a method disclosed herein, or an effective amount of a population of human Treg cells disclosed herein (e.g., CK0804), and (ii) ruxolitinib. In some embodiments, ruxolitinib is administered to the subject continuously, and the human Treg cells are administered to the subject every 2, 3, or 4 weeks. In some embodiments, ruxolitinib is administered orally twice daily as a 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg tablet.

[0211] PMF is a clonal hematopoietic stem cell disorder in which 50% of patients have a constitutively activating mutation in the Janus kinase (JAK) 2 gene JAK2V617F. PMF is generally considered to arise from mutated stem or progenitor hematopoietic cells, but immune dysregulation is common. For example, plasma levels of inflammatory cytokines are increased, and clinical and laboratory symptoms of autoimmunity appear. (See Barosi Curr Hematol Malig Rep 9(4):331-39(2014)). This clonal bone marrow proliferation is characteristically accompanied by reactive myelofibrosis (myelofibrosis) and extramedullary hematopoiesis in the spleen or multiple organs.

[0212] Proinflammatory cytokines are known to be highly elevated in PMF and contribute to the pathogenesis of the disease. Indeed, treatment with ruxolitinib is associated with a dramatic reduction in circulating levels of proinflammatory cytokines such as IL-6 and tumor necrosis factor (TNF)-α.

[0213] A diagnosis of PMF can be made using the criteria set forth in Table 7 (see Barbui et al., Blood Cancer Journal 8(2):15(2018)).

[0214] [Table 7]

[0215] The revised International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and European-Leukemia Network (ELN) response criteria (see Tefferi et al., Blood 122(8):1395-98 (2013)) shown in Table 8 can be used to determine the response of a subject with PMF to the treatment methods described herein. In some embodiments, administration of an effective amount of a cryopreserved Treg cell product enriched for CXCR4 expression (e.g., CK0804) to a subject with PMF reduces or ameliorates the symptoms listed in Table 8.

[0216] [Table 8]

[0217] Systemic lupus erythematosus (SLE) Provided herein is a method for treating or preventing systemic lupus erythematosus (SLE) in a subject, the method comprising administering to the subject an effective amount of a population of activated human Treg cells produced by the methods disclosed herein, or an effective amount of a population of human Treg cells disclosed herein.

[0218] In some embodiments, the methods described herein improve, reduce, or prevent one or more symptoms of SLE in a subject. In some embodiments, after administration of activated human Treg cells to a subject, urinary albumin spillover is reduced, SLE cell infiltration in glomeruli is reduced, and / or hair follicles are preserved. In some embodiments, the methods described herein extend the survival of a subject with SLE.

[0219] SLE is a chronic, multisystem inflammatory autoimmune disease. Lupus can affect many parts of the body, including joints, skin, kidneys, heart, lungs, blood vessels, and / or brain. For example, SLE can manifest as joint pain or arthritis, Raynaud's phenomenon, cheek and other rashes, pleurisy or pericarditis, renal or CNS involvement, and / or cytopenias.

[0220] Inflammatory cancer Provided herein are methods for treating or preventing inflammatory cancer in a subject, the methods comprising administering to the subject an effective amount of a population of activated human Treg cells produced by the methods disclosed herein, or an effective amount of a population of human Treg cells disclosed herein. In some embodiments, the inflammatory cancer is multiple myeloma or inflammatory breast cancer. In some embodiments, a treatment regimen for multiple myeloma comprises administration of an effective amount of a population of human Treg cells and a bispecific protein (e.g., an antibody) useful for treating inflammatory cancer. In some embodiments, the bispecific protein is a bispecific T cell engager. In some embodiments, the bispecific T cell engager binds to CD3 and BCMA.

[0221] In some embodiments, the methods described herein improve, reduce, or prevent one or more symptoms of inflammatory cancer in a subject. In some embodiments, the methods described herein extend the survival of a subject with inflammatory cancer.

[0222] Neuroinflammatory disorders Provided herein is a method for treating or preventing a neuroinflammatory disorder in a subject, the method comprising administering to the subject an effective amount of a population of activated human Treg cells produced by a method disclosed herein, or an effective amount of a population of human Treg cells disclosed herein. In some embodiments, the inflammatory cancer is Guillain-Barre syndrome or amyotrophic lateral sclerosis.

[0223] In some embodiments, the methods described herein improve, reduce, or prevent one or more symptoms of a neuroinflammatory disorder in a subject. In some embodiments, the methods described herein extend the survival of a subject with a neuroinflammatory disorder.

[0224] Guillain-Barré syndrome (GBS) Provided herein is a method for treating or preventing Guillain-Barre Syndrome (GBS) in a subject, the method comprising administering to the subject an effective amount of a population of activated human Treg cells generated by the methods disclosed herein, or an effective amount of a population of human Treg cells disclosed herein.

[0225] Also provided herein is a method of treating GBS in a subject, where the subject does not respond to treatment with intravenous immunoglobulin (IVIG) or plasma exchange.

[0226] GBS is an autoimmune disorder characterized by rapid onset of muscle weakness due to inflammation of the nerves. There are two major subtypes: (1) acute inflammatory demyelinating polyneuropathy (AIDP) and (2) acute axonal neuropathy (AMAN). Although the exact cause of GBS is unknown, there is strong evidence that the immune response to infection triggers an autoimmune response that damages the nerves.

[0227] Experimental autoimmune neuritis (EAN) is an immune-inflammatory demyelinating disorder of the peripheral nervous system and serves as an animal model of AIDP. Therapeutics described herein can be tested in this animal model. It is generally induced in susceptible animal strains by immunization with myelin proteins such as P0 or P2, leading to the destruction of the blood-nerve barrier, infiltration of autoreactive T cells and macrophages, and demyelination of the peripheral nervous system (Soliven, B., Autoimmune neuropathies: insights from animal models. J Peripherer Nerv Syst, 2012. 17 Suppl 2: p. 28-33). EAN can be actively initiated by targeting neuritogenic epitopes of peripheral nerve proteins P0, P2, and peripheral myelin protein 22 (PMP22) (Hughes, RA, et al., Pathogenesis of Guillain-Barre syndrome. J Neuroimmunol, 1999.100(1-2):p.74-97) or by adoptive transfer of sensitized T cells.

[0228] Amyotrophic lateral sclerosis (ALS) Provided herein is a method for treating or preventing amyotrophic lateral sclerosis (ALS) in a subject, the method comprising administering to a subject an effective amount of a population of activated human Treg cells produced by a method disclosed herein, or an effective amount of a population of activated human Treg cells disclosed herein (e.g., 1×10 8 pieces, 3×10 8 pcs or 1×10 9 The method includes administering to a subject a therapeutically effective amount of activated human Treg cells.

[0229] In some embodiments, provided herein is a method for treating or preventing a neuroinflammatory disorder in a subject, the method comprising administering to the subject an effective amount of a population of human Treg cells disclosed herein.

[0230] ALS is a rare neurological disease involving the death of neurons that control voluntary muscles. This results in severe muscle atrophy and loss of the ability to walk and speak. The disease is characterized by a 5-year mortality rate of about 80%. Autoimmune neuroinflammation forms the basis of the pathogenesis and progression of ALS. In fact, ALS patients have enhanced inflammation in the spinal cord, and the degree of microglial activation corresponds to the severity of the disease.

[0231] In ALS, Tregs are dysfunctional and less effective at suppressing the proliferation of responder T lymphocytes. Furthermore, late-stage ALS is characterized by the infiltration of M1-like macrophages / microglia and inflammatory effector T cells. ALS patients have a higher number of Tregs (CD4 + / CD25 + ) tends to be decreased, and the rate of progression is negatively correlated with Treg cell counts. Similarly, low FoxP3 mRNA levels are a predictor of rapid ALS progression. Furthermore, Tregs from ALS patients have a reduced ability to suppress Th17 cell proliferation compared to healthy subjects.

[0232] COVID-19 (Coronavirus Disease)-borne Acute Respiratory Distress Syndrome (CoV-ARDS) Provided herein is a method for treating or preventing COVID-19 (coronavirus disease) mediated acute respiratory distress syndrome (CoV-ARDS) in a subject, the method comprising administering to a subject an effective amount of a population of activated human Treg cells produced by a method disclosed herein, or an effective amount of a population of human Treg cells disclosed herein (e.g., about 1×10 8 Pieces or about 3 x 10 8 In some embodiments, the method comprises administering to the subject about 1×10 activated human Treg cells. 8 Pieces or about 3 x 10 8 activated human Treg cells are administered to the subject on days 0 and 3. In some embodiments, about 1×10 8 Pieces or about 3 x 10 8Human Treg cells are administered to the subject on days 0, 3, and 7. In some embodiments, the human Treg cells are cryopreserved allogeneic cord blood-derived Treg cells (CK0802). In some embodiments, the human Treg cells are administered as a single agent.

[0233] In some embodiments, the subject is infected or suspected of being infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0234] The highly pathogenic SARS-CoV-2 is associated with rapid viral replication, massive inflammatory cell infiltration, and elevated proinflammatory cytokine / chemokine responses, causing acute lung injury leading to acute respiratory distress syndrome (ARDS), pulmonary fibrosis, and death. The early stage of viral infection includes robust viral replication and clinical symptoms including fever, cough, etc. The second stage of viral infection includes high fever, hypoxemia, progression to pneumonia-like symptoms, and a progressive decline in viral titers toward the end. The third stage of viral infection includes a hyperproliferative host inflammatory response, excessive production of cytokines and chemokines, a dysregulated innate immune response, and ARDS. Clinically, ARDS is characterized by acute hypoxemic respiratory failure and bilateral pulmonary infiltrates on chest x-ray.

[0235] Uncontrolled cytokine storm may be responsible for the acuity of respiratory complications in some subjects infected with SARS-CoV-2. In some embodiments, the CoV-ARDS cytokine storm includes an increase in proinflammatory cytokines (e.g., IFN-γ, IL-1, IL-6, IL-12, or TGFβ) and chemokines (e.g., CCL2, CXCL10, CXCL9, and IL-8). Higher viral titers and dysregulated cytokine / chemokine responses orchestrate massive infiltration of inflammatory cells into the lungs. In some embodiments, the CoV-ARDS cytokine storm includes a decrease in anti-inflammatory cytokines (e.g., IL-10). In clinical lung injury models, injection of CB-Treg cells leads to i) a decrease in inflammatory T cells, ii) a decrease in alveolar bleeding, iii) regeneration of the lung epithelium and alveoli, and iv) a decrease in inflammatory cytokines including IL-17 and IL-6, both of which are involved in CoV-ARDS.

[0236] No specific treatment exists except for supportive care, including mechanical ventilation, which has a mortality rate of over 50%. New therapeutic options are urgently needed. Regulatory T cells (Tregs) are a specialized type of T cell that limit inflammation-induced lung damage through multiple mechanisms leading to tissue repair and regeneration.

[0237] In some embodiments, administration of an effective amount of a population of human Treg cells disclosed herein may treat CoV-ARDS or symptoms of CoV-ARDS by resolving inflammation. In some embodiments, administration of a population of activated human Treg cells disclosed herein or an effective amount of a population of activated human Treg cells disclosed herein may induce the release of inhibitory cytokines (e.g., TGF-β, IL-6, IL-10, IL-17, IL-18, or IL-33).

[0238] In some embodiments, the human Treg cells used in these therapeutic methods express CCR4, a homing marker for lung tissue involved in trafficking to sites of CoV-ARDS-associated inflammation.

[0239] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0240] Any of the aspects and embodiments described herein may be combined with any other aspect or embodiment disclosed herein in the Summary of the Invention, the Drawings, and / or the Detailed Description of the Invention, including the following specific non-limiting examples / embodiments of the invention.

[0241] Numbered embodiments Without limiting the scope of the appended claims, the present disclosure describes the following numbered embodiments.

[0242] 1. A method for producing an expanded population of human regulatory T (Treg) cells enriched for CXCR4+ Treg cells from cryopreserved human umbilical cord blood units, comprising: (a) thawing a cryopreserved human umbilical cord blood unit; (b) diluting and washing the thawed cord blood units in a functionally closed system; (c)CD25 + isolating Treg cells using a dual selection method based on cell surface expression; (d) Isolated CD25+ Treg cells were cultured in medium in a gas-permeable culture vessel. (1) an effective amount of interleukin-2 (IL-2); (2) a reagent that specifically binds to CD3 and CD28, and (3) In the presence of anti-CXCR4 magnetic microbeads, CD25 + ex vivo expansion for up to 10 days or up to 12 days, with medium being changed about every 48 hours, to produce a CXCR4-enriched culture of Treg cells; (e) Activated CD25 from the culture medium + CXCR4 +Cells were harvested and CXCR4 + producing an expanded population of human Treg cells enriched for Treg cells.

[0243] 2. The method of embodiment 1, wherein in step (d), (1) ex vivo expansion begins on day 0; (2) administering an effective amount of IL-2 to CD25 cells isolated on day 0; + Add to Treg cells, (3) Reagents that specifically bind CD3 and CD28 are administered to the CD25 isolated on day 0. + A method of adding to Treg cells.

[0244] 3. The method according to embodiment 1 or 2, wherein the reagents that specifically bind to CD3 and CD28 are removed from the culture medium before the anti-CXCR4 magnetic microbeads are added to the culture medium.

[0245] 4. The method according to any one of embodiments 1 to 3, wherein in step (d), anti-CXCR4 magnetic microbeads are added to the culture medium 3 or 4 days after ex vivo expansion is initiated.

[0246] 5. In step (d), CXCR4 is isolated using a double ferromagnetic column. + The method according to any one of embodiments 1 to 4, wherein anti-CXCR4 magnetic microbeads are added to the culture medium for about 30 minutes before isolating the Treg cells.

[0247] 6. Isolated CD25 + The method of any one of embodiments 1-5, wherein Treg cells are enriched for CXCR4 in the third feed of ex vivo expansion.

[0248] 7. The method of any one of the preceding embodiments, wherein step (d) is carried out for 4 or 5 days.

[0249] 8. The method of any one of embodiments 1 to 7, wherein the reagent that specifically binds to CD25 is an anti-CD25 antibody or an antigen-binding fragment thereof.

[0250] 9. The method of any one of embodiments 1 to 8, wherein the reagent that specifically binds to CD25 is conjugated to a solid support.

[0251] 10. The method of embodiment 9, wherein the solid support is a magnetic microbead.

[0252] 11. The method of any one of embodiments 1 to 10, wherein the reagent that specifically binds to CD3 and CD28 comprises an anti-CD3 antibody, or an antigen-binding fragment thereof, and an anti-CD28 antibody, or an antigen-binding fragment thereof.

[0253] 12. The method according to any one of the preceding embodiments, wherein the reagents that specifically bind to CD3 and CD28 comprise anti-CD3-coated beads and anti-CD28-coated beads.

[0254] 13. The method of any one of embodiments 1-12, wherein the effective amount of IL-2 is about 1000 IU / mL.

[0255] 14. CXCR4 Isolation from Cryopreserved Human Umbilical Cord Blood Units + 1. A method for producing an expanded population of human Treg cells enriched for Treg cells, the method comprising: (a) thawing a cryopreserved human umbilical cord blood unit; (b) diluting and washing the thawed cord blood units in a functionally closed system; (c)CD25 + isolating Treg cells using a dual selection method based on cell surface expression; (d) ex vivo expanding the isolated CD25+ Treg cells in a gas-permeable culture vessel in a medium, the ex vivo expanding step comprising: (1) On day 0, anti-CD3 and anti-CD28 coated beads were added to the medium containing CD25+ adding to Treg cells; (2) on day 2, adding about 1000 IU / mL of IL-2 to the medium; (3) on the third or fourth day, removing the anti-CD3 and anti-CD28 coated beads from the medium and adding anti-CXCR4 magnetic microbeads to the medium; and (4) On day 3 or 4, CXCR4 + The anti-CXCR4 magnetic microbeads attached to the Treg cells were removed from the medium and fresh anti-CD3 and anti-CD28 coated beads were added to the CXCR4 + adding to Treg cells; CD25 + ex vivo expansion for up to 10 days or up to 12 days, with medium being changed about every 48 hours, to produce a CXCR4-enriched culture of Treg cells; (e) Activated CD25 from the culture medium + CXCR4 + Cells were harvested and CXCR4 + producing an expanded population of human Treg cells enriched for Treg cells.

[0256] 15. In step (d), IL-2 is administered to the isolated CD25 + The method according to any one of embodiments 1 to 14, wherein the medium containing the Treg cells is added about every 48 hours.

[0257] 16. The method according to any one of embodiments 12 to 15, wherein the anti-CD3 coated beads and the anti-CD28 coated beads are in a 1:1 ratio.

[0258] 17.CD25 + The method of embodiment 8 or 9, wherein the cells and the anti-CD3 and anti-CD28 coated beads are in a 1:1 ratio.

[0259] 18. In step (e), about 1 × 10 6 CD25 + The method according to any one of the preceding embodiments, wherein cells / mL are cultured.

[0260] 19. In step (e), the cells are first cultured in a 10 cm 2 19. The method according to any one of the preceding embodiments, wherein the culture is carried out in a gas permeable incubator having a membrane surface area of ​​1000 μm.

[0261] 20. Next, the culture is 2 20. The method of embodiment 19, wherein the culture medium is transferred to a gas permeable culture vessel having a membrane surface area of ​​1000 μm.

[0262] 21. The method of any one of embodiments 1 to 20, wherein in step (d), the culture is not rocked or stirred when adding IL-2.

[0263] 22. The method according to any one of the preceding embodiments, wherein in step (a), the cryopreserved human umbilical cord blood unit is thawed in a single step in a water bath.

[0264] 23. The method of any one of the preceding embodiments, wherein step (b) does not include manual washing.

[0265] 24. The method of any one of embodiments 1 to 23, wherein step (b) is carried out in a solution comprising PBS, EDTA, and about 0.5% human serum albumin.

[0266] 25. A double ferromagnetic column method is used in step (c) to isolate CD25 + The method according to any one of embodiments 1 to 24, wherein Treg cells are isolated.

[0267] 26. CXCR4 + 26. The method of any one of embodiments 1-25, further comprising cryopreserving the expanded population of human Treg cells enriched for Treg cells.

[0268] 27. CXCR4 as measured by flow cytometry + The expanded population of human Treg cells enriched for Treg cells is (i) ≥ 60% CD4 + CD25 + and (ii) ≥ 60% CD4 + CD25 + CXCR4 + and (iii) ≤10% CD4 - CD8 + and The method according to any one of embodiments 1 to 26.

[0269] 28. CXCR4 produced by the method according to any one of embodiments 1 to 27. + Expanded population of human Treg cells enriched for Treg cells.

[0270] 29. A method for treating or preventing bone marrow failure syndrome in a subject, the method comprising administering to the subject an effective amount of a population of activated human Treg cells produced by the method of any one of embodiments 1 to 23 or the population of embodiment 28.

[0271] 30. The method of embodiment 29, wherein the bone marrow failure syndrome is aplastic anemia, primary myelofibrosis or myelodysplastic syndrome.

[0272] 31. A method for treating or preventing primary myelofibrosis in a subject, the method comprising administering to the subject an effective amount of a population of activated human Treg cells produced by the method of any one of embodiments 1-27 or the population of embodiment 28, wherein a dose of 100 million Treg cells is administered to the subject on day 1 of a 28-day cycle for up to 6 cycles.

[0273] The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods described and claimed herein are made and evaluated and are intended to be, and are purely illustrative and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for herein. Unless otherwise specified, parts are parts by weight, temperature is in degrees Celsius or at ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0274] Example 1: Producing an expanded population of activated regulatory T cells from umbilical cord blood Cryopreserved human cord blood units (CBUs) were obtained from a qualified US public cord blood bank. The CBUs were rapidly thawed. The thawed cord blood units were subjected to automated washing using a Sepax device (Biosafe) with the starting volume set at 25 mL. The final volume was set at 100 mL with a dilution factor of 1.0. The washing reagents used were 5% human serum albumin (HSA) (CSL Behring) and 10% dextran-40 (D-40) (Hospira). After washing, cord blood cells were collected in a cord blood washing bag.

[0275] For washing purposes, the basic washing medium was 20 ml of 25% HSA to 1000 ml of PBS / EDTA buffer, the working washing medium was 300 ml of basic washing buffer and 50 mg of magnesium chloride (MgCl2) and 2500 units of DNase, where the modified medium was X-Vivo15 medium (Lonza) and 10 ml of GlutaMAX-1 and 100 ml of thawed human AB serum. After completing the automated wash, the washed cord blood cells underwent an additional manual wash using working washing medium. Here, the final volume consisted of 200 mL, and the reconstituted cells were centrifuged at 300 g for 10 min at room temperature. Finally, the washed cells were diluted with 100 × 10 PBS in 0.09 ml.6 Resuspend to a concentration of total nucleated cell (TNC) count of 100 x 10 cells.

[0276] Next, CD25 microbeads were added at 100 × 10 6 A ratio of 0.02 ml of human CD25 reagent was added per TNC. The cells and microbeads were incubated together for 30 minutes at 4 degrees Celsius. Following the incubation step, the cells were transferred into a Miltenyi LS column attached to a MidiMACS device, and a magnet was used to capture the anti-CD25 labeled cells. After immunomagnetic selection, the cells were released from the magnetic field.

[0277] Approximately 1×10 6 CD25 + The cells were washed and suspended in X-VIVO containing 1% L-glutamine, 10% human serum albumin (HSA) and interleukin-2 (IL-2, 1000 IU / mL). The solution was then mixed with anti-CD3 / anti-CD28 beads at a 1:1 ratio of cells to beads. The mixture was then transferred to a 10 cm 2 The culture was then transferred to a gas-permeable incubator with a membrane surface area of ​​100 cm. 2 The cells were then transferred to a 100-well gas-permeable incubator and incubated for a total of 14 days, with medium being changed every 48 hours without disturbing the cells. After 14 days, the cells were harvested and the anti-CD3 / anti-CD28 beads were removed with a magnetic particle concentrator. The cells were then resuspended in the final medium.

[0278] Cells were sampled at various points during the production process and their characteristics are shown in Table 9.

[0279] [Table 9]

[0280] As shown in Figure 1, expanded activated Treg cells produced by the above method were stable when stored at room temperature (15-30 °C) or 4 °C. Figure 1 shows the results of a flow cytometry-based assay to assess viability using 7-aminoactinomycin D (7AAD), a fluorescent intercalator that undergoes a spectral shift when bound to DNA, as 7AAD appears to be generally excluded from viable cells. Cells are incubated on ice for 30 min in the presence of 1 microliter of 7AAD stock solution. As soon as possible after the incubation period, stained cells are analyzed by flow cytometry using violet and 488 nm excitation, and fluorescence emission is measured using 440 nm and 670 nm bandpass filters (or their close equivalents). Viable cells show only low levels of fluorescence.

[0281] The phenotype of the expanded activated Treg cells was measured by flow cytometry at the initiation of cell culture (day 0) and on days 8 and 14 after initiation of cell culture. The results are shown in Table 10.

[0282] [Table 10]

[0283] As shown in Figures 2A and 2B, expanded activated Treg cells are suppressive, showing 70-96% suppression. As shown in Figures 4A-4D, expanded activated Treg cells do not express RORγt and show a reciprocal increase in IL-10 expression in response to stress. Figure 4A shows that IL-6 does not affect the suppressive activity of Treg cells. Figure 4B shows that IL-6 does not affect RORγ expression by Treg cells. Figure 4C shows that IL-6 does not affect IL-17A production by Treg cells. Figure 4D shows that IL-6 induces an increase in IL-10 production by Treg cells. Figure 25 shows that IL-6 induces granzyme B production by Treg cells. Furthermore, expanded activated Treg cells can be immunosuppressive across the HLA barrier (Figure 3). Expanded activated Tregs show a Gaussian (polyclonal) distribution of the T cell receptor V repertoire (Figure 6).

[0284] Expanded activated Treg cells maintain suppressive properties even in the presence of steroids. Figures 7A and 7B show that Treg cells maintain suppressive properties in the presence of dexamethasone. The effect of prednisone on the viability of Treg and Tcon cells is shown below.

[0285] [Table 11] As shown below, Treg cells remain suppressive in the presence of prednisone.

[0286] [Table 12]

[0287] Example 2: Cryopreservation of expanded populations of activated regulatory T cells from umbilical cord blood The expanded activated Treg cells produced by the method described in Example 1 were cryopreserved as follows.

[0288] Total of 50 x 10 per 5ml vial 610 x 10 cells per ml 6 The harvested expanded population of activated human Treg cells was centrifuged at 400 g for 10 min at a temperature of 4° C. The total cell number was calculated using an automated cell counter, and the total cell number was determined to be 50×10 6 The number of cryovials was estimated by dividing by the number of cells. The cells were then frozen to a maximum of 50 x 10 per 5 mL cryovial using a frozen stock solution consisting of a pre-formulated solution containing 10% dimethyl sulfoxide (DMSO) (Cryostor®). 6 cells were cryopreserved. While the cells were being centrifuged, the controlled speed freezer was turned on and once the controlled speed freezer reached the appropriate starting temperature, the command "The program is waiting for the user to click here to continue" was displayed. Upon mixing with the frozen stock solution, up to 50 × 10 6 Cryovials containing each of the cells were placed in a controlled rate freezer using a freezing algorithm, allowing the freezing rate of the cells to be regulated to avoid cell death and maintain cell function. After the freezing program was completed, the cryovials were removed from the controlled rate freezer and placed in a liquid nitrogen cryogenic freezer at a temperature of -190°C for long-term cryopreservation.

[0289] Cryopreserved activated Treg cells display a consistent phenotype and are capable of immunosuppression similar to freshly activated Treg cells (Figures 8A-8C). Cryopreserved activated Treg cells display high expression of Helios (Figure 8B) and suppression of expanding conventional T cells (Figure 8C). As further described in Example 3, cryopreserved and freshly expanded activated Treg cells are equivalent in preventing or treating graft-versus-host disease.

[0290] Example 3: Prevention and treatment of graft-versus-host disease with regulatory T cells derived from umbilical cord blood A xenogeneic mouse model of graft-versus-host disease (GVHD) was used to evaluate the function of umbilical cord blood-derived regulatory T cells generated by the methods described in Examples 1 and 2. The model of GVHD is described in Parmar et al., Cytotherapy 16(10:90-100(2013)). To study the effect of Tregs on the prevention of GVHD, NOD / SCID IL-2Rγ null (NSG) mice (Jackson Laboratory, Bar Harbor, ME) were inoculated with 1×10 7 One day before injection of 1 x 10 Treg cells, and 7 Two days prior to intravenous infusion of human PBMCs, mice received sublethal total body irradiation (300 cGy from a 137Cs source delivered over 1 min by a JL Shepherd and Associates Mark I-25 Irradiator, San Fernando, CA). Mice were assessed using a clinical GVHD scoring system (see Reddy et al., Transplantation 69(4):691-93 (2000)). Treatment with fresh cord blood-derived Tregs and cryopreserved Tregs resulted in comparable GVHD scores (Figure 9A) and effects on body weight (Figure 9B).

[0291] Administration of cryopreserved Tregs prevented and treated GVHD in a xenogeneic mouse model. Figure 10A shows the study design for monitoring the effect of a single Treg infusion on GVHD prevention. Figure 10B shows the study design for monitoring the effect of multiple Treg infusions on GVHD treatment. As shown in Figures 11A-11B, administration of activated Tregs can both prevent and treat GVHD. Administration of activated Tregs suppresses the levels of inflammatory cytokines in peripheral blood 14 days after PBMC infusion (Figures 12A-12F). Activated Tregs are distributed to the site of inflammation in treated mice (Figure 13). Furthermore, activated Tregs do not interfere with conventional T cell-mediated anti-leukemia effects (Figure 14).

[0292] Example 4: Treatment of systemic lupus erythematosus with cryopreserved umbilical cord blood-derived regulatory T cells Taking advantage of a xenogeneic mouse model of systemic lupus erythematosus (SLE) (Andrade et al., Arthritis Rheum. 2011 Sep, 63(9):2764-2773), we transplanted systemic lupus erythematosus-derived peripheral blood mononuclear cells into non-SCID gamma-null (NSG) mice by intravenous injection on day 0. 6 Female Rag2 mice transplanted with 100 human SLE peripheral blood mononuclear cells (PBMCs) - / - γc - / - Mice. The mice were allowed to develop disease and 30 days after implantation, they were divided into two groups: i) control and ii) treatment. 1 × 10 7 Ex vivo expanded, cryopreserved, allogeneic, HLA-mismatched CB Tregs were injected intravenously into SLE xenografts once a week via the tail vein for 4 weeks. Serial blood sampling was performed for phenotypic analysis, cytokine assays, and anti-double-stranded (ds)DNA IgG antibody analysis. Serial urine samples were performed for quantification of creatinine and albumin. Histopathological examination of harvested organs was performed at the time of scheduled euthanasia at 13 weeks.

[0293] This SLE model was used to evaluate the function of cord blood-derived regulatory T cells generated by the methods described in Examples 1 and 2. As shown in Figure 15, a single injection of activated Treg cells inhibited CD45 T cells 9 weeks after engraftment of SLE-PBMCs. +Activated Treg cells were injected four times a week and reduced the levels of effector T cells. SLE-PBMCs were injected on day 0, and weekly injections of cord blood (CB) Tregs were administered starting on week +4. Four weekly injections of activated Treg cells improved survival in SLE mice (Figure 16A) and reduced the levels of anti-double-stranded DNA antibodies (dsDNA Ig) (Figure 16B). The presence of anti-double-stranded DNA antibodies is a marker of lupus disease activity. Treg recipients showed sustained weight gain and lower GVHD scores. Four weekly injections of activated Treg cells also reduced the levels of urinary albumin (Figure 17A), urinary creatinine outflow (Figure 17B), and improved renal histology (Figure 18) in SLE mice. As shown in Figure 19, administration of activated Tregs reduces the concentration of human sCD40L in SLE mice. Weekly injections of activated cryopreserved Tregs also reduced the levels of circulating CD8 + A sustained decrease in effector T cells (Figure 20A) and CD8 + This resulted in a reduction in the infiltration of effector T cells (Figure 20B). Histopathological results from two index cases from each group showed that Treg recipients had reduced renal T cell (CD3 + ) and B cells (CD20 + ) and reduced lymphocytic infiltration into the glomeruli and renal parenchyma compared to the control group.

[0294] Example 5: Treatment of multiple myeloma with fresh cord blood-derived regulatory T cells Transwell migration assay 6.5 mm 24-well transwell plates with 8.0 μm pore polycarbonate membrane inserts (Corning, Corning, NY, US) were used. T effector cells (Teff) were cultured in a 24-well plate containing 100% CD3 +MicroBeads (Miltenyi Biotec) were used for isolation. Firefly luciferase / GFP-labeled MM1.S and wild-type RPMI 8226 cells were obtained from the Orlowski laboratory (MD Anderson Cancer Center (MDACC)). U266 and HL-60 cells were purchased from the American Type Culture Collection (Manassas, VA). Nalm6 cells were provided by the Institute of Hematopathology (MDACC). RPMI 8226 and Nalm6 cells were stained with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) according to the manufacturer's instructions. Target cells: GFP-labeled MM1.S (3 × 10 5 cells), GFP-labeled U266 (3 × 10 5 cells), CFSE-stained RPMI 8226 (3 × 10 5 cells), or negative control GFP-labeled HL-60 (1.5 × 10 5 cells) or CFSE-stained Nalm6 (6 × 10 5 Each of the actor cells (CB Tregs) was resuspended in 300 μL of medium and seeded in the upper compartment of the transwell. 6 cells) or positive control CD3 + Teff(1×10 6 Cells) were resuspended in 750 μL of media and added to the lower compartment. A schematic of the experiment is shown in Figure 40A. Migrated target cells were analyzed using a flow cytometer (BD FACSCanto™).

[0295] To understand the influence of CB Treg cells on the trafficking of myeloma cells, transwell experiments were set up and target cells were seeded in the upper compartment of the transwell (Figure 40A). These target cells were myeloma cells: GFP-MM1.S, GFP-U266 or CFSE-RPMI8226. In addition, two leukemia cell lines were used as negative control target cells: GFP-HL60 (acute myeloid leukemia) or CFSE-Nalm6 (pre-B leukemia). Actor cells were seeded in the lower compartment and were CB Treg cells or, as a positive control, Teff cells. Such measures were taken to isolate myeloma-specific influences of CB Treg. CB Treg were able to prevent the migration of MM1.S (Figure 40B, p<0.01)) and RPMI 8226 (Figure 40C, p=0.04) but not U266 (Figure 40D, p=0.14). No effect of CB Tregs was observed on the migration patterns of leukemia cell lines, including HL-60 (FIG. 40E) or Nalm6 (FIG. 40F).

[0296] Heterogeneous multiple myeloma mouse model A xenogeneic mouse model of multiple myeloma was used to evaluate the function of cord blood-derived regulatory T cells generated by the methods described in Examples 1 and 2. Non-SCID γ-null female mice (Jackson Laboratory, Bar Harbor, ME) were inoculated with firefly luciferase-labeled MM1.S cells (ATCC, Manassas, VA) (3×10 6 cells / mouse), 1 × 10 7Mice were injected intravenously via the tail vein with or without ex vivo expanded CB Treg cells. CB Treg cells were injected 1 day before MM1.S cell injection. Mice were then photographed as previously described (Parmar et al., Cytotherapy, 2014.16(1):p.90-100). Mice were bled once a week. Plasma samples were sent to Eve Technologies (Calgary, AB, Canada) for measuring mouse cytokine levels. Lysed blood was stained with anti-human CD45 / APC (Thermo, anti-human CD25 / PE (Becton Dickinson), anti-human CD38 / APC eFluor780 (Thermo Fisher Scientific), and anti-mouse CD45 / Pacific Blue (Thermo Fisher Scientific). Cells were acquired by BD FACSCanto™ II. At the time of euthanasia, bone marrow and spleen were harvested.

[0297] Survival was estimated using the Kaplan-Meier method, and groups were compared using the log-rank test. Two groups were compared by unpaired Student's t-test, and more than two means were compared by one-way analysis of variance followed by Bonferroni's test for multiple comparisons. Values ​​are expressed as the mean and standard error of the mean. P values ​​<0.05 were considered statistically significant. All statistical analyses and graph generation were performed using GraphPad Prism 7.0 (San Diego, CA).

[0298] To understand the effect of CB Tregs on preventing myeloma engraftment, 3 × 10 6 A heteromyeloma mouse model in which MM1.S cells were injected intravenously. In the treatment group, CB Tregs (1 × 10 7The mice were injected with 1000x the number of circulating CD38 cells (100x the number of Treg recipients). Mice were weighed twice weekly, when "myeloma alone" mice were observed to lose weight and significant differences were evident at the time of euthanasia, with weights remaining similar in both groups until week 3 post-tumor inoculation (Figure 21A). Myeloma burden was quantified in peripheral blood, with circulating CD38 cells elevated by day 28 in the control group compared to Treg recipients where the difference became statistically significant by the time of euthanasia. + A similar trend was observed with a slight increase in myeloma cells (FIG. 21B; myeloma alone: ​​0.8%±0.3 vs. myeloma with Tregs: 12.4%±2.9, P=0.002).

[0299] Mice were imaged weekly using non-invasive bioluminescence, and significant uptake of GFP-labeled MM1.S cells was again evident in the control group approximately 3 weeks after tumor inoculation and widespread by week 4, with minimal luminescence detected in CB Treg recipients (Figure 21C). Tumor progression was rapid, and the increase in tumor burden quantified by BLI in CB Treg recipients was significantly delayed compared to that in the control group over the observation period (Figure 21D).

[0300] As myeloma cells thrive in an inflammatory tumor microenvironment and interleukin-6 (IL-6) has been implicated as a major driver of myeloma disease progression (Harmer et al. Front Endocrinol (Lausanne), 2018, 9: p. 788), the influence of CB Tregs on this inflammatory cytokine was investigated. As shown in Figure 23, circulating IL-6 levels were comparable in the two groups until week 4 after tumor inoculation, when a significant increase in plasma IL-6 levels in the "myeloma alone" group was measured and continued to increase until week 5. Ultimately, the increased tumor burden and increased inflammatory burden translated into mortality in the "myeloma alone" group and led to a statistically significant survival advantage for Treg recipients (Figure 22). At the time of euthanasia, tumor cells were measured in harvested organs and compared between the two groups. Myeloma cells were barely detectable in the bone marrow of Treg recipients compared to the "myeloma alone" group (Figure 24A; 0.6% ± 0.1 vs. 90.0% ± 2.2, P < 0.0001). A similar pattern was observed in the spleen (Figure 24B: myeloma + Treg: 1.3% ± 0.4 vs. myeloma alone: ​​12.9% ± 4.2, P = 0.009).

[0301] The data support the hypothesis that a single injection of CB Treg cells prior to injection of myeloma cells confers a sufficient growth advantage that allows suppression of inflammatory signals generated by myeloma cells in vivo, as shown by the lack of IL-6 production that ultimately results in conditions hostile to myeloma engraftment. The physical signs of tumor burden and weight loss, as well as the overlay of circulating and organ-infiltrating myeloma cells, reinforce the systemic anti-inflammatory effect of CB Treg cells.

[0302] Impact on established myeloma disease Method: 3×10 6 GFP-labeled MM.1S cells were injected into NSG mice, followed by 5 × 10 6 CD3 + T conventional (Tcon) cells were injected. In a subset of Tcon-treated mice, 1 × 10 7CB Treg cells were injected on days +16, +23, and +30 (see experimental design table below). Mice were followed every other day for body weight and GVHD scores. Non-invasive bioluminescence imaging (BLI) was performed serially. Blood was collected weekly for cellular and cytokine assays. At the time of euthanasia, blood, spleen, and bone marrow were harvested for histopathology and flow analysis. In subsequent experiments, intraperitoneal injections of a bispecific antibody against CD3 and BCMA (BCMA-BiTE® (bispecific T cell engager)) were administered in the heteromyeloma model in the presence or absence of CB Treg cells. All mice were supplemented with Pan T cells to promote the antitumor effect of BiTE®. The experimental design is shown in Figure 61E.

[0303] [Table 13]

[0304] Results: Both Tcon and Tcon+Treg recipients maintained their body weight compared to myeloma alone or myeloma+Treg groups (Figure 61A). Addition of Treg did not prevent Tcon-mediated anti-myeloma effects and prevented delayed relapse (Figures 61B-D). Addition of Treg+BiTE® resulted in a similar degree of tumor control compared to mice treated with BiTE® alone (Figure 61F). Addition of Treg did not prevent BiTE®-mediated anti-myeloma effects. Addition of Treg mitigated BiTE®-induced weight loss (Figure 61G) with corresponding higher GVHD scores (Figure 61H).

[0305] Example 6: Evaluation of safety and efficacy for administering cord blood-derived regulatory T cells in the treatment of bone marrow failure syndromes and other autoimmune diseases Rationale for the study Adoptive therapy with cord blood-derived regulatory T cells may be able to reduce circulating proinflammatory cytokines and improve outcomes. Previous studies have shown that infusion of cord blood-derived regulatory T cells is safe and may be effective in preventing GVHD, but efficacy in both preclinical and clinical trials appears to be highly dependent on the ratio of Treg to Tcon in vivo. Current strategies to minimize GVHD call for long-term immunosuppressive therapy with drugs such as calcineurin inhibitors (CNIs), cyclosporine, and tacrolimus. However, this prolonged immunosuppression results in delayed immune function and poses the risk of infectious complications and post-transplant lymphoproliferative disorders. Therefore, adoptive therapy with cord blood-derived regulatory T cells may be an attractive alternative for the treatment of GVHD and other autoimmune diseases.

[0306] The cord blood derived regulatory T cell product (CK0801) consists of ex vivo expanded regulatory T cells derived from a single cord blood unit (CBU) and produced according to the methods described herein.

[0307] The purpose of this study is to evaluate whether it is safe and practical to administer CK0801 to patients with treatment-resistant bone marrow failure syndromes, including myelodysplasia, myelofibrosis, and aplastic anemia. Only patients with relapsed / refractory bone marrow failure who have not responded to standard treatments will be enrolled in these studies. The study will determine the highest possible dose that is safe to administer and whether CK0801 improves symptoms of bone marrow failure syndromes.

[0308] Participants eligible to take part in this study are either unable or unwilling to be treated with standard treatments, or have failed standard treatments.

[0309] Main purpose The primary objective is to determine dose-limiting toxicity of CK0801, defined as any event that begins individually during CK0801 infusion.

[0310] Severe (grade 3 or 4) infusion toxicity within 24 hours (NCI-CTCAE V4.0) Regimen-related death within 30 days Severe (grade 3 or 4) cytokine release syndrome within 30 days secondary purpose Preliminary evaluation of disease-specific responses Duration of disease-specific response

[0311] exploratory purpose Peripheral blood and bone marrow immune reconstitution and inflammatory cytokines will be assessed at baseline and at scheduled follow-up in the post-treatment setting. Samples will be collected on days -10, 0, +3, +7, +14, +21, +30, +60, +90, and 1 year after each infusion.

[0312] Groups and interventions [Table 14] research design A standard 3+3 Phase I statistical design was utilized, with 3 patients enrolled at dose level 1:1×10 6 If no dose-limiting toxicity (DLT) is observed, the dose will be increased to the next cohort of 3 patients at dose level 2: (range) >1 × 10 6 / kg~1×10 7 / kg. If no DLT is observed, the dose will be increased to dose level 3: (range) >1 × 10 7 / kg~1.5×10 7 / kg.

[0313] If one DLT is observed at a dose level, three additional patients will be treated at that level. If there are no additional DLTs, that dose level will be defined as the MTD.

[0314] In the event of ≥ 2 DLTs at dose levels 2 or 3, the previous dose level will be defined as the MTD. In the event of ≥ 2 or more DLTs at dose level 1, the Data Safety Monitoring Board (DSMB) will review and evaluate continuation of the study.

[0315] The MTD will be determined when 6 patients are treated at a dose level with <2 DLTs. A maximum of 18 patients will be treated.

[0316] Upon enrollment of subjects into each study cohort (3 or 6 patients), the cohort will be closed until 30 days after the last patient completes Day 0 (CK0801 infusion). Dose escalation may occur only following DSMB review of previously dosed cohorts.

[0317] If the eligibility criteria are met, the subject is consented and enrolled in the study.

[0318] Investigational Drug Sources and Pharmacology CK0801 (Cord Blood-derived Regulatory T Cells) is manufactured at the Cellenkos GMP facility using a single allogeneic unrelated donor cord blood unit selected based on predefined criteria and certified for use in manufacturing. CK0801 is a CD25 Treg / Treg antibody with collection of Tregs and final formulation in Plasma-Lyte A and 0.5% human serum albumin (HSA). + Manufactured using a Treg immunomagnetic selection and 14-day culture expansion process. Final cell product is released only after a formal lot release process that includes review of all available test results. Lot release criteria include 7AAD viability of ≥ 70%, %CD4 + CD25 + Cellular purity, %CD4 <10% - / CD8 + cells, <100 / 3×10 6 These include anti-CD3 / anti-CD28 Ab bead count of 100 cells, Gram stain for "no microorganisms", endotoxin <5EU / kg, sterility (sampled 48-72 hours prior to final formulation) negative, and mycoplasma negative.

[0319] Finding, selecting, and shipping cord blood to manufacturing facilities Cord blood units provided to Cellenkos, Inc. for the generation of CK0801 will be obtained from individually accredited and selected Cord Blood Banks (CBBs) that meet minimum accreditation standards of the Foundation for Accreditation of Cellular Therapy (FACT) or the American Association of Blood Banks (AABB). Eligible CB units can be classified as either accredited or non-accredited and meet pre-determined eligibility criteria.

[0320] Upon agreement, the subject provides a blood sample for HLA typing. The results are provided to the sponsor's clinical coordinator to facilitate the cord blood search and selection process. The sponsor identifies available cord blood units according to a standard search algorithm that are HLA-matched to the recipient (subject) at 3, 4, 5, or 6 of the 6 antigens at the HLA-A, HLA-B, and DRB1 loci, and a list is provided to the Principal Investigator (PI). The sponsor and PI select the appropriate cord blood units based on predefined criteria.

[0321] After the cord blood units are selected, the sponsor's clinical coordinator arranges for shipping and transportation logistics and the units are shipped to Cellenkos' GMP manufacturing facility. Upon arrival at the manufacturing facility, the cord blood units are inspected, checked in, and verified against the CB donor / recipient shipping request. Cord blood units that meet acceptance criteria (including identification, labeling, and temperature) are stored in liquid nitrogen, vapor-phase storage freezers at ≤-150°C until day -14 (start of manufacturing) and coordinated with the subject's planned infusion schedule.

[0322] Prior to infusion, the sponsor's clinical coordinator and on-site clinical team are responsible for arranging for the infusion of CK0801 at the prescribed time point and time frame. CK0801 should be administered within 8 hours of final formulation.

[0323] The sponsor's clinical coordinator will arrange for transportation of CK0801 to the clinical site. The on-site clinical team will be responsible for receiving, accepting, preparing, and managing CK0801.

[0324] Formulation and Stability CK0801 is formulated to the final cell dose in Plasmalyte + 0.5% Human Serum Albumin (HSA) buffer. Injection of CK0801 should occur within 8 hours of final formulation.

[0325] Storage and Handling CK0801 will be transported to the clinical site in a shipping container validated to maintain a temperature of 15°C to 30°C and will be maintained at 15°C to 30°C prior to infusion.

[0326] toxicity Although infusion of cord blood-derived regulatory T cells has previously been shown to be safe, subjects should be monitored during CK0801 infusion as per standard clinical practice. Recommended timing of vital signs on each infusion day: pre-infusion, 15 minutes after start of infusion, 30 minutes after start of infusion, 1 hour after start of infusion, 2 hours after start of infusion, then per standard clinical practice.

[0327] Vital signs include temperature, respiration, blood pressure, and pulse.

[0328] Route of administration CK0801 is administered via a central or peripheral line at a rate not to exceed 5 mL / min. After administration, the bag and line are repeatedly flushed with saline.

[0329] CK0801 injection The study will explore infusions of CK0801 at three different dose levels and will use a standard 3+3 Phase I statistical design.

[0330] No conditioning or lymphodepletion was administered to the patients. Three patients were treated at dose level 1:1 × 10 6 / kg IBW. If no dose-limiting toxicity (DLT) is observed, the dose will be increased to Dose Level 2: (range) 3 × 10 for the next cohort of 3 patients. 6 / kg IBW. If no DLT is observed, the dose will be increased to dose level 3: (range) 1 × 10 7 / kg IBW.

[0331] If one DLT is observed at a dose level, three additional patients will be treated at that level. If there are no additional DLTs, that dose level will be defined as the MTD.

[0332] In the event of ≥ 2 DLTs at dose levels 2 or 3, the previous dose level will be defined as the MTD. In the event of ≥ 2 DLTs at dose level 1, the Data Safety Monitoring Board (DSMB) will review and evaluate continuation of the study.

[0333] The MTD will be determined when 6 patients are treated at a dose level with <2 DLTs.

[0334] Patients are premedicated with diphenhydramine (Benadryl®) 50 mg IV piggyback (IVPB) and acetaminophen 650 mg orally 30 minutes prior to infusion of CK0801. CK0801 is infused by gravity flow over 15-30 minutes via an IV line that must contain no solutions other than 0.9% Sodium Chloride (Normal Saline) USP. CK0801 is compatible with standard blood product tubing. The use of filters is prohibited.

[0335] Study population selection Inclusion criteria 1. Subject meets diagnostic criteria for bone marrow failure syndrome, including aplastic anemia, myelodysplastic syndrome, or myelofibrosis. 2. HLA-matched (≥3 / 6 for HLA-A, HLA-B, and HLA-DRB1) cord blood units available in the CK0801 generation. 3. Participants must be 18 years of age or older. 4. Bilirubin ≤ 2xULN and SGPT (ALT) ≤ 2xULN (unless Gilbert's syndrome is documented). 5. Creatinine clearance >50 mL / min was calculated using the Cockcroft-Gault formula. 6.Zubrod performance status ≦2. 7. Female subjects of childbearing potential (FPCP) must have a negative urine or serum pregnancy test. Note: FPCP are defined as premenopausal and not surgically sterilized. FPCP must agree to use maximally effective contraception or abstain from heterosexual activity throughout the study. Effective contraceptive methods include intrauterine devices, oral and / or injectable hormonal contraception, or two appropriate barrier methods (e.g., cervical cap with spermicide, diaphragm with spermicide). 8. Subject agrees to comply with all procedures required by the protocol, including study-related assessments, visits, and long-term follow-up. 9. Subject is willing and able to provide informed consent. Exclusion criteria 1. Subject received an investigational drug within 4 weeks prior to CK0801 infusion. 2. Subject has undergone radiation therapy or chemotherapy within 21 days prior to CK0801 infusion. 3. Subject has previously received cord blood-derived regulatory T cell therapy. 4. Known HIV seropositivity. 5. Subject has an uncontrolled infection that has not responded to appropriate antimicrobials after 7 days of treatment. The protocol PI is the final arbiter of eligibility. 6. Subject with uncontrolled intercurrent illness that, in the opinion of the Investigator, places the patient at high risk of severe toxicity and / or impairs the activity of CK0801. 7. The subject is pregnant or lactating. 8. Bone marrow failure caused by stem cell transplantation. 9. Subjects unable to provide consent or who, in the Investigator's opinion, are unlikely to fully comply with the protocol requirements.

[0336] Data collection Treatment and toxicity data related to CK0801 infusions will be collected from the day of the first CK0801 infusion until up to 30 days after the last infusion.

[0337] Subjects who experience study-related death or documented disease progression with subsequent alternative treatment will be considered treatment failures and treated as observations censored at the time of the event, with no further data collection. Subjects who withdraw informed consent or discontinue the study for non-compliance will also be censored at that time. Results judgment method Main outcome measures: 1. To evaluate the safety of infusing CK0801 in subjects suffering from bone marrow failure by collecting adverse events and serious adverse events as assessed by CTCAE v4.0, with a participant population of treatment-related adverse events. Dose-limiting toxicity is defined to include any event that begins individually during CK0801 infusion. Severe (grade 3 or 4) infusion toxicity within 24 hours (NCI-CTCAE V4.0) Regimen-related death within 30 days Severe (grade 3 or 4) cytokine release syndrome within 30 days [Time frame: 30 days from injection] Secondary outcome determination method: 2. Preliminary assessment of disease-specific response to treatment and duration of response [Time frame: 12 months] Other pre-specified outcome measures: 3. Assess bone marrow (BM) immune reconstitution and inflammatory cytokines Bone marrow samples will be taken at baseline and at scheduled follow-up in the post-treatment setting to analyze immune reconstitution and inflammatory cytokines. [Time frame: 12 months] 4. Assess immune reconstitution and inflammatory cytokines in peripheral blood (PB) Peripheral blood will be collected at baseline and at scheduled follow-up in the post-treatment setting to analyze immune reconstitution and inflammatory cytokines. [Time frame: 12 months]

[0338] Results of a Phase I Clinical Trial of Allogeneic Cord Blood-Derived Treg Cells in Patients with Bone Marrow Failure (BMF) A schematic of the study design is shown in Figure 41. The timing of the correlative studies is shown in the table below. Figure 28 shows that a Phase 1 clinical trial of CK0801 in subjects suffering from bone marrow failure failed to show an early efficacy signal.

[0339] [Table 15]

[0340] Figure 42 provides a description of subjects undergoing treatment in the Phase 1 clinical trial. A summary of the clinical data is shown in Figures 43 and 44.

[0341] Cohort I The treatment history of patient 1 is shown in Figure 45. The patient is a 63-year-old male diagnosed with primary myelofibrosis. The patient received 1 x 10 6 Patients were treated with 100 Treg cells / kg (67 million cells) infused over 17 minutes. Patients also received ruxolitinib 20 mg PO (orally) BID (twice daily). Patient clinical data is shown in Figure 46A and Figure 46B. Inflammatory cytokine levels are shown in Figure 47 and Figure 48. Patients showed a reduction in JAK2 mutation burden (Figure 46B) and splenomegaly (Figure 49) that correlated with the SDF1α-CXCR4 axis (Figure 48). Bone marrow assessments of patients before (PRE) and after (POST) Treg cell administration are shown in Table 16, Table 17a and Table 17b.

[0342] [Table 16]

[0343] [Table 17-1]

[0344] [Table 17-2]

[0345] The treatment history of patient 2 is shown in Figure 50. The patient is a 46-year-old female diagnosed with myeloproliferative neoplasms (MPN) in adolescents and young adults (AYA). The patient received 1 x 10 6 Patients were treated with 100 Treg cells / kg (60 million cells) infused over 20 minutes. Patients also received ruxolitinib 20 mg PO BID. Inflammatory cytokine levels are shown in Figure 51. Bone marrow assessments of patients pre- (PRE) and post- (POST) Treg cell administration are shown in Tables 18, 19, and 20.

[0346] [Table 18]

[0347] [Table 19]

[0348] [Table 20]

[0349] Patient 3 is a 19-year-old female diagnosed with acquired aplastic anemia and is transfusion dependent. 6 The patient was treated with 100 Treg cells / kg (50 million cells) infused over 25 minutes. The patient was also taking eltrombopag and cyclosporine (CSA). The patient's TPO levels over time are shown in Figure 52. The patient's platelet transfusion requirements over time are shown in Figure 53. The patient's PRBC (packed red blood cell) transfusion requirements over time are shown in Figure 54. The patient's bone marrow assessments pre- (PRE) and post- (POST) Treg cell administration are shown in Tables 21, 22, and 23.

[0350] [Table 21]

[0351] [Table 22]

[0352] [Table 23]

[0353] Cohort II Patient 4 was a 29-year-old man diagnosed with idiopathic severe aplastic anemia. 6 Patients were treated with 100 Treg cells / kg. Patients were also using hATG+CSA+steroids+eltombopag+Peg-filgrastim. Patients' platelet transfusion requirements over time are shown in Figure 55. Patients' PRBC (packed red blood cell) transfusion requirements over time are shown in Figure 56. Bone marrow assessments of patients pre-Treg cell administration (PRE) and post-Treg cell administration (POST) are shown in Tables 24, 25, and 26.

[0354] [Table 24]

[0355] [Table 25]

[0356] [Table 26]

[0357] Patient 5 was a 62-year-old woman diagnosed with primary myelofibrosis (essential thrombocythemia (ET)). 6Patients were treated with 100 Treg cells / kg. Bone marrow evaluations of patients pre- (PRE) and post- (POST) Treg cell administration are shown in Tables 27, 28, and 29.

[0358] [Table 27]

[0359] [Table 28]

[0360] [Table 29]

[0361] Patient 6 is a 74-year-old man diagnosed with primary myelofibrosis (grade 2, hypocellular transfusion-dependent). The patient failed treatment with LCL-161 (Novartis, Basel, Switzerland). The patient received 3 × 10 6 Patients were treated with 100 Treg cells / kg. Patient platelet transfusion requirements over time are shown in Figure 57. Patient PRBC (packed red blood cell) transfusion requirements over time are shown in Figure 58. Bone marrow assessments of patients pre-Treg cell administration (PRE) and post-Treg cell administration (POST) are shown in Tables 30, 31, and 32.

[0362] [Table 30]

[0363] [Table 31]

[0364] [Table 32] conclusion Cohort 1 (dose = 1 × 10 6 cells / kg) and cohort 2 (dose = 3 × 10 6No SAEs were observed in six patients treated with 100 mg / kg of IgG4. Improvement of JAK2 mutant allele in patient #1. Durability of response = 6 months Improvement in MPN score for patient #2. Durability of response at 4 months Improvement in red blood cell and platelet transfusion requirements in patient #3. Response durable for 4 weeks Improvement in red blood cell and platelet transfusions in patient #4. Response durable for 4 weeks -Improvement of chronic pain in patient #5. Durable for 4 weeks -Improvement of red blood cell and platelet transfusions in patient #6. Evaluation performed at 4 weeks -Improvement of bone marrow cellularity in patients #1 and #2 Improvement of bone marrow dysplasia in patients #3 and #4 - In all cases except patient #4 (stable), the M:E ratio decreases

[0365] Example 7: Evaluation of safety and efficacy for administering cord blood-derived regulatory T cells in the treatment of treatment-resistant Guillain-Barre syndrome This study will examine whether it is safe and practical to administer CK0801 (a regulatory T cell product derived from umbilical cord blood) to patients with Guillain-Barre syndrome (GBS). Additionally, the highest possible dose that is safe to administer will be determined. Similarly, the study will also examine whether CK0801 can improve the symptoms of GBS.

[0366] Target layer The target population for this study is patients who do not respond to standard treatment with intravenous immunoglobulin (IVIG) therapy or plasma exchange.

[0367] Registration Up to 18 adult patients (aged 18-70 years) will be enrolled. Eligibility Inclusion criteria: 1. Subject meets diagnostic criteria for Guillain-Barre Syndrome (GBS). 2. HLA-matched (≥3 / 6 for HLA-A, HLA-B, and HLA-DRB1) cord blood units available in the CK0801 generation. 3.Target age range: 18-70 years old. 4. Subject has a GBS Disability Scale score of 4 that remains unchanged after 1 week of IVIG or PE treatment. 5. Subject has completed IVIG / PE treatment ≥ 4 weeks prior to CK0801 infusion. 6. Subject had a changed Erasmus GBS outcome score (mEGOS score) of ≥ 7 at the time of presentation and unchanged one week after IVIG or PE treatment. 7. Bilirubin ≤ 2xULN and ALT ≤ 2xULN (excluding Gilbert's syndrome). 8. Creatinine clearance >50 mL / min was calculated using the Cockroft-Gault equation for adult patients aged 18–70 years. 9. Female subjects of childbearing potential (FPCP) must have a negative urine or serum pregnancy test. Note: FPCP are defined as premenopausal and not surgically sterilized. FPCP must agree to use maximally effective contraception or abstain from heterosexual activity throughout the study. Effective contraceptive methods include intrauterine devices, oral and / or injectable hormonal contraception, or two appropriate barrier methods (e.g., cervical cap with spermicide, diaphragm with spermicide). 10. Subject agrees to comply with all procedures required by the protocol, including study-related assessments, visits, and long-term follow-up. 11. Subject is willing and able to provide written informed consent. If the subject is temporarily unable to sign the agreement due to disease-related complications (e.g., upper limb paralysis), a legally authorized representative (LAR) will be used. Subjects will sign the consent as soon as they are able. Exclusion criteria: 1. Subject received immunotherapy, chemotherapy, biologic or investigational medication within 4 weeks prior to CK0801 infusion. 2. Subject has previously received CB Treg therapy. 3. Subject has an uncontrolled infection that has not responded to appropriate antimicrobials after 7 days of treatment. The protocol PI is the final arbiter of eligibility. 4. Subject has been vaccinated against a live virus (e.g., measles, mumps, rubella, chickenpox). 5. The subject is pregnant or lactating. 6.HIV seropositive 7. Subjects unable to provide consent or who, in the Investigator's opinion, are unlikely to fully comply with the protocol requirements.

[0368] Groups and interventions [Table 33]

[0369] Medication (Phase I3+3) The study will involve three doses of CK0801. A minimum of three patients will be treated at each dose level. The dose that patients receive will depend on when they enter the study, as after each dose level is completed, the next patient will receive the next highest dose level. Dosage Level 1: CK0801 IV 1×10 6 / kg ideal weight Dosage Level 2: CK0801 IV 3×10 6 / kg ideal weight Dosage Level 3: CK0801 IV 1×10 7 / kg ideal weight

[0370] Primary Endpoint The primary endpoint of this study was dose-limiting toxicity. Severe (grade 3 or 4) infusion toxicity within 24 hours. Severe (grade 3 or 4) cytokine release syndrome within 30 days Regimen-related death within 30 days Results judgment method Main outcome measures: 1. The number of adverse events and serious adverse events will be collected to provide a preliminary assessment of the safety of infusing CK0801 in patients with Guillain-Barre Syndrome (GBS) who are not responding to standard treatment with intravenous immunoglobulin. [Time frame: 30 days from injection] 2. Dose-limiting toxicity is defined to include any of the following events, each with onset at the time of CK0801 infusion: Severe (grade 3 or 4) infusion toxicity within 24 hours (NCI-CTCAE V4.0) - regimen-related death within 30 days, Severe (grade 3 or 4) cytokine release syndrome (CRS) within 30 days [Time frame: 30 days from injection] Other pre-specified outcome measures: 3. Evaluation of peripheral blood (PB) profiling after CK0801 infusion To evaluate whether CK0801 infusion on day 0 caused changes in the peripheral blood characteristics of patients [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 4. Evaluation of peripheral blood inflammatory cytokines after CK0801 infusion To evaluate whether patients developed proinflammatory cytokines in peripheral blood upon infusion of CK0801 on day 0 [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 5. Assessment of potential changes in GBS disability scores (questionnaire) A questionnaire assessing seven disability scores ranging from health status (0) to death (6). [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 6. Assessment of potential changes in the Overall Neuropathy Limitations Scale (ONLS) (questionnaire) The Global Neuropathy Limitations Scale (ONLS) is a questionnaire that determines symptoms in a patient's arms (numbness, tingling, weakness) and legs (walking, running, altered gait, need for a wheelchair) when performing normal daily activities. The arm scale ranges from 0 (normal) to 5 (impairment in both arms preventing all purposeful movement) and the leg scale ranges from 0 (unaffected walking / stair climbing / running) to 7 (restricted to wheelchair or bed most of the day and unable to perform any purposeful movement of legs). [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 7. Assessment of potential changes in the Global Disability Scale constructed by Rasch (questionnaire) A questionnaire that measures daily activities and their relationship to the patient's health. Scores range from 0 to 2, with 0 = unable to perform the activity and 2 = easy to perform the activity. The questionnaire includes activities such as walking indoors or outdoors, washing the upper or lower body, dressing, eating, washing dishes, and shopping. The overall total raw score ranges from 1 to 48, which correlates to a centriole metric of 0 to 100. [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 8. Assessment of potential changes in MRC (Medical Research Council) total score (questionnaire) The MRC total score is the sum of the MRC scores of six muscle groups, including shoulder abductors, elbow flexors, wrist extensors, knee extensors, and bilateral foot dorsiflexors, and ranges from 60 (normal) to 0 (tetraplegia). [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 9. Evaluation of potential modifications to the MRC model constructed by Rasch (questionnaire) The MRC total score is the sum of the MRC scores of six muscle groups, including shoulder abductors, elbow flexors, wrist extensors, knee extensors, and bilateral foot dorsiflexors, and ranges from 48 (normal) to 0 (tetraplegia). [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 10. Assessment of potential changes in the Fatigue Severity Scale (FSS) (questionnaire) A questionnaire measuring fatigue-related activities on a scale from 9 (no signs of fatigue) to 63 (most disabling fatigue) [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 11. Assessment of potential changes in the Rasch-constructed fatigue severity scale (RFSS) (questionnaire) A questionnaire measuring fatigue-related activities on a scale from 0 (no signs of fatigue) to 21 (most disabling fatigue) [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 12. Assessment of potential changes in the EuroQol E-5D health questionnaire (questionnaire) The EuroQol E-5D health questionnaire is a validated brief health questionnaire that tests a patient's mobility, self-care activities, other usual activities (household chores, leisure activities, etc.), pain / discomfort levels, and anxiety / depression. The scale ranges from 0 (worst health the patient can imagine) to 100 (best health the patient can imagine). [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 13. Assessment of potential changes in the patient's condition based on a comparison of the entry questionnaire, Form A, and the follow-up questionnaire, Form B (questionnaires at weeks 1 and 2) The entry questionnaire establishes a screening level baseline of the patient's overall condition including comorbidities affecting breathing or mobility, other family members with GBS, antecedent events (e.g., cold, gastroenteritis), type of pain (e.g., myalgia, arthralgia, neuropathic pain), location of pain, weakness in arms or legs, status of reflexes, sensory impairment, ataxia, forced vital capacity. The form allows the user to predict whether the patient will require ventilation or be able to walk within 6 months. [Time frame: Screening, Day 0, Week 1, and Week 2] 14. Assessment of potential changes in the patient's condition based on a comparison of the entry questionnaire, Form A, and the follow-up questionnaire, Form B (questionnaires at weeks 4, 12, and 24) This form (questionnaire) uses the same information as the entry questionnaire and provides a mechanism for documenting changes in the patient's condition since enrollment. [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 15. Assessment of potential changes in the patient's condition based on a comparison of the entry questionnaire, Form A, and the follow-up questionnaire, Form C (questionnaires at weeks 1, 2, 4, 12, and 24) This form (questionnaire) uses the same information as the entry questionnaire and provides a mechanism for documenting changes in the patient's condition since enrollment. [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24] 16. Assessment of potential changes in the patient's condition based on a comparison of the entry questionnaire, Form A, and the follow-up questionnaire, Form T (questionnaires at weeks 1, 2, 4, 12, and 24) This form (questionnaire) uses the same information as the entry questionnaire and provides a mechanism for documenting changes in the patient's condition since enrollment. [Timeframe: Screening, Day 0, Week 1, Week 2, Week 4, Week 12, Week 24]

[0371] Example 8: Evaluation of the safety and efficacy of administration of cord blood-derived regulatory T cells in the treatment of acquired idiopathic aplastic anemia and hypoplastic myelodysplastic syndromes Target layer The target population for this study are patients who are ineligible for sibling donor hematopoietic stem cell transplantation (MSD HSCT) or predicted to respond poorly to immunosuppressive therapy (IST).

[0372] Registration Up to 18 adult patients will be enrolled. dosage Dose level 1: CK0804 IV 1×10 8 Cells Dose level 2: CK0804 IV 3×10 8 Cells

[0373] Primary Endpoint The primary endpoint of this study was time to infusion reaction, cytokine release syndrome, and / or death within 30 days.

[0374] Secondary Endpoint The secondary endpoint of this study was hematologic improvement.

[0375] Example 9: Evaluation of the safety and efficacy of administration of cord blood-derived regulatory T cells in the treatment of amyotrophic lateral sclerosis Target layer Adult ALS patients (≥18 years old) with acquired ALS Ability to provide informed consent -Subjects with onset of the disease ≤2 years ≥ 60% predicted forced vital capacity Subjects have a total ALSFRS-R score of ≥ 24 and a score of at least 2 points on all 12 items of the scale. Patients whose progression from onset to screening is >0.3 points / month Registration 52 adult patients. Investigational Drug CK0803 (cryopreserved, multiple-dose, cord blood-derived regulatory T cells enriched for CD11a) dosage Induction: 4 x weekly IV CK0803 doses at the following dose levels: Cohort 1: CK0803 IV 1×10 8 Treg cells Cohort 2: CK0803 IV 3×10 8 Treg cells Maintenance: 6 additional doses every 4 weeks for both cohorts Groups and interventions

[0376] [Table 34]

[0377] The treatment timeline is shown in Figure 26. Phase IB: Main purpose Safety and tolerability Treatment-related adverse events based on CTCAE v4.03 (AEs, SAEs, DLTs) secondary purpose Effectiveness ● ALS Functional Rating Scale-Revised (ALSFRS-R) ●Forced vital capacity (FVC) Handheld Dynamometry (HHD) for Muscle Strength ○Explorative Inflammatory biomarkers and immune reconstitution Endpoint Investigation Clinical Response: 1. A 6-point improvement over 6 months on the ALS Functional Rating Scale-Revised (ALSFRS-R).

[0378] 2. Rate of change in functional status 3. Change in slope of Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score 4. Changes in forced vital capacity (FVC), and 5. Changes in muscle strength

[0379] ALSFR responder analysis (proportion of subjects who improved after treatment compared to before treatment) • Prespecified responder analyses will examine both the percent improvement per month and absolute point improvement in post-treatment ALSFRS-R slope compared with pre-treatment slope. • Patient assessment of 25%, 50%, 75% and 100% improvement. Clinically meaningful = 25% ● Clinically significant = 50% Statistical significance defined as a one-sided p value <0.05 using Fisher's exact test.

[0380] Assessments performed at weeks 4, 8, 12, 16, and 24 Clinical Trial Design: Phase Ib Clinical Trial Research design: ●This study will be a Phase I, 3+3 design, single ascending dose, safety and tolerability study of CK0803. • A fixed-dose strategy tests two dose levels. ●Low dose: 1×10 8 cells; ●High dose: 3×10 8 cells; A minimum of 6 patients and a maximum of 12 patients will be enrolled in this study. Cohorts of 12 subjects will be randomized to one of three treatment sequences with three subjects per sequence as shown in the table above with total (minimum) patients = 12. Clinical trial design: Exploratory study. Peripheral blood ●T cell compartment: Tregs, effector T cells, antiviral activity Inflammatory cytokines in serum: Interleukin (IL) 1, IL-1β, IL-2, IL-4, IL-6, IL7, IL-8, IL-10, IL-13, IL-17, IL-18 ●Interferon gamma, ST2, REG3a, OPN, follistatin, elafin, TGF-beta, TNF-alpha, TNFR-1 C-reactive protein (CRP) Macrophage chemotactic protein-1 (MCP-1) ●8-Hydroxy-2'-deoxyguanosine (8-OHdG) Malondialdehyde (MDA) ●Ratio: glutathione disulfide, GSSG / reduced glutathione, GSH Additional exploratory cytokines: SCF, G-CSF, GM-CSF, HGF, VEGF, SDF1a, MCP1, MCP2, TARC, MIP3a, TECK, CTACK, CCL28, FGF, PDGF, EGF, TGF-α, TLR Cerebrospinal fluid ●Phosphorylated neurofilament heavy chain (pNFH) ● Chit-1 Prostaglandin E2 ●VEGF IL-6 ●GMCSF ●IL-2, IL-8, IL-15, IL-17 MIP-1β ●FGF G-CSF MIP-1α ●MCP-1 IFN-γ X-ray inspection Glial activation measured in vivo11 [C]-PBR28PET is increased in pathologically relevant areas in people with ALS and correlates with clinical measurements. (Alshikho,ANN NEUROL 2018) Integrated PET-MR and 1 H-MRS imaging shows associations between markers of neuronal integrity and neuroinflammation and may provide valuable insights into disease mechanisms in ALS. (Ratai, NeuroImage:Clinical 20(2018)357-364)

[0381] Example 10: Evaluation of the safety and efficacy of cord blood-derived regulatory T cell administration in the treatment of COVID-19 (coronavirus disease)-mediated acute respiratory distress syndrome (CoV-ARDS) The clinical trial design for a Phase IB / IIa study of cryopreserved multiple-dose cord blood-derived regulatory T (Treg) cells (CK0802) for the treatment of CoV-ARDS is shown in Figure 27. There are three treatment arms: treatment arm 1: placebo, treatment arm 2: 1 × 10 8 CK0802 cells, treatment group 3: 3 × 10 88 CK0802 cells. The dosing regimen is 3 doses infused on days 0, 3 (+ / -1) and 7 (+ / -1). CK0802 is administered intravenously. The study population is intubated adults with moderate to severe acute respiratory distress syndrome (ARDS) induced by COVID-19. A minimum of 15 patients and a maximum of 45 patients will be enrolled.

[0382] the purpose Main purpose The purpose of this protocol is to determine whether regulatory T cell infusion expanded from a banked blood unit (CK0802) can safely reduce morbidity and mortality in intubated patients suffering from moderate to severe ARDS secondary to COVID-19 infection.

[0383] Endpoints and Correlations Primary Endpoint The two main results are Regimen-related severe ≥ grade 3 toxicity within 48 hours of CK0802 infusion (NCI-CTCAE (National Cancer Institute Common Terminology Criteria for Adverse Events) V4.0) • Successful treatment at 28 days, defined as S28 = [alive and not intubated 28 days after the date of first infusion].

[0384] Secondary Endpoint Secondary outcomes recorded from the date of first infusion up to 28 days after the date of first infusion included: ●Time to Exercise ●Oxygenation demand (PaO2:FiO2 ratio) changes from day 0 to day +11 Number of days without a ventilator Number of days without organ failure Number of days out of ICU during the first 28 days 28-day all-cause mortality

[0385] Planned non-endpoint correlation analyses Laboratory correlations and general evaluation on days 0, 3, 7, 11, 21, and 28 ●Sequential Organ Failure Assessment (SOFA) score [Vincent et al., Intensive Care Med, 1996.22(7):p.707-10] Inflammatory markers: serum ferritin, procalcitonin, D-dimer and C-reactive protein (CRP), interleukin-6 (IL-6) Peripheral blood lymphocyte subset analysis Ventilator status parameters (if intubated) and ABG (if available)

[0386] Research results CK0802 (cryopreserved umbilical cord blood derived regulatory T cells) refers to allogeneic, off-the-shelf regulatory T cells that are cryopreserved and ready to be used as an intravenous infusion for the treatment of ARDS.

[0387] Sources and Pharmacology Tregs are isolated from allogeneic unrelated umbilical cord blood (CB) units obtained from qualified public, licensed, or unlicensed US CB banks based on pre-determined selection criteria. CB units are thawed and processed according to standard procedures in a 37°C water bath using 10% dextran 40 and 5% human serum albumin as washing solutions. CB cells are resuspended in MgCl2rHuDNAse / sodium citrate cocktail prior to immunomagnetic selection to prevent aggregation. Enrichment of CD25+ Treg cells is achieved by positive selection using directly conjugated anti-CD25 magnetic microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) and a MACS separation device. After selection, CD25+ cells were cultured in GREX flasks at approximately 1 × 10 in X-VIVO 15 medium (Cambrex BioScience, Walkersville, MD) suspended in 10% human AB serum (heat-inactivated; Valley Biomedical Products and Services, Inc., Winchester, VA), L-glutamine (2 mM), and 10% IgG. 6 CD25+ cells are suspended at a concentration of 1.0×106 viable nucleated cells / mL. CD25+ cells are cultured with anti-CD3 / anti-CD28 monoclonal antibody (mAb)-coated Dynabeads (Invitrogen) at a bead-to-cell ratio of 1:1 for 14±1 days. On day 0, cultures are supplemented with 1000 IU / ml IL-2 (Proleukin, Chiron Corporation, Emeryville, CA). Cells are maintained at a density of 1.0×106 viable nucleated cells / mL and cultured at 37°C in 5% CO2 for 14 days.

[0388] On day 14 of culture, cells are harvested, Dynabeads are removed by magnetic separation, and Treg cells are resuspended in Plasmalyte + 0.5% I buffer. The Treg product (CK0802) must pass release criteria for infusion, including: 7AAD viability of ≥ 70%, CD4 purity of ≥ 60%, and IgG antibody titer of ≥ 100%. + CD25 + Cells, <10% CD4 - / CD8 + cells, 3 x 10 6 Anti-CD3 / anti-CD28 mAB bead count of <100 per cell, Gram stain with "no organisms", and endotoxin of <5EU / kg.

[0389] The harvested cells are then aliquoted into clinical cryobags, cryopreserved using a controlled rate freezer, and labeled as a CK0802 product containing cell dose.

[0390] CK0802 injection In this study, CK0802 dose level 1 = 1.0 × 10 8 cells and dose level 2: 3.0 × 10 8 The infusion of cells is considered. Patients are pre-medicated with 50 mg of Benadryl® via IVPB (IV piggyback) 30 minutes prior to infusion of CK0802. CK0802 should be infused by gravity over a period of at least 30 minutes, preferably within 1 hour. CK0802 is compatible with standard blood product tubing and filters.

[0391] Placebo injection Cryopreserved excipients are infused into 30 ml cryobags. Patients are premedicated with Benadryl® 50 mg in an IVPB (IV piggyback) 30 minutes prior to infusion of placebo. Placebo should be infused by gravity within 30 minutes, preferably within 1 hour after thawing. Placebo is compatible with standard blood product tubing and filters.

[0392] Study population The study will recruit subjects who meet all of the inclusion / exclusion criteria detailed below. Inclusion criteria 1. Have a documented RT-PCR-based diagnosis of SARS-CoV-2 infection. 2. Moderate to severe ARDS as defined by the Berlin criteria [Force et al., JAMA, 2012.307(23):p.2526-33]: ratio of arterial oxygen pressure (PaO2) to fraction of inspired oxygen (FiO2) ≤ 200 mmHg assessed with a positive end-expiratory pressure (PEEP) of > 5 cmH2O. 3. Intubation for less than 120 hours 4. Age ≥ 18 5. A duly appointed health care representative who is capable of providing informed consent or has the authority to provide informed consent. Exclusion criteria 1. In the investigator's opinion, you are unlikely to survive more than 48 hours from screening. 2. Physical examination findings and / or medical history that, in the opinion of the study investigator, may confound the results of the study or pose additional risk to the patient from participation in the study. 3.Currently receiving extracorporeal membrane oxygenation (ECMO) or high-frequency oscillatory ventilation (HFOV) 4. Pregnant women 5.Patients with active bacteremia or other concurrent active moderate to severe infections at the start of treatment enrollment that, in the opinion of the investigator, may affect the safety of CK0802 treatment. 6. Patients intubated for >120 hours 7. Known hypersensitivity to DMSO or porcine or bovine proteins 8. Any end-stage organ disease that, in the opinion of the investigator, may affect the safety of CK0802 treatment 9. Steroids are lympholytic and may be harmful to infused Treg cells. Steroid therapy at or above stress doses is excluded: hydrocortisone >50 mg every 6 hours or other systemic steroids >0.5 mg / kg / day methylprednisolone intravenously or >60 mg methylprednisolone orally daily. 10. Administration of investigational cell therapy drugs

[0393] Evaluation during the study Clinical evaluation Baseline assessment on the day of injection of the first dose of assigned treatment group: CK0802 or placebo, then daily assessments thereafter for 14 days after injection of the first dose of assigned treatment group: CK0802 or placebo. Ventilation parameters: Daily average record with range (min and max) Plateau pressure ●Tidal volume Mean airway pressure ●FiO2 ●PEEP ●PaO2 / FiO2 ratio ●C-STAT / Compliance (static compliance of the lungs) Arterial Blood Gases: Daily average record with range (min and max) Arterial pH ●Oxygen pressure (PaO2) ●Partial pressure of carbon dioxide (PaCO2) ●Bicarbonate (HCO3) ●Oxygen saturation (O2Sat) Vital signs: Daily average record with range (min and max) Body temperature ● Blood Pressure (BP): Systolic and diastolic BP measurements ●Respiration rate Heart rate

[0394] SOFA Score The Sequential Organ Failure Assessment (SOFA) score predicts ICU mortality based on laboratory and clinical data.

[0395] SOFA Score [Table 35]

[0396] Example 11: Effect of cryopreservation on the cytosuppressive activity of regulatory T cells derived from umbilical cord blood Cryopreserved cord blood (CB) Treg cells (CK0802) were shown to have comparable suppressive function compared to fresh CB Treg cells. Tcon cells showed a high rate of proliferation in the presence of costimulatory CD3 / 28 beads as revealed by serial dilutions of CellTrace™ Violet dye in the positive control group (Figure 5A), whereas no such proliferation was captured in the negative control group in the absence of CD3 / 28 beads (Figure 5B). Expanded CB Treg cells were either derived from fresh cultures (Figure 5C) or thawed from cryopreserved aliquots (Figure 5D), and a comparable degree of suppression of the expanding Tcon cells was demonstrated by the lack of dilution of CellTrace™ Violet dye.

[0397] Example 12: Effect of ruxolitinib on the activity of regulatory T cells derived from umbilical cord blood Ruxolitinib improved cord blood-derived Treg cell function both in vitro and in vivo. These findings were unexpected, as previous reports described a deleterious effect of ruxolitinib on Treg cells in patients.

[0398] As shown in Figure 31, the addition of ruxolitinib to thawed cryopreserved cord blood (CB) Treg cells restored the suppressive function of Treg cells in vitro. When thawed CB Treg cells are placed into secondary culture, the Treg cells lose suppressive function over time. The addition of ruxolitinib can restore suppressive function.

[0399] Ruxolitinib and CB Treg cells show synergistic effects in suppressing the release of interferon gamma (IFNγ) from pathogenic lupus cells. Peripheral blood mononuclear cells from subjects with systemic lupus erythematosus (SLE-PBMC) secrete high levels of the inflammatory cytokine IFNγ. The levels of IFN-γ are reduced by the addition of ruxolitinib or CB Treg cells. However, when added together, the combination of CB Treg cells and ruxolitinib synergistically suppresses the release of IFNγ from SLE-PBMC (Figure 32). Camptothecin is used as a control to show that non-specific inflammatory stimuli do not increase IFNγ secretion from CB Treg cells.

[0400] A xenogeneic mouse graft-versus-host disease (GVHD) model was treated with ruxolitinib and activated CB Treg cell regimen as shown in Figure 33. NSG mice received sublethal irradiation on day -1 followed by 1x10 7 donor peripheral blood (PB) mononuclear cells (MNCs) were injected. Oral ruxolitinib was administered at 1 mg daily, followed by 1 × 10 7 Mice were continuously fed with or without CB Treg cells, tagged with CellTrace™ Violet dye (ThermoFisher) and administered on days +4, +7, +11 and +18. Mice were followed every other day for weight, GVHD score and survival. Serial blood samples were taken to analyze cellular compartments and cytokine assays.

[0401] Combination treatment reduced GVHD scores (Figure 34A) and improved survival in mouse models (Figure 34B). Ruxolitinib improved CB Treg persistence in mouse models (Figures 35A-C). Ruxolitinib, as a single agent and in combination with CB Treg cells, reduced the number of human cells (Figure 35A). Ruxolitinib increased the percentage of CD4 and CD25 co-expressing cells when administered in combination with CB Treg cells (Figure 35B). Ruxolitinib increased the percentage of circulating CB Treg cells when administered in combination with CB Treg cells compared to CB Treg cells administered alone (Figure 35C).

[0402] Ruxolitinib enhanced the survival signal pathways of IL-7 and IL-15 and attenuated the inhibitory signal pathway of IL-4 on CB Treg cells in a xenogeneic mouse GVHD model. When ruxolitinib was administered in combination with CB Treg cells, plasma IL-7 (Figure 36A) and plasma IL-15 (Figure 36B) levels increased. Increased availability of IL-7 enhanced Treg survival, stabilized Treg molecular signature, enhanced surface IL-2Rα expression, and improved IL-2 binding of Treg cells (Schmaler et al. Proc Natl Acad Sci USA. 112(43):13330-5, 2015). IL-15 impaired the upregulation of RORγt and IL-17 expression and improved Treg proliferation (Tosiek et al. (2016) Nat Commun 7:10888). When ruxolitinib was administered in combination with CB Treg cells, plasma IL-4 levels were reduced (Figure 36C). IL-4 production by Th2 cells is inhibited by Tregs (Pace et al. J Immunol 2005;174:7645-7653).

[0403] The combination of ruxolitinib and CB Treg cells reduced the secretion of inflammatory cytokines in a xenogeneic mouse GVHD model. Plasma levels of IL-1a (Figure 37A), IL-17 (Figure 37B), and IFNa2 (Figure 37C) were reduced by the addition of ruxolitinib to CB Treg cells. Levels of FGF-12 (Figure 37D) and macrophage-derived chemokine (MDC) (Figure 37E) were equally reduced by administration of CB Treg cells alone, ruxolitinib alone, and the combination of ruxolitinib and CB Treg cells.

[0404] The combination of ruxolitinib and CB Treg cells increased the secretion of anti-inflammatory cytokines in the xenogeneic mouse GVHD model. Plasma levels of IL-1RA (Figure 38A), IL-1a3 (Figure 38B) and IL-12p70 (Figure 38C) were increased. The combination of ruxolitinib and CB Treg cells improved the hematological parameters of the xenogeneic mouse GVHD model. The administration of both ruxolitinib and CB Treg cells increased the levels of platelets (Figure 39B). On day 14, a significant decrease in hemoglobin levels in the ruxolitinib alone group is evident compared to the increase in hemoglobin levels in the CB Treg cells + ruxolitinib group (Figure 39A).

[0405] Example 13: Effect of umbilical cord blood-derived regulatory T cells on chimeric antigen receptor T cells The xenogeneic lymphoma model was 0.3 × 10 6 All mice were injected with GFP-labeled Raji cells on day 0, followed by i) mock CAR T, ii) no CART, or iii) 0.3 × 10 CD19-CAR T cells on day +5. 6 The ES cells were generated using NSG mice injected with 1 × 10 cells at days +11, +18, and +25. 7Additional injections of CB Treg cells were added to the no CAR T group and to the CD19-CAR T group with 3 mice per group. Mice were followed for body weight, GVHD score and survival. Serial imaging was performed to assess tumor burden using non-invasive bioluminescence. Blood was collected serially for cell analysis and cytokine assays.

[0406] As shown in Figure 59A, in vivo expansion of GFP-labeled Raji cells was evident in all mice every +4 days. CD19-CAR T, but not mock CAR T cells, reduced tumor burden at day +11. However, at day +14, all mice, including CD19-CAR T cell recipients, showed progression, whereas CD19-CAR T+CB Treg cell recipients showed no evidence of bioluminescence. Superior survival in CD19-CAR T+CB Treg cell recipients was evident when compared to other treatment groups (Figure 60A). At the time of euthanasia, various organs were evaluated for detection of CD19-CART cells, which were only recovered in CD19-CART+CB Treg cell recipients (Figure 60B). CD19-CAR T recipients showed increased inflammatory cytokines in PB samples at day +16, including IFN-γ (Figure 59B) and TNF-α (Figure 59C), which were reduced in the CD19-CAR T+CB Treg group. Furthermore, a reciprocal increase in the anti-inflammatory cytokine IL-1RA was observed in the CD19-CAR T+CB Treg group compared to CD19-CAR T alone (Figure 59D).

[0407] The addition of CB Treg cells to CD19-CAR T cells in a xenogeneic lymphoma model led to suppression of the cytokine storm and improved the targeting efficacy of CAR T cells.

[0408] Example 14: Analysis of alternative selection methods for CXCR4-expressing cord blood-derived regulatory T cells Concentrated CXCR4 +Two methods were analyzed to select CXCR4-expressing cord blood-derived Treg cells to generate Treg cell cultures. The first method is called "Day 0 (double) enrichment" (Figure 62). This method is characterized by the selection of day 0 CD25 + The second method involves cell selection, followed by cell washing, followed by incubation with CXCR4 microbeads, and then a second positive enrichment. The second method is called "Day 3 (sequential) enrichment" (Figure 63). This method is + It involves cell selection, followed by cell culture, followed by cell washing to separate the CD3 / 28 microbeads, followed by incubation with CXCR4 microbeads, and then a second positive enrichment.

[0409] Three cultures of Treg cells were generated from different cord blood units on different days by the method described in Figure 63. The characteristics of these cells on the day of the CXCR4 enrichment step are shown in Table 36.

[0410] [Table 36]

[0411] Figure 64A, Figure 64B and Figure 64C show summary data from cultures 1, 2 and 3, showing that day 3 (D3) Treg cells can be efficiently enriched by magnetic separation of CXCR4. Figure 64A shows the CXCR4 concentration in D3 CXCR4 unseparated and positive fraction Treg cells. hiFigure 64B shows the frequency of CXCR4 in D3 CXCR4 unseparated and positive fraction Treg cells (see also Table 38). Paired t-test showed a difference between the two groups (17±7.211, P=0.0551). Figure 64B shows summary data showing the mean / median fluorescence for CXCR4 in D3 CXCR4 unseparated and positive fraction Treg cells (see also Table 38). Paired t-test showed a statistically significant difference between the two groups (4000±1424, P=0.0397). Figure 64C shows summary data showing the fold change of CXCR4 in D3 CXCR4 unseparated and positive fraction Treg cells (see also Table 39). Paired t-test showed a statistically significant difference between the two groups (2.007±0.5886, P=0.0275).

[0412] [Table 37]

[0413] [Table 38]

[0414] [Table 39]

[0415] Figure 65 shows the effect of the CXCR4 enrichment step on Treg CXCR4 enriched cells, comparing them to CXCR4 negative cells as well as to pre-selection populations from representative cultures. Figure 65A shows a comparison of the mean fluorescence intensity of CXCR4 expression of the pre-selected population on day 3 (median MFI=2064) vs. the mean fluorescence intensity of CXCR4 expression of the CXCR4 negative population on day 3 (median MFI=54) vs. the mean fluorescence intensity of CXCR4 expression of the positively selected population on day 3 (median MFI=5347). Figure 65B shows a histogram representation of the mean fluorescence intensity of CXCR4 expression of the cell populations containing pre-selection Treg cells vs. the CXCR4 negative Treg cell population vs. the CXCR4 enriched Treg cells on day 3 in one graph. Figure 65C shows a contour plot of the mean fluorescence intensity of CXCR4 expression of the cell populations containing pre-selection Treg cells vs. the CXCR4 negative Treg cell population vs. the CXCR4 enriched Treg cells on day 3 in one graph.

[0416] Figures 66A-C show the effect of CXCR4 magnetic enrichment for CXCR4 expression in D3 CB Treg cells. Figure 66A shows the effect of CXCR4 magnetic enrichment for CXCR4 expression in D3 CB Treg cells compared to unseparated D3 CB Tregs in culture 1. CXCR4 Figure 66B shows that D3 CB Tregs in culture 2 showed a 2.75-fold increase in CXCR4 expression compared to unseparated D3 CB Tregs. CXCR4 Figure 66C shows that D3 CB Tregs showed a 2.59-fold increase in CXCR4 expression compared to unseparated D3 CB Tregs in culture 3. CXCR4 showed a 3.68-fold increase in CXCR4 expression.

[0417] FIG. 67 shows a graph depicting the number of cells in the categories "CXCR4 enriched," "CXCR4 negative," and "Treg control" at the various culture days shown in FIG. 67 (data from all three cultures (culture 1, culture 2, and culture 3) combined).

[0418] Figures 68A and 68B show data regarding the phenotype of the culture products at day 12. Figure 68A shows a dot plot showing the results from CXCR4 magnetic enrichment for D12 (day 12) CB Treg cells from a representative culture. Figure 68B shows a dot plot of intracellular staining for FOXP3 and Helios in CXCR4 enriched Treg cells at day 12.

[0419] Figure 69 shows a graph depicting the results from a cell suppression assay performed on day 12 of a representative culture, in which CXCR4-enriched Tregs exert superior suppression compared to the Treg control cell population. 商標 Donor CD4 stained with Violet dye + T responder cells were cultured with CD3 / 28 beads at a 1:1 ratio for 96 hours under the following conditions: i) no cells added, 96% of responder cells actively proliferating, ii) donor CD4 responder cells, 41% of responder cells actively proliferating. + Treg control cells added at a 1:1 ratio to T responder cells, and iii) 7.7% of responder cells were actively proliferating donor CD4 + Treg enriched CXCR4 cells were added to T responder cells at a 1:1 ratio.

[0420] Figure 70B shows a photomicrograph of cell numbers in the lower chamber in a transwell migration assay, demonstrating the superior migration capacity of CXCR4-enriched Treg cells in response to the bone marrow stromal chemoattractant SDF 1α. Figure 70A shows a schematic of the experimental design of the transwell migration assay. Figure 70B shows the results of a micrograph of cell numbers in the lower chamber in a transwell migration assay, demonstrating the superior migration capacity of CXCR4-enriched Treg cells in response to the bone marrow stromal chemoattractant SDF 1α. 6 When cells are layered with 500 ng / mL SDF1α in the upper chamber of a 0.5 micron transwell, CXCR4-enriched Treg cells migrate faster in response to SDF1α at a concentration of 500 ng / mL compared to Treg control and CXCR4-negative Treg cell populations.

[0421] Example 15: A Phase Ib, Open-Label Safety Study of Add-On Therapy with CK0804 in Patients with Myelofibrosis Who Have a Suboptimal Response to Ruxolitinib. This is a single-arm study consisting of a 9-patient safety run-in followed by an expansion cohort of an additional 15 patients for a total of 24 patients. Each patient will receive a single infusion of CK0804 (cryopreserved allogeneic cord blood-derived Treg cells, enriched for CXCR4-expressing Treg cells) at a fixed dose of 100 million cells, administered every 28 days for up to 6 doses. Table 40 shows the objectives and endpoints. Table 41 presents key study design elements.

[0422] [Table 40]

[0423] [Table 41-1] [Table 41-2]

Claims

1. CXCR4 from cryopreserved human umbilical cord blood units ＋ 1. A method for producing an expanded population of human regulatory T (Treg) cells enriched for Treg cells, comprising: (a) thawing a cryopreserved human umbilical cord blood unit; (b) diluting and washing the thawed cord blood unit in a functionally closed system; (c) CD25 ＋ isolating Treg cells using a double selection method based on cell surface expression; (d) the isolated CD25 ＋ Treg cells were cultured in a gas-permeable culture vessel in medium. (1) an effective amount of interleukin-2 (IL-2); (2) a reagent that specifically binds to CD3 and CD28, and (3) in the presence of anti-CXCR4 magnetic microbeads, CD25 ＋ ex vivo expansion for up to 10 days or up to 12 days, with the medium being changed about every 48 hours, to produce a CXCR4-enriched culture of Treg cells; (e) the activated CD25 ＋ CXCR4 ＋ Cells were harvested from the culture and subjected to CXCR4 ＋ Producing an expanded population of human Treg cells enriched for Treg cells.

2. In step (d), (1) the ex vivo expansion is initiated on day 0; (2) administering an effective amount of IL-2 to the isolated CD25 on day 0; ＋ Add to Treg cells, (3) The reagent that specifically binds to CD3 and CD28 is added to the isolated CD25 on day 0. ＋ The method of claim 1, wherein the antibody is added to Treg cells.

3. The method according to claim 1 or 2, wherein the reagents that specifically bind to CD3 and CD28 are removed from the culture medium before the anti-CXCR4 magnetic microbeads are added to the culture medium.

4. The method according to any one of claims 1 to 3, wherein in step (d), the anti-CXCR4 magnetic micro-beads are added to the culture medium 3 or 4 days after the ex vivo expansion is initiated.

5. In step (d), CXCR4 is isolated using a dual ferromagnetic column. ＋ The method according to any one of claims 1 to 4, wherein said anti-CXCR4 magnetic micro-beads are added to said culture medium for about 30 minutes prior to isolating Treg cells.

6. The isolated CD25 ＋ The method of any one of claims 1 to 5, wherein Treg cells are enriched for CXCR4 in the third feed of ex vivo expansion.

7. The method of any one of claims 1 to 6, wherein step (d) is carried out over 4 or 5 days.

8. The method of any one of claims 1 to 7, wherein the reagent that specifically binds to CD25 is an anti-CD25 antibody or an antigen-binding fragment thereof.

9. The method of any one of claims 1 to 8, wherein the reagent that specifically binds to CD25 is conjugated to a solid support.

10. The method of claim 9 , wherein the solid support is a magnetic microbead.

11. The method of any one of claims 1 to 10, wherein the reagents that specifically bind to CD3 and CD28 comprise an anti-CD3 antibody or an antigen-binding fragment thereof, and an anti-CD28 antibody or an antigen-binding fragment thereof.

12. The method of any one of claims 1 to 11, wherein the reagents that specifically bind to CD3 and CD28 comprise anti-CD3 coated beads and anti-CD28 coated beads.

13. The method of any one of claims 1 to 12, wherein the effective amount of IL-2 is about 1000 IU / mL.

14. CXCR4 from cryopreserved human umbilical cord blood units ＋ 1. A method for producing an expanded population of human Treg cells enriched for Treg cells, comprising: (a) thawing the cryopreserved human umbilical cord blood unit; (b) diluting and washing the thawed cord blood unit in a functionally closed system; (c) CD25 ＋ isolating Treg cells using a double selection method based on cell surface expression; (d) the isolated CD25 in a gas-permeable culture vessel in a medium. ＋ Expanding Treg cells ex vivo, comprising: (1) On day 0, anti-CD3 and anti-CD28 coated beads were added to the CD25 in the medium. ＋ adding to Treg cells; (2) on day 2, adding about 1000 IU / mL of IL-2 to the medium; (3) on the third or fourth day, removing the anti-CD3 and anti-CD28 coated beads from the medium and adding anti-CXCR4 magnetic microbeads to the medium; and (4) On the third or fourth day, CXCR4 ＋ The anti-CXCR4 magnetic microbeads attached to the Treg cells were removed from the medium, and fresh anti-CD3 and anti-CD28 coated beads were replenished with the CXCR4 ＋ adding to Treg cells, CD25 ＋ ex vivo expansion for up to 10 days or up to 12 days, with the medium being changed about every 48 hours, to produce a CXCR4-enriched culture of Treg cells; (e) the activated CD25 ＋ CXCR4 ＋ Cells were harvested from the culture and subjected to CXCR4 ＋ Producing an expanded population of human Treg cells enriched for Treg cells.

15. In step (d), IL-2 is administered to the isolated CD25 ＋ The method of any one of claims 1 to 14, wherein the medium containing Treg cells is added about every 48 hours.

16. The method according to any one of claims 12 to 15, wherein the anti-CD3 coated beads and the anti-CD28 coated beads are in a 1:1 ratio.

17. The CD25 ＋ 10. The method of claim 8 or 9, wherein the cells and the anti-CD3 and anti-CD28 coated beads are in a 1:1 ratio.

18. In step (e), about 1×10 ６ CD25 ＋ The method of any one of claims 1 to 17, wherein cells / mL are cultured.

19. In step (e), the cells are first cultured in a 10 cm ２ The method according to any one of claims 1 to 18, wherein the culture is carried out in a gas-permeable culture vessel having a membrane surface area of ​​1000 μm or more.

20. The culture is then cultured at 100 cm ２ 20. The method of claim 19, wherein the culture medium is transferred to a gas permeable incubator having a membrane surface area of ​​1000 nm to 1000 nm.

21. The method of any one of claims 1 to 20, wherein in step (d), the culture is not mixed and resuspended when adding the IL-2.

22. 22. The method of any one of claims 1 to 21, wherein in step (a) the cryopreserved human umbilical cord blood unit is thawed in a single step in a water bath.

23. The method of any one of claims 1 to 22, wherein step (b) does not include manual washing.

24. 24. The method of any one of claims 1 to 23, wherein step (b) is carried out in a solution comprising PBS, EDTA, and about 0.5% human serum albumin.

25. A dual ferromagnetic column method is used in step (c) to isolate CD25 ＋ The method according to any one of claims 1 to 24, wherein Treg cells are isolated.

26. CXCR4 ＋ The method of any one of claims 1 to 25, further comprising cryopreserving the expanded population of human Treg cells enriched in human Treg cells.

27. CXCR4 as measured by flow cytometry ＋ The expanded population of human Treg cells enriched for Treg cells. (i) ≥ 60% CD4 ＋ CD25 ＋ and (ii) ≥ 60% CD4 ＋ CD25 ＋ CXCR4 ＋ and (iii) ≦10% CD4 － CD8 ＋ That is, 27. The method according to any one of claims 1 to 26.

28. CXCR4 produced by the method according to any one of claims 1 to 27. ＋ Expanded populations of human Treg cells enriched for Treg cells.

29. A pharmaceutical composition comprising an effective amount of a population of activated human Treg cells produced by the method of any one of claims 1 to 27, or the population of claim 28, for treating or preventing bone marrow failure syndrome in a subject.

30. The pharmaceutical composition described in claim 29, wherein the bone marrow failure syndrome is aplastic anemia, primary myelofibrosis or myelodysplastic syndrome.

31. 29. A pharmaceutical composition comprising an effective amount of a population of activated human Treg cells produced by the method of any one of claims 1 to 27 or the population of claim 28, for treating or preventing primary myelofibrosis in a subject, wherein a dose of 100 million Treg cells is administered to the subject on day 1 of a 28 day cycle for up to 6 cycles.

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