Compositions comprising regulatory t cells and methods of making and using the same

Abstract

To provide a population of cord blood-derived regulatory T cells expanded ex vivo. To provide a method for producing a population of cord blood-derived regulatory T cells expanded ex vivo, and a method for using the same.SOLUTION: Provided is a population of human Treg cells comprising at least about 1 * 10 8 human Treg cells, wherein (i) ≥ 60% are CD4 + CD25 + and (ii) ≤ 10% are CD4 - CD8 +, wherein said human Treg cells are immunosuppressive.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 064,129, filed August 11, 2020, U.S. Provisional Patent Application No. 63 / 038,345, filed June 12, 2020, U.S. Provisional Patent Application No. 62 / 990,913, filed March 17, 2020, and U.S. Provisional Patent Application No. 62 / 906,283, filed September 26, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates generally to the field of immunoregulatory T cells (Tregs). More specifically, the present disclosure provides an enriched cord blood-derived population of Tregs, and methods for making and using same. [Background technology]

[0003] Tregs naturally suppress and regulate immune responses. Many autoimmune diseases, inflammatory disorders, and malignancies are directly caused or exacerbated by the lack / deficiency of Tregs and / or exhaustion of their suppressive function, or the total number of endogenous Tregs, which in turn allows the unlimited proliferation of autoreactive cytotoxic T cells, leading to cell and tissue damage, which in turn leads to the symptoms of some diseases. Replacing and supplementing such defective Tregs with allogeneic healthy umbilical cord blood-derived Tregs can lead to clinical improvement of the underlying disease and reestablish homeostasis by suppressing the harmful effects of autoreactive cytotoxic T cells. There remains a need in the art to develop additional treatments for these autoimmune diseases, inflammatory disorders, and malignancies. Summary of the Invention

[0004] Provided herein are at least about 1 x 10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are CD4 + CD25 +, and (ii) ≤10% CD4 - CD8 + Further provided herein is a population of human Treg cells, wherein the human Treg cells are immunosuppressive. 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are CD4 + CD25 + , (ii) ≥ 60% CD4 + CD25 + CXCR4 + , and (iii) ≤10% CD4 - CD8 + Further provided herein is a population of human Treg cells, wherein the human Treg cells are immunosuppressive. 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are 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 x 10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are 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 is at least about 1 x 10 9In some embodiments, the human Treg cells are determined to be immunosuppressive by an assay using carboxyfluorescein succinimidyl ester intracellular dye or CellTrace™ Violet intracellular dye.

[0005] Further provided herein is a method for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit, the method comprising: a) thawing the cryopreserved human umbilical cord blood unit; b) diluting and washing the thawed umbilical 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 10 days, up to 12 days, or up to 14 days. + Treg cells are expanded ex vivo, with the medium replaced approximately every 48 hours, and activated CD25 + e) generating and expanding a population of Treg cells; and f) inducing activated CD25 + harvesting the cells to produce an expanded population of activated human Treg cells.

[0006] In some embodiments, the cryopreserved human umbilical cord blood unit is thawed in a single step in a water bath.

[0007] In some embodiments, the dilution and washing steps do not include manual washing. In some embodiments, the dilution and washing steps are performed in a solution comprising PBS, EDTA, and about 0.5% human serum albumin.

[0008] In some embodiments, selection criteria for the starting material for cryopreserved human cord blood units include donor qualifications, total nucleated cell count, pre-freeze cell viability, cryopreserved volume, collection date, storage conditions, cytomegalovirus seropositivity, race and ethnicity, maternal donor history, family medical history, and donor maternal infectious disease profile. In some embodiments, a single cord blood unit is used. In some embodiments, two to four pooled cord blood units are used. In some embodiments, more than four pooled cord blood units are used.

[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. In some embodiments, a double ferromagnetic column method is used to detect CD25. + Isolate Treg cells.

[0010] In some embodiments, the reagents that specifically bind CD3 and CD28 include anti-CD3-coated beads and anti-CD28-coated beads. In some embodiments, the anti-CD3-coated beads and anti-CD28-coated beads are in a 1:1 ratio. In some embodiments, in the culturing step of the methods described herein, CD25 + The cells and anti-CD3 and anti-CD28 coated beads are in a 1:1 ratio. In some embodiments, the culture step of the methods described herein involves culturing approximately 1 x 10 6 CD25 + cells / ml are cultured.

[0011] In some embodiments, the effective amount of IL-2 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 isolated CD25 + Treg cells are suspended in medium containing IL-2 immediately prior to the culture step of the methods described herein.

[0012] In some embodiments, in the culturing step of the methods described herein, CD25 + The cells were initially grown in a 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 mixed and resuspended during the culturing step of the methods described herein.

[0013] In some embodiments, about 1×10 9 ~About 10×10 9 Activated CD25 + Cells are harvested after 10, 12, or 14 days of culture.

[0014] Further provided herein is a method for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit, the method comprising: a) thawing the cryopreserved human umbilical 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 0.5% human serum albumin in a functionally closed system without manual washing; and c) isolating the thawed cord blood unit from the cryopreserved human umbilical 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 approximately 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. + Treg cells are expanded ex vivo, with the medium replaced approximately every 48 hours, and activated CD25 + Generates a population of Treg cells and expresses CD25 +e) growing Treg cells and anti-CD3 and anti-CD28 coated beads in a 1:1 ratio and without mixing and resuspending the culture; and f) extracting activated CD25 from the culture medium. + harvesting the cells to produce an expanded population of activated human Treg cells.

[0015] In some embodiments, the methods of the invention further comprise cryopreserving the expanded population of activated human Treg cells.

[0016] Further provided herein are populations of activated human Treg cells produced by the methods described herein.

[0017] In some embodiments, the Treg cells in any of the populations disclosed herein are at least 90% CXCR4 + In some embodiments, the Treg cells in any of the populations disclosed herein are at least 95% CXCR4 + , at least 95% CD45RA + and at least 80% CD45RO + In some embodiments, the Treg cells are at least 95% CXCR4 + , at least 95% CD45RA + , at least 80% CD45RO + , at least 95% CD95 + , at least 95% HLADR + , at least 95% alpha4beta7 + , at least 15% are CXCR3hi + , at least 95% CCR6 + , at least 95% CD54 + , at least 95% CD11A + , at least 85% CD45RARO + , at least 80% CTLA4 + , at least 80% are GPR83 + and at least 80% CD62L +In some embodiments, the human Treg cells in any of the populations disclosed herein exhibit high expression of FOXP3 and low expression of RORγt. In some embodiments, the human Treg cells in any of the populations disclosed herein maintain their polyclonal T cell receptor Vβ (TCR Vβ) repertoire. In some embodiments, the human Treg cells in any of the populations disclosed herein are cryopreserved prior to use.

[0018] Also provided herein is a method for cryopreserving an expanded population of activated human regulatory T (Treg) cells produced from at least one cryopreserved human umbilical cord blood unit, the method comprising: a) thawing the cryopreserved human umbilical cord blood unit; b) diluting and washing the thawed cord blood unit in a functionally closed system; and c) detecting CD25 + and (d) isolating endogenous Treg cells using a double selection method based on cell surface expression, and (e) culturing the isolated CD25 Treg cells in a gas-permeable culture vessel 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 10 days, up to 12 days, or up to 14 days. + Treg cells are expanded ex vivo, with the medium replaced approximately every 48 hours, and activated CD25 + e) generating and expanding a population of Treg cells; and f) inducing activated CD25 + harvesting the cells to produce an expanded population of activated human Treg cells; f) releasing the activated cultured human Treg cells for clinical use based on defined criteria; and g) cryopreserving the released activated cultured human Treg cells with a characteristic phenotype.

[0019] Further provided herein is a method for treating or preventing graft-versus-host disease 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. Also provided herein is a method for treating or preventing graft-versus-host disease in a subject, the method comprising administering to the subject (i) 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, and (ii) ruxolitinib.

[0020] Also provided herein is a method for treating or preventing a 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 methods disclosed herein or an effective amount of a population of human Treg cells disclosed herein. In some embodiments, the bone marrow failure syndrome is aplastic anemia, primary myelofibrosis, or myelodysplastic syndrome. Further provided herein is a method for treating or preventing primary myelofibrosis in a subject, the method comprising administering to the subject (i) 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, and (ii) ruxolitinib.

[0021] Further 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.

[0022] Also provided herein is a method for treating or preventing multiple myeloma 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.

[0023] Further provided herein are methods 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 the methods disclosed herein or an effective amount of a population of human Treg cells disclosed herein. In some embodiments, the neuroinflammatory disorder is Guillain-Barré syndrome, amyotrophic lateral sclerosis, multiple sclerosis, or a demyelinating neuropathy.

[0024] Further provided herein are methods for treating or preventing a respiratory disease, disorder, or condition associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection 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. In some embodiments, the respiratory disease, disorder, or condition is COVID-19 (coronavirus disease)-mediated acute respiratory distress syndrome (CoV-ARDS).

[0025] Further provided herein are methods for treating or preventing cytokine release syndrome (CRS) 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. In some embodiments, the CRS is associated with chimeric antigen receptor T-cell therapy.

[0026] In some embodiments, an effective amount of the population of human Treg cells is administered intravenously to the subject.

[0027] In some embodiments, an effective amount of a population of human Treg cells is about 1 x 10 of a subject's body weight. 6 ~Approx. 1×10 7 In some embodiments, an effective amount of a population of human Treg cells is about 1 x 10 8 Treg cells ~ approximately 3 x 10 8 These are Treg cells.

[0028] In some embodiments, following administration of an effective amount of the population of human Treg cells, the level of circulating inflammatory cytokines in the subject is reduced compared to the level of circulating inflammatory cytokines in the subject prior to administration.

[0029] In some embodiments, prior to treatment, the subject's serum biomarkers are tested to determine whether the subject will respond to an effective amount of a population of human Treg cells. In some embodiments, after treatment, the subject's serum biomarkers are tested to determine correlations with clinical response. In some embodiments, serum biomarkers are serially tested to determine whether subsequent retreatment with Treg cells is necessary.

[0030] In some embodiments, the population of human Treg cells is prepared from one or more cord blood units of a blood type that is compatible with the subject to be treated by the methods disclosed herein. In some embodiments, the population of human Treg cells is prepared from cord blood units that are at least 3 out of 6 HLA (human leukocyte antigen) matches to the subject to be treated by the methods disclosed herein. In some embodiments, the population of human Treg cells is prepared from cord blood units that are not HLA-matched to the subject to be treated by the methods disclosed herein.

[0031] Further provided herein is the use of the population of human Treg cells disclosed herein in the preparation of a medicament. [Brief explanation of the drawings]

[0032] [Figure 1]This is a line graph showing the results of an assay that measured the viability (7AAD) of freshly activated Treg cells stored at room temperature (15-30°C) or 4°C (N=9). [Figure 2A-2B] Figure 2A shows a series of graphs demonstrating that expanded, activated Treg cells are immunosuppressive. For suppression assays, conventional T cells (Tcon) (CD4+CD25-) were thawed and stained with CellTrace™ Violet (ThermoFisher) according to the manufacturer's instructions. Cord blood Tregs and Tcon cells were arranged 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 collected on day 14 compared to freshly isolated cord blood on day 0 in HLA-matched pairs (p=0.03) and HLA-mismatched pairs (p=0.03, 2-sided t-test). (n=2) [Figure 3]

[0023] Figure 1 shows a line graph demonstrating 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 x 10 cells to induce GVHD. In the treatment group, umbilical cord blood Tregs from HLA A2-negative donors were injected at a dose of 1 x 10 cells one day before 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 compared with the PBMC-only group. [Figures 4A-4D]A series of graphs and plots are shown showing that expanded activated Treg cells remain suppressive, do not express RORγt, and exhibit 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, 40, or 200 ng / ml of IL-6. Cells were fed every 48 hours, and flow cytometry-based analysis was performed for intracellular staining of RORγt and cytokine release assays of IL-10 and IL-17. [Figures 5A-5D] Figure 5 shows graphs demonstrating that cryopreserved cord blood (CB) Treg cells have equivalent 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] This is a series of graphs showing that expanded cord blood Tregs exhibit a Gaussian (polyclonal) distribution of T cell receptor Vβ 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). The 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. [Figures 7A-7B] Expanded cord blood Tregs maintain suppressive properties in the presence of dexamethasone (also called "Dex" or "steroid"). "Tcon" refers to conventional T cells. "Treg" refers to regulatory T cells. The top and bottom left panels are steroid-free. The top and bottom right panels contain 100 μg / mL of steroid. [Figures 8A-8C] Cryopreserved activated Treg cells exhibit a consistent phenotype, demonstrating their ability to suppress immune responses 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 exhibit 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-9B] Figure 9A shows the results of a study using a xenogeneic mouse graft-versus-host disease (GVHD) model. Using this xenogeneic GVHD model (Parmar et al., Cytotherapy 16(10:90-100(2013)), freshly activated Treg cells or cryopreserved (frozen) activated Treg cells were administered at a dose of 1 x 107 cells one day before donor peripheral blood mononuclear cells at a dose of 1 x 107 cells for GVHD prophylaxis. Figure 9A shows the effect of freshly activated Treg cells or cryopreserved (frozen) activated Treg cells on GVHD scores. Figure 9B shows the effect of freshly 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. [Figures 10A-10B]The design of a study using a xenogeneic mouse graft-versus-host disease (GVHD) model is shown. Figure 10A shows the GVHD prevention study design, in which NSG mice are sublethally irradiated on day -1, followed by injection of 1 x 107 umbilical cord blood (CB) Treg cells, and then injected with 1 x 107 PBMC cells on day 0. Mice are then followed every other day for measurements of body weight and GVHD score. Peripheral blood and serum samples are collected at baseline and then at weekly intervals starting on day +7. Figure 10B shows the GVHD treatment study design, in which NSG mice are sublethally irradiated on day -1, followed by injection of 1 x 107 PBMC cells on day 0. Injections of 1 x 107 CB Treg cells are administered on days +4, +11, +18, and +25. Mice are then followed every other day for measurements of body weight, GVHD score, and survival rate. Peripheral blood and serum samples are collected at baseline and then at weekly intervals from day +7. "PBMC" refers to peripheral blood mononuclear cells. "Frozen Treg" refers to cryopreserved Treg. "NSG" refers to non-SCID gamma null mice. [Figures 11A-11B] Figure 11 shows the effect of administration of cryopreserved activated Tregs on body weight fluctuation (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 in which Treg cells were not administered. [Figures 12A-12F] The results of peripheral blood cytokine analysis of control, prophylaxis, and treatment groups at baseline, +7 days, and +14 days after PBMC infusion in a xenogeneic mouse graft-versus-host disease (GVHD) model are shown in Figure 12A, Figure 12B: TNFα, Figure 12C: GM-CSF, Figure 12D: MIP-1β, Figure 12E: FLT-3L, and 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 without the continued absence of PBMCs (CB Tregs only), but remained detectable in PBMC recipient mice (CB Treg+PBMCs). In mice with expanded 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 lines were injected at a dose of 3 × 10 cells into four groups of NSG mice: 1) control mice (PBS & AML): HL60 + PBS; 2) Treg mice (AML + Treg): HL60 + Treg (1 × 10 cells); 3) Tcon mice (AML + Tcon): HL60 + Tcon (1 × 10 cells); and 4) Tcon + Treg mice (AML + Tcon + Treg): HL60 + Tcon (1 × 10 cells) + Treg (1 × 10 cells). To understand the effect of injected Tcon and Treg on tumor volume burden, mice were photographed at weekly intervals. Mice succumbed to tumors in control (PBS-treated) and mice treated with CB Tregs alone. Recipients of Tcon were able to eliminate tumors but died of GVHD. Recipients of Tcon and Treg were able to prolong survival due to tumor control and lack of GVHD. [Figure 15]This figure shows a line graph demonstrating that a single injection of activated Treg cells reduced the levels of CD45+ effector T cells 9 weeks after engraftment of SLE-PBMCs in a xenogeneic mouse model of systemic lupus erythematosus (SLE). NSG mice were injected with SLE-PBMCs (3 x 106 cells) and CB Tregs (1 x 107 cells) 1 week after SLE-PBMC injection. "PBMC" refers to peripheral blood mononuclear cells. [Figure 16A] 1 shows a graph demonstrating that in a xenogeneic mouse model of systemic lupus erythematosus (SLE), four weekly injections of activated Treg cells (1 x 10 cells) starting four weeks after injection of SLE-PBMCs (3 x 10 cells) improved survival. [Figure 16B] 1 shows a bar graph demonstrating 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). [Figures 17A-17B] 17A and 17B show plots demonstrating that four weekly injections of activated Treg cells reduced urinary albumin levels (FIG. 17A) and 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). [Figures 20A-20B] Graphs are shown showing that weekly injections of activated cryopreserved Tregs resulted in a sustained decrease in circulating CD8+ effector T cells (FIG. 20A), as well as a decrease in the 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). "PBMC" refers to peripheral blood mononuclear cells. [Figures 21A-21D]Figure 21A shows a series of graphs and images depicting the effects of Treg administration in a xenogeneic mouse model of multiple myeloma. Figure 21A is a line graph depicting the effect on mouse weight over time. CB Treg recipients maintained their weight, while a decline in the "myeloma alone" group indicates that weight loss began around week 4 after tumor inoculation. Figure 21B is a line graph depicting the effect on circulating myeloma cells in peripheral blood over time. Weekly blood samples were collected and isolated cells analyzed for circulating human CD38+ cells. A significant increase in circulating myeloma cells was evident in the "myeloma alone" group compared to the Treg recipients (p=0.002). Figure 21C shows a series of images depicting visualization of tumor burden. As monitored by weekly bioluminescence imaging, minimal evidence of MM1S cells was visualized in the CB Treg recipients compared to the extensive tumors in the "myeloma alone" mice. Figure 21D is a line graph depicting quantification of tumor burden over time. Bioluminescence imaging showed significantly higher signals on days 17, 24, and 31. Triangles indicate CB Treg i.v. injection, and arrows indicate MM1S cell i.v. injection. [Figure 22] 1 shows a graph showing that administration of activated Tregs improves survival in a xenogeneic mouse model of multiple myeloma. In this heterogeneous 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 as determined by the log-rank test. [Figure 23] A bar graph shows that administration of activated Tregs reduces plasma IL-6 levels in a xenogeneic mouse model of multiple myeloma. In this mouse model, injection of cord blood (CB) Tregs one day before myeloma cell injection prevented myeloma engraftment and led to improved overall survival, which 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 on 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. [Figures 24A-24B] Figure 24 shows bar graphs demonstrating that administration of activated Treg cells reduced myeloma burden in the bone marrow (Figure 24A) and spleen (Figure 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 the population of MM.1S cells was analyzed by flow cytometry. [Figure 25] 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 for 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 administering 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. [Figure 30] 1 is a table providing cord blood selection criteria for various products containing populations of activated human Treg cells. "CK0802.a4b7" refers to "CK0802.α4β7." [Figure 31]

[0023] Figure 1 is a line graph showing the percentage of suppression by activated Treg cells in the absence or presence of 0.05 μM ruxolitinib 96 hours after the initiation of co-culture of Treg cells, Tcon cells, and ruxolitinib. The x-axis shows the ratio of Treg cells to Tcon cells. Ruxo = ruxolitinib. [Figure 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. [Figure 33] Schematic diagram of treatment of a xenogeneic murine graft-versus-host disease (GVHD) model with ruxolitinib and activated Treg cell regimen. PBMC = peripheral blood mononuclear cells. [Figure 34A-34B] Graphs are shown showing the effect of treatment with (1) activated Treg cells (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on GVHD scores (FIG. 34A) or survival (FIG. 34B) in a xenogeneic mouse GVHD model. Rux or R = ruxolitinib. PBMC = peripheral blood mononuclear cells. [Figures 35A-35C] Figure 35 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 the persistence of activated Treg cells 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. [Figures 36A-36C] Figure 36 shows a series of bar graphs depicting the effects 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. [Figures 37A-37E]Figure 37 shows a series of bar graphs depicting the effects of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on inflammatory 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. [Figures 38A-38C] Figure 38 shows a series of bar graphs depicting the effects of treatment with (1) activated Treg cells, (2) ruxolitinib, or (3) activated Treg cells and ruxolitinib on anti-inflammatory 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-39B]

[0039] Figure 39 shows a series of bar graphs depicting the effects 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. [Fig. 40B-40F]A series of bar graphs depicting the effect of CB Treg cells on the migration of myeloma and leukemia target cells are shown. Figure 40B shows that CB Tregs and Teff cells completely block the migration of MM1S (a myeloma cell line) (p<0.001). Figure 40C shows that CB Tregs and Teff cells completely block the migration of RPMI8226 (a myeloma cell line) (p=0.04). Figure 40D shows that CB Tregs reduce, but not significantly reduce, the migration of U266 (a 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 (an acute myeloid leukemia cell line). Figure 40F shows that CB Tregs and Teff cells have no effect on the migration of Nalm6 (a pre-B cell leukemia cell line). **P<0.05 determined by unpaired Student's t-test at each time point. The y-axis in Figures 40B-40D represents cell number x 103 / μL. [Figure 41] Figure 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). [Figure 42] FIG. 1 summarizes clinical data from a phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 43] 1 is a table summarizing clinical data from a phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 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. [Figure 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. [Figures 46A-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]

[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: 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] FIG. 1 summarizes the treatment history of patient 2 in a phase 1 clinical trial of allogeneic cord blood-derived Treg cells in patients with BMF. [Figure 51] 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 is a graph showing 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] 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] 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] 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. [Figures 59A-59D] Data are presented from studies of xenogeneic lymphoma mouse models treated with i) mimetic 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-60B]

[00130] 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 for the various groups. Figure 60B shows CD19-CAR T cells / μL in various organs. [Figures 61A-61H]Figure 61A shows a series of graphs and images depicting the effect of administering multiple doses of Tregs in a xenogeneic mouse model of multiple myeloma. Figure 61A is a line graph depicting 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 umbilical 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 depicting quantification of tumor burden by BLI. Figure 61D is an image depicting 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 engagers 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. DETAILED DESCRIPTION OF THE INVENTION

[0033] 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. Therefore, Tregs are important for homeostasis and immune control, as well as for protecting the host against the development of autoimmunity. Furthermore, both infused and innate Tregs are susceptible to: i) the proliferation of effector T cells that produce excess IL-2, which is essential for Treg survival; and ii) the creation of inflammatory areas due to homing signals released by damaged antigen-presenting cells / dendritic cells present in tissues.

[0034] Although several types of Tregs have been described, 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 lo Tregs 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)).

[0035] In their endogenous state, Treg cells play a critical 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 from the onslaught of autoreactive cytotoxic / effector T cells.

[0036] 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).

[0037] To address this challenge, the present disclosure uses Tregs derived from umbilical cord blood. Umbilical cord blood has low immunogenicity 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 a different ratio of blood cells due to its high suppressive properties. Furthermore, umbilical cord blood cells are primitive, less immunoreactive, naive, exhibit a higher proliferation index, and can function across human leukocyte antigen (HLA) boundaries. Umbilical cord blood is a unique source because Tregs derived from umbilical cord blood are naive, highly suppressive, and lack plasticity compared to other Treg sources. Similarly, umbilical cord blood cells are constantly stimulated by numerous cytokines during the stress of childbirth, making them less sensitive to potentially toxic environmental substances.

[0038] 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 are methods for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit. Disclosed also are populations of activated human Treg cells produced by the methods described herein. Further disclosed herein are methods 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 are methods for cryopreserving an expanded population of activated human Treg cells produced from at least one cryopreserved human umbilical cord blood unit. Further disclosed herein are populations of immunosuppressive Treg cells.

[0039] 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 invention 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" should be understood to mean the inclusion of an entire entity or group of entire entities, but not the exclusion of any other entire entity or group of entire entities. Unless the context requires otherwise, the singular includes the plural and the plural includes the singular. Any examples following the terms "eg" or "for example" are not meant to be exhaustive or limiting.

[0040] As used herein, the terms "a," "an," and "the" are understood to be singular or plural unless specifically stated otherwise or clear from the context.

[0041] As used herein, the term "or" is understood to be inclusive unless specifically stated otherwise or clear from the context.

[0042] 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).

[0043] A population of "activated" Treg cells can 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 the cell densities specified herein, in the presence of T cell receptor (TCR) stimulation with CD3 / 28 beads, which allows stimulated Treg cells to provide consistent suppression of inflammation.

[0044] 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 includes 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'), diabodies, linear antibodies, and V H These include, but are not limited to, single domain antibodies which comprise only the domain (VHH).

[0045] As used herein, the terms "patient" or "subject" are used interchangeably herein to refer to any mammal, including humans, domestic animals and livestock, as well as zoo animals, sport 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.

[0046] As used herein, unless otherwise specified, 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.

[0047] As used herein, "prevent" means to prevent, in whole or in part, the producing or occurring of, or ameliorating or controlling, or reducing or arresting, the thing or event to be prevented, e.g., a disease, disorder, or condition.

[0048] As used herein, phrases such as "therapeutically effective amount" and "effective amount" refer to the amount needed to be administered to a patient, or to a patient's cells, tissues, or organs, to achieve a therapeutic effect, such as ameliorating or replacing a curative 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.

[0049] 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.

[0050] Methods for generating expanded populations of regulatory T cells Because Treg cells are present at low frequencies in circulating blood or umbilical cord blood, the generation of clinically relevant Treg cell doses requires the use of CD4 + CD25 + Ex vivo enrichment and expansion of Treg cells with this phenotype is required.

[0051] In any of the methods described herein, the cord blood bank and donor may be certified prior to use of the human cord blood in the methods described herein. In some embodiments, the human cord blood unit is provided by a public cord blood bank in the United States, the European Union, or other regions that meets the donor qualification criteria. Certification of the cord blood unit may include verifying 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, pre-freeze cell viability, 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, etc. 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.

[0052] In some embodiments, the cell starting material (CBU) is thawed, washed, and purified using immunomagnetic selection to identify CD25 + Enriched for mononuclear cells (MNC). CD25 + MNCs are placed in a gas-permeable culture device containing interleukin-2 (IL-2) and anti-CD3 / anti-CD28 beads. The cells are culture-expanded for up to 10 days, up to 12 days, or up to 14 days. In some embodiments, the cells are culture-expanded for 8-10 days or 10-12 days. On days 8, 9, 10, 11, 12, or 14, the expanded cells are harvested and washed, and the CD3 / CD28 beads are immunomagnetically removed. The bead-depleted cells are then formulated and packaged.

[0053] In some embodiments, disclosed herein are methods for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit, the method comprising: a) thawing the cryopreserved human umbilical cord blood unit; b) diluting and washing the thawed cord blood unit in a functionally closed system or a closed system; and c) detecting CD25 + Isolating endogenous Treg cells using a dual selection method based on cell surface expression; and d) culturing the isolated CD25 Treg cells in a gas-permeable culture vessel 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. + Treg cells are expanded ex vivo, with the medium replaced approximately every 48 hours, and activated CD25 + e) generating and expanding a population of Treg cells; and f) inducing activated CD25 + and harvesting the cells 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 the 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)).

[0054] 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.

[0055] In some embodiments, cryopreserved human cord blood units are thawed in a single step in a water bath (e.g., at 37°C + / - 1 degree). In some embodiments, thawing a 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 partially thawed. The cells are then immediately removed for a washing process.

[0056] In some embodiments, the thawed cord blood unit undergoes 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 a Sepax system (Biosafe). The Sepax system is a centrifuge and pump device intended for use in cell therapy, where specific blood components must be isolated. Its principle is based on centrifugation, allowing separation of blood particles according to their density and size. Blood components are collected in individual bags and are immediately available for transfusion. The automated cell processing system can accommodate a starting volume of up to 100 ml and a final volume of 50–150 ml. The dilution ratio between the initial volume and the dilution volume is adjustable from 0.5–2.0 times. Washing cycles can include one standard wash or, in certain circumstances, two high-level washes. The automated cell processing system is programmed to automatically perform initial product dilution, osmolality recovery, washing, centrifugation, supernatant extraction, and cell resuspension. Typically, the starting volume is set to 25 ml, the final volume to 100 ml, and the dilution factor 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.

[0057] 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, about 50 mg of magnesium chloride (MgCl), and about 2500 units of DNase. In some embodiments, the modified medium comprises X-Vivo15 medium (Lonza), 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.

[0058] In some embodiments, the washing step does not include manual washing.

[0059] In some embodiments, the automatically washed cord blood cells undergo an additional manual wash using working wash medium to a final volume of 200 ml, and the reconstituted cells are centrifuged at 300 g for 10 minutes at room temperature. Finally, the washed cells are reconstituted in 0.09 ml at 100 x 10 6 Resuspend at a concentration of 100 cells.

[0060] 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, a cellulose derivative, an acrylic resin, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, a copolymer 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.

[0061] 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 microbeads are removed. + The cells are kept in suspension within the column and do not actually "bind" the column matrix. This suspension minimizes stress on the cells and avoids cell clumping, allowing for efficient sterile washing. The LS column is primed with working wash medium and loaded with microbead-labeled CD25. + The cells are allowed to pass through an LS column attached to a 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 acts 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 form the final CD25 selection. + In some embodiments, CD25 cells are obtained using a double ferromagnetic column (e.g., LS column) method. + Isolate the cells.

[0062] 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 anti-CD3-coated beads and anti-CD28-coated beads (i.e., "anti-CD3 / anti-CD28-coated beads"). In some embodiments, the anti-CD3-coated beads and anti-CD28-coated beads are in a 1:1 ratio in the reagent that specifically binds to CD3 and CD28. In some embodiments, the reagent that specifically binds to CD28 comprises anti-CD3-coated beads and anti-CD28-coated beads (i.e., "anti-CD3 / anti-CD28-coated beads"). In some embodiments, the ratio of anti-CD3-coated beads to anti-CD28-coated beads is 1:1 in the reagent that specifically binds to CD3 and CD28. In some embodiments, the ratio of anti-CD25-coated beads to anti-CD28-coated beads is 1:1 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.

[0063] 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, isolated CD25 + Treg cells are suspended in medium containing IL-2 immediately prior to the culture step of the methods described herein.

[0064] In some embodiments, during the culturing step, the medium is changed about every 48 hours without disturbing the cells. In some embodiments, the culture is not mixed and resuspended during the culturing step of the methods described herein.

[0065] In some embodiments, about 1×10 6 CD25 + The cells / ml are cultured in the presence of a reagent that specifically binds to CD3 and CD28 in a method to produce an expanded population of activated human Treg cells. In some embodiments, CD25 + The cells were initially grown in a 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 nm.

[0066] In some embodiments, about 0.5×10 9 ~Approx. 12×10 9 , or approximately 1 × 10 9 ~about 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 step of: + CD25 + resulting 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, but is rapidly released for administration.

[0067] Further provided herein is a method for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit, the method comprising: a) thawing the cryopreserved human umbilical 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 without manual washing; and c) isolating the thawed cord blood unit from the cryopreserved human umbilical 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 approximately 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. + Treg cells are expanded ex vivo, with the medium replaced approximately every 48 hours, and activated CD25 + Generates a population of Treg cells and expresses CD25 + e) growing Treg cells and anti-CD3 and anti-CD28 coated beads in a 1:1 ratio and without mixing and resuspending the culture; and f) extracting activated CD25 from the culture medium. + harvesting the cells to produce an expanded population of activated human Treg cells.

[0068] After harvesting, Treg cells may be tested for contamination, viability, purity, cell number counted, and / or examined using flow cytometry.

[0069] In some embodiments, the active ingredient (DS) is a regulatory T cell phenotype (CD4 + CD25 + In some embodiments, the DS is a liquid cell suspension comprising or consisting 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 an 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.

[0070] In some embodiments, the conditional CD8 + A cell depletion step can optionally be performed to remove CD4 T cells from the population of activated Treg cells before the final preparation. - CD8 + Used to reduce the content of cytotoxic / suppressor T cells. Prior to harvest, CD8 is removed from the culture medium using a reagent that specifically binds to CD8 (i.e., an anti-CD8 antibody or its antigen-binding fragment). +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, silicone, plastic, nitrocellulose, natural sponge, silica gel, controlled pore glass, metal, cross-linked dextran, and agarose gel.

[0071] CD8 + Following cell depletion, the method described herein results in 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.

[0072] At the end of cell culture, the concentration was 3 x 10 6 If there are more than 100 per cell, an additional step of removing the anti-CD3 / anti-CD28 coated beads can be performed.

[0073] Criteria for releasing expanded populations of activated human Treg cells with a characteristic phenotype for clinical use include 7-amino-actinomycin-D (7-AAD) survival of 70% or more, CD4 + CD25 +These include purity, "organism-free" Gram stain, and endotoxin less than 5EU / kg.

[0074] In some embodiments, large volumes of product can be produced with extensive expansion of up to 1000-fold or more, with final populations of cells reaching approximately 0.5 x 10 cells harvested after up to 14 days of culture. 9 ~12×10 9 The final product is a homogenous and well-defined Treg cell line ranging from 100 to 1000 Treg cells. In some embodiments, the final product can remain stable for up to 8 hours when stored at room temperature and 96 hours when stored at 4°C.

[0075] In some embodiments, additional steps are performed to enrich for cell surface expression of CXCR4, α4β7, or CD11a.

[0076] In some embodiments, the manufacturing process includes some or all of the following steps:

[0077] Step 1: Thawing the cord blood unit (CBU) (day 0) Input: CBU Output: CBU after thawing The frozen CBUs are removed from liquid nitrogen (LN2) vapor storage and placed in a plastic overwrap bag to prevent port contamination during thawing. The enclosed cryobags are quickly thawed by placing them in a 37°C water bath and gently kneading the bags to ensure uniform thawing. The output, thawed CBUs, are sampled for: Nucleated cell (NC) count Viability % (trypan blue) The test results are used to monitor the process.

[0078] 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 a Sepax (GE Healthcare) single-use disposable kit. The cells are diluted and washed in a Sepax system containing 10% low molecular weight dextran (LMD) in 0.9% NaCl. The output of the Sepax wash (post-wash CBU) is approximately 100 mL and is sampled for: Nucleated cell (NC) count Viability % (trypan blue) The test results are used to monitor the process.

[0079] Step 3: Pre-selection wash (day 0) Input: CBU after cleaning Output: CB mononuclear cells (MNC) After washing, the CBU cells are centrifuged at 400 x g (centrifugal force) for 10 minutes at room temperature. After gently removing the supernatant by aspiration, the cells (CB MNCs) are resuspended in approximately 8-10 mL of Miltenyi PBS / EDTA buffer. The output, CB MNCs, are not sampled.

[0080] Step 4: CD25 antibody incubation (day 0) Input:CB MNC Output: CB MNCs after incubation CB mononuclear cells are incubated with Miltenyi anti-CD25 microbeads for 15 minutes at 4-8°C with intermittent manual mixing. After incubation, the cell and anti-CD25 microbead mixture is washed and resuspended in approximately 10 mL of Miltenyi PBS / EDTA buffer supplemented with Pulmozyme and MgCl2. The output, post-incubation CB MNCs, is not sampled.

[0081] Step 5: CD25 positive selection (day 0) Input: CB MNCs after incubation Output: CD25 + MNC Following an incubation step with Miltenyi CD25 antibody reagent, the incubated CB MNCs are transferred into a Miltenyi LS column attached to a MidiMACS device, which captures anti-CD25 labeled cells using a magnet. After immunomagnetic selection, the cells are released from the magnetic field and the output CD25 + The MNC is sampled for: Nucleated cell (NC) count Viability % (trypan blue) Viability % (7-AAD flow cytometry) %CD4 - CD8 + (Flow cytometry) %CD4 + CD25 + (Flow cytometry) The test results are used to monitor the process.

[0082] Step 6: Initiation of culture growth (Day 0) Input: CD25 + MNC Output: Day 0 of culture CD25 + The selected MNCs are washed and suspended in X-Vivo15 containing 1% glutamine and 10% human AB serum containing interleukin-2 (IL-2, 1000 IU / mL), then mixed with CD3 / CD28 beads at a 1:1 cell to bead ratio. The cell and bead mixture is placed in a 10 cm2 tube with a surface area of ​​10 cm2. 2 The cells are transferred into a gas-permeable growth (10M) system at 37°C and incubated in 5% CO2. The cell suspension is not shaken or stirred. No sampling is performed at this step. The gas-permeable growth (10M) system consists of a cylindrical, sterile, single-use, disposable plastic device. After transferring cells and media into the gas-permeable growth system, the cells reside at the bottom of the vessel and the surface is gas-permeable. The gas-permeable membrane of the 10M system is 10 cm 2The system is placed in a conventional incubator but can be removed intermittently as needed for sampling, media removal, media addition, or cell harvesting.

[0083] Step 7: Addition of IL-2 (Day 2 or Day 3) Input: Day 0 of culture Output: Day 2 / 3 culture + IL-2 On day 2 or 3 (less than 66 hours after the final media / IL-2 exchange), fresh IL-2 is added at 1000 IU / mL to cultured cells in a gas-permeable growth (10M) system to replenish presumed consumed IL-2. No sampling is performed at this step. 2 The cells in the gas-permeable growth (100M) system are returned to incubation at 37° C. and 5% CO 2 . There is no shaking or agitation of the cell suspension.

[0084] Step 8: Migrate and Feed (Day 4, 5, or 6) Input: Day 2 / 3 culture + IL-2 Output: Day 4 / 5 / 6 culture + fresh medium + IL-2 On days 4, 5, or 6 (less than 66 hours after the final media / IL-2 exchange), an aliquot of cultured cells in the gas-permeable growth (10M) system is removed and sampled for the following: Nucleated cell (NC) count Viability % (trypan blue) NC counts and % viability are used to monitor the process of culture growth. The remaining cultured cells in the gas-permeable growth (10M) system are transferred to a gas-permeable growth (100M) system containing fresh medium (X-Vivo15 containing 1% glutamine and 10% human AB serum, and 1000 IU / mL IL-2) added to a volume of 1000 mL. The cells in the gas-permeable growth (100M) system are returned to incubation at 37°C and 5% CO2. The cell suspension is not shaken or stirred. The gas-permeable growth (100M) system consists of a cylindrical, sterile, single-use, disposable plastic device. After transferring cells and media into the gas-permeable growth system, the cells reside at the bottom of the vessel and the surface is gas-permeable. The gas-permeable membrane of the 100M system is 100 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.

[0085] Step 9: Addition of IL-2 (Day 7 or 8) Input: Day 4 / 5 / 6 culture + fresh medium + IL-2 Output: Day 7 / 8 culture + IL-2 On day 7 or 8 (less than 66 hours after the final media / IL-2 exchange), fresh IL-2 is added to the cultured cells in the G-Rex 100M system to replenish the presumably consumed IL-2. No sampling is performed at this step. The cells in the gas-permeable (100M) system are returned to incubation at 37°C and 5% CO2. The cell suspension is not rocked or agitated.

[0086] Step 10: Addition of IL-2 (Day 9 or 10) Input: Day 7 / 8 culture + IL-2 Output: Day 9 / 10 culture + IL-2 On day 9 or 10 (less than 66 hours after the final media / IL-2 exchange), fresh IL-2 is added to the cultured cells in the gas-permeable growth (100M) system to replenish the presumably consumed IL-2. No sampling is performed at this step. The cells in the gas-permeable growth (100M) system are returned to incubation at 37°C and 5% CO2. The cell suspension is not shaken or agitated.

[0087] Step 11: Addition of IL-2 (Day 11 or 12) Input: Day 9 / 10 culture + IL-2 Output: Day 11 / 12 culture + IL-2 On day 11 or 12 (less than 66 hours after the final media / IL-2 exchange), cultured cells are sampled for: Mycoplasma ·Sterility Test results for mycoplasma (final report; release criteria is negative for mycoplasma species) and sterility (interim report; release criteria is a report of "no growth" for the final formulation and samples obtained 48-72 hours prior to lot release) are used for release of the final product on day 14. After sampling, fresh IL-2 is added to the cultured cells in the gas-permeable growth (100M) system to replenish the presumably consumed IL-2. The cells in the gas-permeable growth (100M) system are returned to incubation at 37°C and 5% CO2. The cell suspension is not shaken or agitated.

[0088] Step 12: Pre-harvest sample (day 14) Input: Day 11 / 12 culture + IL-2 Output: 14 days before collection, sampling On day 14, prior to harvesting the culture-expanded T-Reg cells, the cell suspension is sampled for: Mycoplasma The mycoplasma test is repeated at this point, but the results are not usually available before product burst, but the results of the mycoplasma test on days 11 / 12 are used for burst. After sampling for mycoplasma, 750 mL of the 1000 mL total cell suspension volume in the gas permeable growth (100M) system is removed and the remaining culture is sampled for the following: Nucleated cell (NC) count Viability % (trypan blue) %CD4 - CD8 + (Flow cytometry) NC count and % viability are used to monitor the process. %CD4 - CD8 + CD8 +Used to determine the need for immunomagnetic depletion of cells (conditional step S-1). %CD4 - CD8 + If the cell population represents more than 10% of the culture-expanded cells. If CD8 depletion is required, conditional step S-1 is performed after harvest on day 14 (step 13).

[0089] Step 13: Harvesting (Day 14) Input: 14 days before collection, sampling Output: T-Reg harvest After sampling, the remaining 250 mL volume in the gas-permeable growth (100M) system is transferred to a 500 mL conical tube by rinsing the gas-permeable growth flask to optimize cell recovery, and the volume is brought to 400 mL with injection buffer (Plasma-Lyte A with 0.5% HSA). The 500 mL conical tube is centrifuged twice at 400 x g for 10 min at room temperature to wash the cells with Plasma-Lyte A with 0.5% HSA. The cell suspension is brought to a volume of 10 mL with Plasma-Lyte A with 0.5% HSA in a 15 mL conical tube for bead removal (step 14).

[0090] Conditional Step S-1: CD8 depletion Input: T-Reg harvest Output: After CD8 depletion %CD4 obtained from sampling in step 12 - CD8 + Flow cytometry results showed that CD4 - CD8 + If the cell population is shown to represent more than 10% of culture-expanded cells, CD8 depletion is performed. To deplete CD8, the T-Reg harvest is incubated with Miltenyi CD8 microbeads for 15 minutes at 4-8°C with gentle agitation, then transferred to a Miltenyi LS column and immunomagnetically selected using a MidiMACS device. The output after CD8 depletion is sampled for: Nucleated cell (NC) count ·Survival rate% %CD4 - CD8 + (Flow cytometry)

[0091] Step 14: Washing and removal of CD3 / CD28 beads (day 14) Input: Day 14 of collection Output: T-Reg collected material, de-beaded 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 the cells but not the CD3 / CD28 beads) is collected in another 15 mL conical tube ("Debeaded #1") before releasing the magnet. After releasing the magnet, the remaining beads and cells are resuspended in 2 mL of Plasma-Lyte A containing 0.5% HSA and placed in the Dynal MPC-1 magnet for 2 minutes. The supernatant is collected and transferred to the "Debeaded #1 tube." The "Debeaded #1" tube is then placed in the Dynal MPC-1 magnet for 2 minutes. The supernatant is collected in another 15 mL conical tube ("Debeaded #2") before releasing the magnet. The cell suspension in the "Debeaded #2" tube is now approximately 17 mL in volume and is sampled for the following: Nucleated cell (NC) count Viability % (trypan blue) %CD4 - CD8 + (Flow cytometry) %CD4 + CD25 + (Flow cytometry) Viability % (7-AAD, flow cytometry) Residual beads The output of this step, the T-Reg harvest, de-beaded material, is the active ingredient (drug substance). Nucleated cell (NC) count and % viability (trypan blue) are used to monitor the process. %CD4 - CD8 + (flow cytometry; release criteria ≤10%), %CD4 + CD25 +(flow cytometry; release criterion is ≥60%), % viability (7-AAD dye exclusion), and residual bead assay (release criterion is ≥3 × 10 6 Fewer than 100 beads per nucleated cell) are used for rapid release of the final product.

[0092] Step 15: Formulation and packaging (Day 14) Input: T-Reg harvested material, de-beaded material Output: T-Reg end product The T-Reg harvest, de-beaded, is transferred from the 15 mL conical tube to a 300 mL transfer pack. The conical tube is rinsed with 10 mL of Plasma-Lyte A + 0.5% HSA, and the 300 mL transfer pack is added to this rinse. The cell suspension in the transfer pack will have a volume of approximately 54 mL and will be sampled for: Gram staining Endotoxin ·Sterility Gram stain (by light microscopy; release criteria of "no organisms seen") and endotoxin (using the Endosafe PTS system; release criteria <5EU / mL) results are available for the rapid release of the final product. Sterility test results are not available for rapid release at this time point, but interim sterility test results from the 11 / 12 day time point will be used for rapid release. After sampling, the transfer set attached to the transfer pack is removed by sealing. After sampling, the volume of the cell suspension (final product) in the final product container is approximately 50 mL. These manufacturing steps are also summarized in the tables provided below, which present a flowchart of the manufacturing process (Table 1) that continues through to the final formulation and does not define any in-process / intermediate product or active ingredient retention steps, and a flowchart of the conditional CD8 cell depletion steps (Table 2). Because the process is continuous from the steps leading to the manufacture of the active ingredient (DS) through to the final formulation and packaging of the end product (DP), the manufacture of both the DS and DP is shown. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0093] 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).

[0094] In some embodiments, a method for cryopreserving an expanded population of activated human regulatory T (Treg) cells produced from at least one cryopreserved human umbilical cord blood unit comprises: a) thawing the cryopreserved human umbilical 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 dual selection method based on cell surface expression; and d) culturing the isolated CD25 Treg cells in a gas-permeable culture vessel 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. + Treg cells are expanded ex vivo, with the medium replaced approximately every 48 hours, and activated CD25 + e) generating and expanding a population of Treg cells; and f) inducing 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.

[0095] In some embodiments, the method further comprises releasing the activated cultured human Treg cells for clinical use based on the criteria defined between step e) and step f).

[0096] 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 placement in a controlled-speed freezer using a specifically defined program. Cryopreserved products can be stored at -180°C for at least several months. Upon thawing the cryopreserved products, Treg cells maintain their cell surface and intracellular phenotype with high expression of FOXP3 (forkhead box P3) and Helios and can retain their suppressive function, as demonstrated by in vitro cell suppression assays (Figures 8A-8C) and in vivo data in different animal models (Figures 9A-9B).

[0097] In some embodiments, about 10 x 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 x 10 cells are cryopreserved. 6 cells ~ approx. 1 x 10 8 Cells can be cryopreserved in a single cryogenic bag with a maximum volume of 10 ml to 100 ml.

[0098] In some embodiments, for purposes of cryopreservation, 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 a total cell count of 50×10 6Subsequently, frozen stock solutions can be prepared using pre-formulated solutions containing 5% or 10% dimethyl sulfoxide (DMSO) (Cryostor®) to obtain up to 50 x 10 cells per 5 ml cryovial. 6 Cells may be cryopreserved. While the cells are being centrifuged, the controlled-speed freezer is turned on, and once the controlled-speed freezer reaches the appropriate starting temperature, the command "Program waiting for user - click here to continue" appears. Upon mixing with the freezing stock solution, up to 50 x 10 cells may be cryopreserved. 6 Cryovials containing cells are placed in a controlled-rate freezer using a freezing algorithm, allowing the rate at which cells are frozen to be regulated, avoiding cell death and maintaining cell function. After the freezing program is complete, the cryovials are removed from the controlled-rate freezer and placed in a liquid nitrogen cryogenic freezer at temperatures as low as -190°C for long-term cryopreservation.

[0099] The expanded Treg population can be cryopreserved in several aliquots to generate a clinical dose suitable for therapeutic administration.

[0100] Regulatory T cell populations and pharmaceutical compositions Disclosed herein are populations of human Treg cells produced by the methods described herein. The populations are suitable for use in allogeneic cell therapy. In some embodiments, the human Treg cells are immunosuppressive.

[0101] 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.

[0102] 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.

[0103] 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% 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 approximately 15% are CXCR3hi + , at least about 95% are CCR6 + , at least about 95% are CD54 + , at least 95% CD11A + , at least about 85% are CD45RARO + , at least about 80% are CTLA4 + , at least about 80% is 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.

[0104] 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: α4β7 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.

[0105] In some embodiments, the population of human Treg cells is greater than or equal to about 60% CD4+ CD25 + Treg cells and approximately less than 10% of CD4 - CD8 + They have a flow cytometry phenotype of T-cytotoxic / suppressor cells.

[0106] 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 Vβ (TCR Vβ) repertoire. In some embodiments, the population of human Treg cells is cryopreserved prior to use.

[0107] In some embodiments, the population of human Treg cells expresses intracellular Helios. In some embodiments, human Treg cells produced by the methods disclosed herein retain their immunosuppressive function and phenotype under stressful conditions. In some embodiments, 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, human Treg cells produced by the methods disclosed herein, due to their epigenetic signature and the nature of the selection / expansion protocols described herein, are resistant to interleukin-17 (IL-17) secretion and are less likely to "flip" into pro-inflammatory T17 cells compared to peripheral blood Tregs.

[0108] The biological activity of interest for Treg cells in the populations described herein is their immunosuppressive function, which can be measured by an in vitro suppression assay using the intracellular dye CFSE (carboxyfluorescein succinimidyl ester) or CellTrace™ Violet. In this assay, Treg cells are cocultured with normal peripheral blood T responder (Tresp) cells at various ratios, and proliferating cells are detected using flow cytometry to detect the uptake 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. If effective suppression by Treg cells is present, suppression within first-generation dividing 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 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.

[0109] In some embodiments, human Treg cell populations exhibit paracrine functions such as increased production of the inhibitory cytokine interleukin-10 (IL-10), but not transforming growth factor β (TGFβ). In some embodiments, human Treg cell populations secrete granzyme B in response to IL-6 treatment (see, e.g., Figure 25).

[0110] Provided herein are at least about 1 x 10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are CD4 + CD25 + , and (ii) ≤10% CD4 - CD8+ and the human Treg cells are immunosuppressive. Further provided herein is a population of at least about 1 x 10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are 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 x 10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are 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 at least about 1 x 10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are CD4 + CD25 + and (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 x 10 9 human Treg cells or at least about 1 x 10 10 In some embodiments, the population of human Treg cells disclosed herein comprises about 1 x 10 8 ~1×10 10 pieces, about 1×10 8 ~1×10 9 pieces, or approximately 1 x 109 ~1×10 10 Contains human Treg cells.

[0111] 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 three of six HLA (human leukocyte antigen) matched (e.g., three of six, four of six, five of six, or six of six HLA matches) to the subject to whom the product is administered. The CK0801 product is administered to the subject as a single infusion, with a dose based on the subject's weight. This product contains immunosuppressive Treg cells.

[0112] 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), and (ii) ≤10% CD4 - CD8 + (T-cytotoxic / suppressive phenotype). In some embodiments, the CK0801 product is administered to a subject to treat an inflammatory bone marrow disorder or Guillain-Barre syndrome.

[0113] In some embodiments, the population of human Treg cells is cryopreserved and / or formulated as a multi-dose product (e.g., CK0802, CK0802.CXCR4, CK0802.α4β7, or CK0802.CD11a). In some embodiments, CK0802, CK0802.CXCR4, CK0802.α4β7, or CK0802.CD11a is formulated in an injectable cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). CK0802, CK0802.CXCR4, CK0802.α4β7, and CK0802.CD11a 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 include immunosuppressive Treg cells.

[0114] In some embodiments, the CK0802 product is CD25 + The CK0802 product is isolated via selection and after a 14-day culture period. In some embodiments, the release criteria for the CK0802 product are: (i) 100 x 10 in 10 mL 6 Tregs / bag (10 x 10 6 Tregs / ml), (ii) ≥ 60% CD4 + CD25 + (T-regulatory phenotype), and (iii) ≤10% CD4 - CD8 + (T-cytotoxic / 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.

[0115] In some embodiments, the CK0802.CXCR4 product is CD25 +Through selection and further enrichment for CXCR4, and after a culture period of 10-12 days, the release criteria for the CK0802.CXCR4 product are (i) 100 x 10 in 10 mL; 6 Tregs / bag (10 x 10 6 Tregs / ml), (ii) ≥ 60% CD4 + CD25 + (T-regulatory phenotype), (iii) ≥60% CD4 + CD25 + CXCR4 + (bone marrow-homing subtype), and (iv) ≤10% CD4 - CD8 + (T-cytotoxic / suppressive phenotype). In some embodiments, the CK0802.CXCR4 product is administered to a subject to treat myelofibrosis, aplastic anemia, or immune thrombocytopenia. In some embodiments, the CK0802.CXCR4 product is administered to a subject monthly for up to six months.

[0116] In some embodiments, the CK0802.α4β7 product is CD25 + Through selection and further enrichment for α4β7, and after a culture period of 8-10 days, the release criteria for CK0802.α4β7 product is (i) 100 x 10 in 10 mL. 6 Tregs / bag (10 x 10 6 Tregs / ml), (ii) ≥ 60% CD4 + CD25 + (T-regulatory phenotype), (iii) ≥60% CD4 + CD25 + α4β7 + (gastrointestinal homing subtype), and (iv) ≤10% CD4 - CD8 +(T-cytotoxic / suppressive phenotype). In some embodiments, the CK0802.α4β7 product is administered to a subject to treat gastrointestinal graft-versus-host disease or inflammatory bowel disease. In some embodiments, the CK0802.α4β7 product is administered to a subject in the following dosing regimen: (i) induction: weekly for up to 4 weeks, and (ii) maintenance: monthly for up to 6 months.

[0117] In some embodiments, the CK0802.CD11a product is CD25 + Through selection and further enrichment for CD11a, and after a culture period of 8-10 days, the release criteria for the CK0802.CD11a product are (i) 100 x 10 in 10 mL 6 Tregs / bag (10 x 10 6 Tregs / ml), (ii) ≥ 60% CD4 + CD25 + (T-regulatory phenotype), (iii) ≥60% CD4 + CD25 + CD11a + (neuron-homing subtype), and (iv) ≤10% CD4 - CD8 + (T-cytotoxic / suppressive phenotype). In some embodiments, the CK0802.CD11a product is administered to a subject to treat amyotrophic lateral sclerosis, multiple sclerosis, or a demyelinating neuropathy. In some embodiments, the CK0802.CD11a product is administered to a subject in the following dosing regimen: (i) induction: weekly for up to 4 weeks, and (ii) maintenance: monthly for up to 6 months.

[0118] The cord blood unit selection criteria for various populations of human Treg cells are provided in Figures 29 and 30.

[0119] The cellular starting material for CK0802 is a single unit of umbilical cord blood (CBU) from a normal, healthy, unrelated donor. For the production of clinically relevant Treg cell doses, CD4 + CD25 +In some embodiments, the 14-day manufacturing process includes ex vivo enrichment and expansion of Treg cells with a CD4 phenotype. + 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 transported to the clinical site for infusion.

[0120] CK0802 is polyclonal, expresses a broad V-beta repertoire, and expresses highly 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.

[0121] 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 the 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 10x10 in injectable cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). 6 It is a suspension of live cells containing the active pharmaceutical ingredient CK0802 suspended at a cell concentration of Treg cells / mL.

[0122] An example of the composition of a CK0802 formulation is provided in Table 2. [Table 2]

[0123] Further disclosed herein are pharmaceutical compositions comprising a population of activated human Treg cells and one or more pharmaceutically or veterinarily acceptable carriers, diluents, excipients, or vehicles.

[0124] The terms "pharmaceutically acceptable" and "veterinarily acceptable" refer to a pharmaceutically or veterinarily acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each component must be "pharmaceutically acceptable" or "veterinarily acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation. It must also be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. (Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition, Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005, and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Pre-formulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004).

[0125] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their 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).

[0126] It will be understood that the therapeutic entities of the present disclosure will be administered with appropriate carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, and the like. Numerous 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 by Blaug and 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 of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be suitable for treatment and therapy according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation, the formulation is physiologically compatible, and the administration route is tolerable.See also Baldrick P. "Pharmaceutical excipient 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 for additional information related to formulations, excipients, and carriers familiar to pharmaceutical chemists.

[0127] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where 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 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 a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. The prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or 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.

[0128] Sterile injectable solution can be prepared by incorporating the required amount of active ingredient into suitable solvent, and optionally with one or combination of the above-listed ingredients, and then filter sterilization.Generally, dispersion is prepared by incorporating this active compound into a sterile vehicle that contains basic dispersion medium and other necessary ingredients from above-listed ingredients.For the sterile powder that prepares sterile injectable solution, the preparation method is vacuum drying and freeze-drying, thereby obtaining the powder of active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0129] In some embodiments, the active ingredient can 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 can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0130] As used herein, unit dosage form refers to a physically discrete unit suitable as a single dose for the subject to be treated, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present disclosure are determined and directly depend on the inherent characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such active compounds for the treatment of individuals.

[0131] An example of the composition of the final product (consisting of the active ingredient suspended in the 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 cells 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 the phenotype of regulatory T cells. [Table 3]

[0132] In some embodiments, the final formulated product is provided for use in a sealed 300 mL polyvinyl chloride (PVC) plastic blood bag with a port that can be accessed with the plastic spike of a conventional intravenous (IV) administration set used for administration to a patient.

[0133] In some embodiments, excipients used to formulate the final product may include: [Table 4]

[0134] 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, comprising a composition described herein (e.g., a container, pack, or dispenser) together with instructions for use or administration. Also provided are articles of manufacture comprising a container containing any of the populations of activated human Treg cells described herein and instructions for use.

[0135] Treatment 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-Barré 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).

[0136] 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.

[0137] In some embodiments, cord blood-derived Tregs may exhibit one or more of the following properties to generate an anti-inflammatory effect: 1) blocking direct engagement with recipient antigen-presenting cells (APCs) and interaction with T effector (Teff) cells (i.e., by suppressing pro-inflammatory immune cells through direct interaction); 2) release of inhibitory cytokines including transforming growth factor β (TGFβ), interleukin-10 (IL-10), and interleukin-35 (IL-35); 3) depletion of IL-2 supply to Teffs leading to apoptosis; and / or 4) playing a role in granzyme / perforin production (i.e., secreting granzyme B or perforin, thereby inhibiting the proliferation of 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.

[0138] The dosage of Treg cells in the final product can be expressed as the number of cells per kg of subject body weight. Determining 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 x 10 of the subject's body weight. 5~ Approximately 1×10 8 Treg cells / kg, or approximately 1 x 10 6~ Approximately 1×10 7 In some embodiments, the cell dose for any of the methods described herein may be: Dose level 1: approximately 1 x 10 6 Treg cells / kg Dose level 2: approximately 3 x 10 6 Treg cells / kg Dose level 3: approximately 1 x 10 7 Treg cells / kg

[0139] In some embodiments, a fixed dose can be administered that is not dependent on the subject's weight. In some embodiments, the dose is about 1 x 108 Activated human Treg cells ~approximately 3 x 10 8 For example, the dose may be about 1 x 10 Treg cells. 8 , about 3×10 8 , or approximately 1 × 10 9 The cells may be activated human Treg cells.

[0140] In some embodiments, an effective amount of the population of activated human Treg cells is administered intravenously to the subject.

[0141] In some embodiments, a single effective dose of the population of human Treg cells is administered to a subject. In some embodiments, multiple effective doses of the population of activated human Treg cells are administered to a 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 approximately every 4-6 weeks. In some embodiments, Treg cells may be administered weekly for a 4-week period, followed by monthly administration for a period of at least 6-9 months (i.e., 6, 7, 8, or 9 months).

[0142] In some embodiments, after administration of an effective amount of the population of activated human Treg cells, the level of circulating inflammatory cytokines in the subject is reduced compared to the level of circulating inflammatory cytokines in the subject before administration. In some embodiments, the circulating inflammatory cytokine is interleukin-6 (IL-6), interferon gamma (IFNγ), or tumor necrosis factor-alpha (TNFα).

[0143] In some embodiments, prior to treatment, the subject's serum biomarkers are tested to determine whether the subject will respond to an effective amount of a population of activated human Treg cells. In some embodiments, after treatment, the subject's serum biomarkers are tested to determine correlation with clinical response. In some embodiments, serum biomarkers are tested serially to determine whether subsequent re-treatment with Treg cells is necessary.

[0144] In some embodiments, diphenhydramine is administered to a 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 about 30 minutes prior to administration of an effective amount of a population of activated human Treg cells.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] Also provided herein are methods for treating or preventing GVHD in a subject, the methods comprising administering to the subject (i) 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, 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.

[0149] 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 host 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) typically 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 recipients. (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) develops after the first 100 days after transplantation and is characterized by systemic inflammation and tissue destruction affecting multiple organs, particularly the intestine, 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 thought to be primarily a Th1 / Th17-driven process, while cGVHD is thought 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 can 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 leukemia cells (see Edinger et al., Nat Med 9(9):1144-50 (2003)).

[0150] Current strategies to minimize GVHD require long-term immunosuppressive therapy with drugs such as calcineurin inhibitors (CNIs), cyclosporine, and tacrolimus. However, this long-term immunosuppression results in delayed immune function and poses the risk of infectious complications and post-transplant lymphoproliferative disease. In some embodiments, provided herein are methods 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.

[0151] A xenogeneic mouse model of GVHD can be used to assess the function of cord blood-derived regulatory T cells in the treatment of GVHD. (See Parmar et al., Cytotherapy 16(10:90-100(2013)).

[0152] Bone marrow failure syndrome (BMF) Provided herein are methods for treating or preventing bone marrow failure syndrome (BMF) in a subject, the methods 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. In some embodiments, an effective amount of a fresh single dose of a Treg cell product (e.g., CK0801) is administered to treat or prevent BMF.

[0153] 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.

[0154] Bone marrow fibrosis (BMF) refers to a reduction in the production of one or more major hematopoietic lineages, 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 syndrome, 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.

[0155] 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.

[0156] AA is characterized by pancytopenia in peripheral blood (PB), and bone marrow (BM) aplasia in AA is characterized by CD8+ / - deficiency of BM hematopoietic progenitors. + Cytotoxic T cells, CD4 + BMF is a syndrome characterized by attacks by autoreactive cytotoxic T cells, such as Th1 and Th17 cells (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)).

[0157] Mechanisms of immune-mediated hematopoietic destruction include interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and the uptake of autologous CD34 by T cell populations. + These include Th1-polarized responses that result in direct toxicity to cells and excessive production 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 this context, AA is a specific autoimmune disease, with BM being the primary target organ, due to an excess of cytotoxic autoreactive T cells combined with defective and deficient regulatory T cells, which causes abnormal T cell immune homeostasis.

[0158] Also provided herein are methods of treating acquired idiopathic aplastic anemia in a subject, wherein 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).

[0159] The diagnosis of acquired AA is This 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)).

[0160] The AA response criteria (see Killick et al., Br J Haematol 172(2):187-207 (2016)) can be used to determine the response of subjects with AA to the treatments described herein, as shown in the table below. [Table 5]

[0161] 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.

[0162] 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 can 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)). Dysregulated immune function is a recognized fact in MDS. (See Fozza et al., Exp Hematol 37(8):947-55 (2009)). Among possible mechanisms, inhibition of T cell-mediated hematopoiesis has been recognized as a typical feature, especially in low-risk, 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 particularly operative in the hypoplastic form of MDS (HMDS) (See Tuzuner et al., Br J Haematol 91(3):612-17(1995)), which 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)).

[0163] Patients with MDS have a decreased CD4 to CD8 ratio, multiple activated CD8 + These include proliferation of T cell clones 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 account for the large number of apoptotic bone marrow cells seen in these patients. (See Selleri et al., Cancer 95(9):1911-22(2002)).

[0164] 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 9 It is established based on (ii) the presence of significant myelodysplasia or excess blasts or typical cytogenetic abnormalities, and (iii) the exclusion of other differential diagnoses. (See Barrett et al., Semin Hematol 37(1):15-29 (2000)). Common peripheral blood findings include macrocytic anemia, reticulocytopenia, neutropenia with hypocellular neutrophils (pseudo-Pelger-Huett), circulating immature myeloid cells including myeloblasts, and thrombocytopenia.

[0165] The International Working Group (IWG) response criteria (see Cheson et al., Blood 108(2):419-25 (2006)) can be used to determine the response of subjects with MDS to the treatments described herein, as shown in the table below. [Table 6]

[0166] 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 + is.

[0167] Also provided herein are methods for treating or preventing PMF in a subject, the methods comprising administering to the subject an effective amount of (i) 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, 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.

[0168] PMF is a clonal hematopoietic stem cell disorder in which 50% of patients harbor 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, resulting in clinical and laboratory manifestations of autoimmunity. (See Barosi Curr Hematol Malig Rep 9(4):331-39(2014)). This clonal bone marrow proliferation is characteristically accompanied by reactive myelofibrosis (myofibrosis) and extramedullary hematopoiesis in the spleen or multiple organs.

[0169] 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.

[0170] 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)). [Table 7]

[0171] 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 the table below, can be used to determine the response of subjects with PMF to the treatments described herein. [Table 8-1] [Table 8-2]

[0172] 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.

[0173] In some embodiments, the methods described herein improve, alleviate, or prevent one or more symptoms of SLE in a subject. In some embodiments, after administering activated human Treg cells to a subject, albumin spillover in urine is reduced, SLE cell infiltration in glomeruli is reduced, and / or hair follicles are preserved. In some embodiments, the methods described herein prolong the survival of a subject with SLE.

[0174] SLE is a chronic, multisystem inflammatory autoimmune disease. Lupus can affect many parts of the body, including the 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, kidney or CNS involvement, and / or cytopenia.

[0175] 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 administering 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.

[0176] 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 prolong the survival of a subject with inflammatory cancer.

[0177] Neuroinflammatory disorders Provided herein are methods for treating or preventing a neuroinflammatory disorder 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 Guillain-Barré syndrome or amyotrophic lateral sclerosis.

[0178] 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 prolong the survival of a subject with a neuroinflammatory disorder.

[0179] Guillain-Barré syndrome (GBS) Provided herein is a method for treating or preventing Guillain-Barré 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.

[0180] Also provided herein are methods of treating GBS in a subject, wherein the subject does not respond to treatment with intravenous immunoglobulin (IVIG) or plasma exchange.

[0181] GBS is an autoimmune disorder characterized by the rapid onset of muscle weakness due to nerve inflammation. 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.

[0182] Experimental autoimmune neuropathy (EAN) is an immune-mediated inflammatory demyelinating disorder of the peripheral nervous system and serves as an animal model of AIDP. The treatment methods 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, the infiltration of autoreactive T cells and macrophages, and the demyelination of the peripheral nervous system (Soliven, B., Autoimmune neuropathies: insights from animal models. J Peripher 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):pp. 74-97) or by adoptive transfer of sensitized T cells.

[0183] Amyotrophic lateral sclerosis (ALS) Provided herein are methods for treating or preventing amyotrophic lateral sclerosis (ALS) in a subject, the methods 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., 1 x 10 8 pieces, 3×10 8 pcs or 1 x 10 9 The method includes administering to a subject a therapeutically effective amount of activated human Treg cells.

[0184] 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.

[0185] ALS is a rare neurological disorder 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 approximately 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.

[0186] 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 high incidence of Tregs (CD4 + / CD25 + ) tend to decrease, and the rate of progression is negatively correlated with Treg cell count. Similarly, low FoxP3 mRNA levels are a predictor of rapid ALS progression. Furthermore, Tregs isolated from ALS patients have a reduced ability to suppress Th17 cell proliferation compared to healthy subjects.

[0187] COVID-19 (Coronavirus Disease)-mediated Acute Respiratory Distress Syndrome (CoV-ARDS) Provided herein are methods for treating or preventing COVID-19 (coronavirus disease) mediated acute respiratory distress syndrome (CoV-ARDS) in a subject, the methods comprising administering to a subject an effective amount of a population of 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 x 10 8 pieces or approximately 3 x 10 8 In some embodiments, the method comprises administering to a subject about 1 x 10 activated human Treg cells. 8 pieces or approximately 3 x 10 8 activated human Treg cells are administered to a subject on days 0 and 3. In some embodiments, about 1 x 10 8 pieces or approximately 3 x 10 8 Human Treg cells are administered to a 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.

[0188] In some embodiments, the subject is infected with or suspected of being infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0189] The highly pathogenic SARS-CoV-2 virus 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 involves robust viral replication and clinical symptoms, including fever and cough. The second stage of viral infection involves high fever, hypoxemia, progression to pneumonia-like symptoms, and a progressive decline in viral titer toward the end of the infection. The third stage of viral infection involves 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 radiography.

[0190] Uncontrolled cytokine storm may contribute to the susceptibility of some subjects infected with SARS-CoV-2 to respiratory complications. 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 preclinical lung injury models, injection of CB-Treg cells resulted in: i) a decrease in inflammatory T cells, ii) a decrease in alveolar hemorrhage, iii) regeneration of the lung epithelium and alveoli, and iv) a decrease in inflammatory cytokines, including IL-17 and IL-6, associated with CoV-ARDS.

[0191] 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.

[0192] 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 suppressive cytokines (e.g., TGF-β, IL-6, IL-10, IL-17, IL-18, or IL-33).

[0193] In some embodiments, the human Treg cells used in these treatment methods express CCR4, a homing marker for lung tissue involved in trafficking to sites of CoV-ARDS-associated inflammation.

[0194] 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.

[0195] 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.

[0196] The following examples are presented to provide those of 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 contemplated, 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. [Example]

[0197] Example 1: Producing expanded populations of activated regulatory T cells from umbilical cord blood Cryopreserved human umbilical 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 automatically washed using a Sepax device (Biosafe) with a starting volume of 25 ml, a final volume of 100 ml, and 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 into the cord blood wash bag.

[0198] For washing purposes, the basic wash medium was 20 ml of 25% HSA in 1000 ml of PBS / EDTA buffer, the working wash medium was 300 ml of basic wash buffer, 50 mg of magnesium chloride (MgCl2), and 2500 units of DNase, and the modified medium was X-Vivo15 medium (Lonza), 10 ml of GlutaMAX-1, and 100 ml of thawed human AB serum. After the automated wash was completed, the washed cord blood cells were subjected to an additional manual wash using the working wash medium to make up a final volume of 200 ml, and the reconstituted cells were centrifuged at 300 g for 10 minutes at room temperature. Finally, the washed cells were diluted to 100 × 10 in 0.09 ml of working wash medium. 6 The cells were resuspended to a concentration of 0.01 mg / mL for a total nucleated cell (TNC) count of 0.01 mg / mL.

[0199] 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 at 4°C for 30 minutes. Following the incubation step, the cells were transferred to 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.

[0200] 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 10cm 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 gas-permeable incubator and incubated for a total of 14 days, with the 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 using a magnetic particle concentrator. The cells were then resuspended in the final medium.

[0201] Cells were sampled at various points during the manufacturing process and their characteristics are shown in Table 9. [Table 9]

[0202] As shown in Figure 1, the 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. Because 7AAD generally appears to be excluded from viable cells, viability is assessed using 7-aminoactinomycin D (7AAD), a fluorescent intercalator that undergoes a spectral shift upon binding to DNA. 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 exhibit only low levels of fluorescence.

[0203] 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 the initiation of cell culture. The results are shown in Table 10. [Table 10]

[0204] As shown in Figures 2A and 2B, expanded activated Treg cells are suppressive, exhibiting 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 increased 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 HLA barriers (Figure 3). Expanded activated Tregs exhibit a Gaussian (polyclonal) distribution of the T cell receptor Vβ repertoire (Figure 6).

[0205] Expanded activated Treg cells maintain their suppressive properties even in the presence of steroids. Figures 7A and 7B show that Treg cells maintain their suppressive properties in the presence of dexamethasone. The effect of prednisone on the viability of Treg and Tcon cells is shown below. [Table 11] [Table 12]

[0206] 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.

[0207] Total of 50 x 10 per 5ml vial 6 10 x 10 cells per ml 6The expanded population of harvested activated human Treg cells was centrifuged at 400 g for 10 minutes at 4°C. The total cell number was calculated using an automated cell counter, and the total cell number was determined to be 50 x 10 cells. 6 The number of cryovials was estimated by dividing by the number of cells. Subsequently, a maximum of 50 x 10 cells per 5 ml cryovial was obtained 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 you to click here to continue" was displayed. Upon mixing with the frozen stock solution, up to 50 × 10 cells were cryopreserved. 6 Cryovials containing each cell were placed in a controlled-rate freezer using a freezing algorithm, allowing the rate at which the cells were frozen to be regulated, avoiding cell death and maintaining 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.

[0208] Cryopreserved activated Treg cells exhibit a consistent phenotype and are capable of immunosuppression similar to freshly activated Treg cells (Figures 8A-8C). Cryopreserved activated Treg cells exhibit high Helios expression (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.

[0209] 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 GVHD model 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 before intravenous infusion of human PBMCs, mice received sublethal total body irradiation (300 cGy from a 137Cs source delivered over 1 minute by a JL Shepherd and Associates Mark I-25 Irradiator, San Fernando, CA). Mice were evaluated 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 body weight effects (Figure 9B).

[0210] 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 distribute 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).

[0211] Example 4: Treatment of systemic lupus erythematosus with cryopreserved umbilical cord blood-derived regulatory T cells Utilizing a xenogeneic mouse model of systemic lupus erythematosus (SLE) (Andrade et al., Arthritis Rheum. 2011 Sep;63(9):2764-2773), we transplanted 3 × 10 peripheral blood mononuclear cells derived from systemic lupus erythematosus 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 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 intravenously injected into SLE xenografts via the tail vein once a week 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 examined for creatinine and albumin quantification. Histopathological examination of harvested organs was performed at the planned euthanasia time point at 13 weeks.

[0212] 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 Treg expression 9 weeks after engraftment of SLE-PBMCs. +The administration of activated Treg cells 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 demonstrated sustained weight gain and lower GVHD scores. Four weekly injections of activated Treg cells also reduced urinary albumin levels (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 reduced the concentration of human sCD40L in SLE mice. Furthermore, weekly injections of activated cryopreserved Tregs increased circulating CD8 + A persistent decrease in effector T cells (Fig. 20A) and CD8 + This resulted in a decrease in the infiltration of effector T cells (Figure 20B). Histopathological results from the two index cases in each group showed that Treg recipients had a decreased T cell (CD3 + ) and B cells (CD20 + ) and showed a reduction in lymphocyte infiltration into the glomeruli and renal parenchyma compared with the control group.

[0213] 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 isolated from 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 Hematopathology Laboratory (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); and CFSE-stained RPMI8226 (3 × 10 5 cells); or negative control GFP-labeled HL-60 (1.5 × 10 5 cells) or CFSE-stained Nalm6 (6 × 10 5 CB Treg cells (1 × 10 cells) were 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 (0.01%) were resuspended in 750 μL of medium 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™).

[0214] To understand the influence of CB Treg cells on myeloma cell trafficking, we set up a transwell experiment and seeded target cells 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: CB Treg cells or, as a positive control, Teff cells. This measure was taken to isolate the myeloma-specific effects of CB Tregs. CB Tregs were able to prevent the trafficking of MM1.S (Figure 40B; p<0.01) and RPMI8226 (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).

[0215] 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 x 10 7Mice were intravenously injected 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. Subsequently, mice were imaged as previously described (Parmar et al., Cytotherapy, 2014.16(1):pp.90-100). Mice were bled once a week. Plasma samples were sent to Eve Technologies (Calgary, AB, Canada) for measurement of 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 using a BD FACSCanto™ II. At the time of euthanasia, bone marrow and spleens were collected.

[0216] Survival rates were 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 means of three or more groups were compared by one-way analysis of variance followed by the Bonferroni test for multiple comparisons. These values ​​are expressed as the mean and standard error of the mean. A P value of <0.05 was considered statistically significant. All statistical analyses and graph generation were performed using GraphPad Prism 7.0 (San Diego, CA).

[0217] To understand the effect of CB Tregs on preventing myeloma engraftment, 3 × 10 cells were used to allow tumor development (control group). 6 A heteromyeloma mouse model in which MM1.S cells were intravenously injected. In the treatment group, CB Tregs (1 × 10) were added 1 day before the injection of myeloma cells. 7Mice were injected with 1000 cells (1000 cells). Mice were weighed twice weekly, and weights remained similar in both groups until week 3 after tumor inoculation, when weight loss was observed in "myeloma alone" mice, with a significant difference evident at the time of euthanasia (Figure 21A). Myeloma burden was quantified in peripheral blood, with circulating CD38 expression levels by day 28 in the control group compared to Treg recipients, with the difference becoming 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).

[0218] Mice were imaged weekly using noninvasive 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, as quantified by BLI, in CB Treg recipients was significantly delayed compared with that in the control group over the observation period (Figure 21D).

[0219] Because myeloma cells thrive in the inflammatory tumor microenvironment and interleukin-6 (IL-6) has been implicated as a key driver of myeloma disease progression (Harmer et al. Front Endocrinol (Lausanne), 2018, 9:788), we investigated the influence of CB Tregs on this inflammatory cytokine. 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 was measured in the "myeloma alone" group and continued to increase until week 5. Ultimately, the increased tumor burden and 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 tissue was measured in harvested organs and compared between the two groups. Myeloma cells were barely detectable in the bone marrow of Treg recipients compared with 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).

[0220] The data support the hypothesis that a single injection of CB Treg cells before myeloma cell injection confers a sufficient growth advantage that allows suppression of inflammatory signals generated by myeloma cells in vivo, as indicated by the lack of IL-6 production that ultimately creates 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 effects of CB Treg cells.

[0221] Impact on established myeloma disease Method: 3×10 6 GFP-labeled MM.1S cells were injected into NSG mice, followed by 5 × 10 on day +14. 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 monitored every other day for body weight and GVHD scores. Non-invasive bioluminescence imaging (BLI) was performed serially. Blood was collected weekly for cellular analysis and cytokine assays. At the time of euthanasia, blood, spleen, and bone marrow were collected 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 to a heteromyeloma model in the presence or absence of CB Treg cells. To promote the anti-tumor effect of BiTE®, all mice were supplemented with Pan T cells. The experimental design is shown in Figure 61E. [Table 13]

[0222] Results: Both Tcon and Tcon + Treg recipients maintained their body weight compared with the myeloma alone or myeloma + Treg groups (Figure 61A). The addition of Treg did not interfere with the Tcon-mediated anti-myeloma effect and prevented delayed relapse (Figures 61B-61D). The addition of Treg + BiTE® resulted in a similar degree of tumor control compared with mice treated with BiTE® alone (Figure 61F). The addition of Treg did not interfere with the BiTE®-mediated anti-myeloma effect. The addition of Treg attenuated BiTE®-induced weight loss (Figure 61G), with corresponding higher GVHD scores (Figure 61H).

[0223] 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 potentially effective in preventing GVHD, although efficacy in both preclinical and clinical studies appears to be highly dependent on the in vivo ratio of Tregs to T cells. Current strategies to minimize GVHD require long-term immunosuppressive therapy with drugs such as calcineurin inhibitors (CNIs), cyclosporine, and tacrolimus. However, this prolonged immunosuppression results in delayed immune function and increases 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.

[0224] 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.

[0225] 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 treatment will be enrolled in these studies. The study will determine the highest possible dose that is safe to administer and whether CK0801 improves the symptoms of bone marrow failure syndromes.

[0226] Participants eligible for this study are unable or unwilling to be treated with standard treatments, or have failed standard treatments.

[0227] Main purpose The primary objective is to determine the dose-limiting toxicity of CK0801, defined as 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

[0228] secondary purpose Preliminary evaluation of disease-specific responses Duration of disease-specific response

[0229] 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.

[0230] Groups and interventions [Table 14]

[0231] research design A standard 3+3 Phase I statistical design was utilized, with three patients enrolled at dose level 1:1 × 10 6 If no dose-limiting toxicity (DLT) is observed, the dose will be increased to dose level 2: (range) >1 × 10 / kg for the next cohort of 3 patients. 6 / kg~1×10 7 If no DLT is observed, the dose will be escalated to dose level 3: (range) >1 × 10 7 / kg~1.5×10 7 / kg.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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 after DSMB review of previously dosed cohorts.

[0236] If the eligibility criteria are met, the subject will be consented and enrolled in the study.

[0237] 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 T cell line with a collection of Tregs and the final product in Plasma-Lyte A and 0.5% human serum albumin (HSA). + Manufactured using a Treg immunomagnetic selection and 14-day culture expansion process. The final cell product is released only after a formal lot release process, which includes review of all available test results. Lot release criteria include 7AAD viability of ≥ 70%, %CD4 + CD25 + Cell purity, %CD4 <10% - / CD8 + cells, <100 / 3×10 6 Testing included anti-CD3 / anti-CD28 Ab bead count of 100 cells, Gram stain for "no microorganisms", <5EU / kg endotoxin, sterility (sampled 48-72 hours prior to final formulation) negative, and mycoplasma negative.

[0238] Cord blood discovery, selection, and shipping to manufacturing facilities Cord blood units provided to Cellenkos, Inc. for the production of CK0801 will be obtained from independently 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 predetermined eligibility criteria.

[0239] Upon consent, 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 six antigens in the HLA-A, HLA-B, and DRB1 loci. A list is provided to the principal investigator (PI). The sponsor and PI select the appropriate cord blood units based on predetermined criteria.

[0240] After cord blood units are selected, the sponsor's clinical coordinator arranges shipping and transportation logistics, and the units are shipped to Cellenkos' GMP manufacturing facility. Upon arrival at the manufacturing facility, 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), coordinated with the subject's planned infusion schedule.

[0241] Prior to the infusion, the sponsor's clinical coordinator and on-site clinical team are responsible for arranging the infusion of CK0801 at the prescribed time point and time frame. CK0801 must be administered within 8 hours of final formulation.

[0242] The sponsor's clinical coordinator will arrange for the transportation of CK0801 to the clinical site. The on-site clinical team will be responsible for receiving, accepting, preparing, and administering CK0801.

[0243] Formulation and Stability CK0801 is formulated to the final cell dose in Plasmalyte + 0.5% human serum albumin (HSA) buffer. Infusion of CK0801 should occur within 8 hours of final formulation.

[0244] 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 maintained at 15°C to 30°C prior to infusion.

[0245] toxicity Although infusion of cord blood-derived regulatory T cells has previously been shown to be safe, subjects should be monitored during the CK0801 infusion, per standard clinical practice. Recommended timing of vital signs on each infusion day: pre-infusion, 15 minutes after initiation of infusion, 30 minutes after initiation of infusion, 1 hour after initiation of infusion, 2 hours after initiation of infusion, and then per standard clinical practice.

[0246] Vital signs include temperature, respiration, blood pressure, and pulse.

[0247] Administration route 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 normal saline.

[0248] CK0801 injection The study will examine infusions of CK0801 at three different dose levels and will use a standard 3+3 Phase I statistical design.

[0249] 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 x 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 x 10 7 / kg IBW.

[0250] 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.

[0251] If there are ≥ 2 DLTs at dose levels 2 or 3, the previous dose level will be defined as the MTD. If there are ≥ 2 DLTs at dose level 1, the Data Safety Monitoring Board (DSMB) will review and evaluate continuation of the study.

[0252] The MTD will be determined when 6 patients are treated at a dose level with <2 DLTs.

[0253] Patients are premedicated with diphenhydramine (Benadryl®) 50 mg IV piggyback (IVPB) and acetaminophen 650 mg orally 30 minutes prior to the infusion of CK0801. CK0801 is infused by gravity flow over 15-30 minutes through 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.

[0254] Study population selection Inclusion criteria 1. Subjects who meet diagnostic criteria for bone marrow failure syndrome, including aplastic anemia, myelodysplastic syndrome, or myelofibrosis. 2. HLA-matched (≥3 / 6 in HLA-A, HLA-B, and HLA-DRB1) cord blood units available in the CK0801 generation. 3. The target audience is 18 years of age or older. 4. Bilirubin ≤ 2xULN and SGPT(ALT) ≤ 2xULN (unless Gilbert 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 birth control, 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.

[0255] Exclusion criteria 1. Subject received an investigational drug within 4 weeks prior to CK0801 infusion. 2. Subject has received 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 antimicrobial agents 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. Subject is pregnant or breastfeeding. 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 protocol requirements.

[0256] 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.

[0257] Subjects who experience study-related death or documented disease progression with subsequent alternative treatment will be considered treatment failures and treated as censored observations at the time of the event, without further data collection. Subjects who withdraw informed consent or discontinue the study due to noncompliance will also be censored at that time.

[0258] Results judgment method Main outcome measures: 1. 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 count 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 [Timeframe: 30 days from injection] Secondary outcome determination method: 2. Preliminary assessment of disease-specific response to treatment and duration of response [Timeframe: 12 months] Other pre-specified outcome measures: 3. Assess bone marrow (BM) immune reconstitution and inflammatory cytokines Bone marrow samples will be collected at baseline and at scheduled follow-up in the post-treatment setting to analyze immune reconstitution and inflammatory cytokines [Timeframe: 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. [Timeframe: 12 months]

[0259] Results of a phase 1 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 with bone marrow failure failed to show an early efficacy signal. [Table 15]

[0260] Figure 42 provides a description of subjects receiving treatment in the Phase 1 clinical trial. A summary of the clinical data is shown in Figures 43 and 44.

[0261] 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. 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 are shown in Figures 46A and 46B. Inflammatory cytokine levels are shown in Figures 47 and 48. Patients showed a decrease in JAK2 mutation burden (Figure 46B) and splenomegaly (Figure 49), which correlated with the SDF1α-CXCR4 axis (Figure 48). Bone marrow evaluations of patients before (PRE) and after (POST) Treg cell administration are shown in the table below. [Table 16] [Table 17-1] [Table 17-2]

[0262] 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 (orally) BID (twice daily). Inflammatory cytokine levels are shown in Figure 51. Bone marrow assessments of patients before (PRE) and after (POST) Treg cell administration are shown in the table below. [Table 18] [Table 19] [Table 20]

[0263] Patient 3 was a 19-year-old female diagnosed with acquired aplastic anemia and was 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 before (PRE) and after (POST) Treg cell administration are shown in the table below. [Table 21] [Table 22] [Table 23]

[0264] 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 receiving hATG+CSA+steroids+eltombopag+Peg-filgrastim. The patient's platelet transfusion requirements over time are shown in Figure 55. The patient's PRBC (packed red blood cell) transfusion requirements over time are shown in Figure 56. The patient's bone marrow assessments before (PRE) and after (POST) Treg cell administration are shown in the table below. [Table 24] [Table 25] [Table 26]

[0265] Patient 5 was a 62-year-old woman diagnosed with primary myelofibrosis (essential thrombocythemia (ET)). 6 Patients were treated with Treg cells / kg. Bone marrow evaluations of patients before (PRE) and after (POST) Treg cell administration are shown in the table below. [Table 27] [Table 28] [Table 29]

[0266] 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 6Patients were treated with 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- (PRE) and post- (POST) Treg cell administration are shown in the table below. [Table 30] [Table 31] [Table 32]

[0267] conclusion Cohort 1 (dose = 1 x 10 6 cells / kg) and Cohort 2 (dose = 3 x 10 6 No SAEs were observed in the six patients treated with 1000 mg / kg of EGFR. 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 Patient #3's red blood cell and platelet transfusion requirements improved. Response durable for 4 weeks. Patient #4 improved with red blood cell and platelet transfusions. Response durable for 4 weeks. ●Improvement of chronic pain in patient #5. Durable for 4 weeks Patient #6's red blood cell and platelet transfusions improved. Evaluation performed at 4 weeks. ●Improvement of bone marrow cellularity in patients #1 and #2 ●Improvement of myelodysplasia in patients #3 and #4 ● In all cases except patient #4 (stable), the M:E ratio decreased

[0268] Example 7: Evaluation of safety and efficacy for administering cord blood-derived regulatory T cells in the treatment of treatment-resistant Guillain-Barré syndrome This study will investigate whether it is safe and practical to administer CK0801 (a regulatory T cell product derived from umbilical cord blood) to patients with Guillain-Barré syndrome (GBS). Additionally, the highest possible dose that is safe to administer will be determined. Similarly, this study will also investigate whether CK0801 can improve GBS symptoms.

[0269] 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.

[0270] Registration Up to 18 adult patients (aged 18-70 years) will be enrolled.

[0271] Eligibility Inclusion criteria: 1. Subject meets diagnostic criteria for Guillain-Barré syndrome (GBS). 2. HLA-matched (≥3 / 6 in HLA-A, HLA-B, and HLA-DRB1) cord blood units available in the CK0801 generation. 3. Target ages 18-70. 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 remained unchanged 1 week after IVIG or PE treatment. 7. Bilirubin ≤ 2xULN and ALT ≤ 2xULN (excluding Gilbert 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 birth control, 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 consent form due to disease-related complications (e.g., upper limb paralysis), a legally authorized representative (LAR) will be used. Subject will sign the consent form as soon as possible.

[0272] Exclusion criteria: 1. Subject received immunotherapy, chemotherapy, biologic or investigational drug 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 antimicrobial agents 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. Subject is pregnant or breastfeeding. 6.HIV seropositive 7. Subjects unable to provide consent or who, in the investigator's opinion, are unlikely to fully comply with protocol requirements.

[0273] Groups and interventions [Table 33]

[0274] 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 a patient receives 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

[0275] Primary endpoint The primary endpoint of this study is 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

[0276] Results judgment method Main outcome measures: 1. Collect the number of adverse events and serious adverse events to provide a preliminary assessment of the safety of infusing CK0801 in patients with Guillain-Barré syndrome (GBS) who are unresponsive to standard treatment with intravenous immunoglobulin. [Timeframe: 30 days from injection] 2. Dose-limiting toxicity is defined to include any of the following events (each beginning 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 [Timeframe: 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 any changes in the patient's peripheral blood characteristics [Timeframe: Screening, Day 0 and 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 inflammatory cytokines in the peripheral blood following CK0801 infusion on day 0 [Timeframe: Screening, Day 0 and 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 healthy (0) to dead (6) [Timeframe: Screening, Day 0 and Week 1, Week 2, Week 4, Week 12, Week 24] 6. Assessment of potential changes in the Overall Neuropathy Limitation Scale (ONLS) (questionnaire) The Global Neuropathy Limitations Scale (ONLS) is a questionnaire that determines a patient's symptoms in their arms (numbness, tingling, weakness) and legs (walking, running, gait changes, need for a wheelchair) when performing normal daily activities. The arm scale ranges from 0 (normal) to 5 (impairment in both arms prevents 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 and Week 1, Week 2, Week 4, Week 12, Week 24] 7. Assessment of potential changes in the Rasch-constructed global disability scale (questionnaire) A questionnaire measuring the relationship between daily activities and 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 upper or lower body, getting dressed, 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 and 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 and 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 and 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 and Week 1, Week 2, Week 4, Week 12, Week 24] 11. Assessment of potential changes in the Rasch-Reconstructed 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 and 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 patients' mobility, self-care activities, other usual activities (housework, 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 and 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 in Form A and the follow-up questionnaire in 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, antecedents (e.g., cold, gastroenteritis), pain type (e.g., myalgia, arthralgia, neuropathic pain), pain location, arm or leg weakness, reflex status, sensory impairment, ataxia, and forced vital capacity. This form allows users to predict whether the patient will require ventilation or be able to walk within six months. [Timeframe: Screening, Day 0 and Week 1, and Week 2] 14. Assessment of potential changes in the patient's condition based on a comparison of the entry questionnaire in Form A and the follow-up questionnaire in Form B (questionnaires at weeks 4, 12, and 24) This form (questionnaire) provides a mechanism for documenting changes in the patient's condition since enrollment, using the same information as the entry questionnaire. [Timeframe: Screening, Day 0 and 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) provides a mechanism for documenting changes in the patient's condition since enrollment, using the same information as the entry questionnaire. [Timeframe: Screening, Day 0 and 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) provides a mechanism for documenting changes in the patient's condition since enrollment, using the same information as the entry questionnaire. [Timeframe: Screening, Day 0 and Week 1, Week 2, Week 4, Week 12, Week 24]

[0277] 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 population The target population for this study is patients who are ineligible for sibling donor hematopoietic stem cell transplantation (MSD HSCT) or predicted to respond poorly to immunosuppressive therapy (IST).

[0278] Registration Up to 18 adult patients will be enrolled.

[0279] dosage Dose level 1: CK0802.CXCR4 IV 1×10 8 individual cells Dose level 2: CK0802.CXCR4 IV 3×10 8 individual cells

[0280] Primary endpoint The primary endpoint of this study was time to cytokine release syndrome, an infusion reaction, and / or death within 30 days.

[0281] Secondary endpoint The secondary endpoint of this study was hematologic improvement.

[0282] 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 population 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 greater than 0.3 points / month

[0283] Registration 52 adult patients.

[0284] investigational drug CK0802.CD11a (CD11a-enriched cryopreserved, multiple-dose, cord blood-derived regulatory T cells)

[0285] dosage Induction: Weekly IV CK0802.CD11a dose x 4 at the following dose levels: Cohort 1: CK0802.CD11a IV 1×10 8 Treg cells Cohort 2: CK0802.CD11a IV 3×10 8 Treg cells

[0286] Maintenance: 6 additional doses every 4 weeks for both cohorts Groups and interventions [Table 34]

[0287] 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 ○Efficacy * ALS Functional Rating Scale-Revised (ALSFRS-R) *Forced vital capacity (FVC) *Handheld Dynamometry (HHD) for Muscle Strength ○Exploration *Inflammatory biomarkers and immune reconstitution

[0288] Endpoint Investigation Clinical response: 1. A 6-point improvement over 6 months on the ALS Functional Rating Scale-Revised (ALSFRS-R). 2. Rate of change in functional status 3. Change in the slope of the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score 4. Changes in forced vital capacity (FVC), and 5. Changes in muscle strength

[0289] ALSFR responder analysis (proportion of subjects who improved after treatment compared to before treatment) Pre-specified responder analyses will examine both the percent improvement per month and absolute point improvement in the post-treatment ALSFRS-R slope compared with the pre-treatment slope. · Patient ratings of 25%, 50%, 75%, and 100% improvement. Clinically significant = 25% Clinically significant = 50% Statistical significance defined as a one-sided p value <0.05 using Fisher's exact test. Assessments performed at weeks 4, 8, 12, 16, and 24

[0290] Clinical trial design: Phase Ib clinical trial Research design: This will be a Phase 1, 3+3 design, single ascending dose, safety and tolerability study of CK0802.CD11a. A fixed-dose strategy tests two dose levels. *Low dose: 1×10 8 individual cells; *High dose: 3×10 8 individual 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 a total (minimum) of 12 patients.

[0291] 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, IL-7, 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 vivo 11 [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 the association between markers of neuronal integrity and neuroinflammation and may provide valuable insight into disease mechanisms in ALS. (Ratai, NeuroImage:Clinical 20(2018)357-364)

[0292] 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 multi-dose cord blood-derived regulatory T (Treg) cells (CK0802) for the treatment of CoV-ARDS is shown in Figure 27. There were three treatment arms: Treatment arm 1: placebo. Treatment arm 2: 1 x 10 8 CK0802 cells; treatment group 3: 3 × 10 8 There are CK0802 cells. The dosing regimen is three 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.

[0293] 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.

[0294] Endpoints and Correlations Primary endpoint The two main results are: Regimen-related severe Grade 3 or greater toxicity within 48 hours of CK0802 infusion (NCI-CTCAE (National Cancer Institute Common Terminology Criteria for Adverse Events) V4.0) • 28-day successful treatment, defined as S28 = [alive and not intubated 28 days after the date of first infusion].

[0295] Secondary endpoint Secondary outcomes recorded from the date of first infusion up to 28 days after the date of first infusion included: Time to extubation 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 rate

[0296] Planned non-endpoint correlation analyses Laboratory correlations and general assessments [ Time Frame: Days 0, 3, 7, 11, 21, 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)

[0297] Research results CK0802 (cryopreserved umbilical cord blood-derived regulatory T cells) refers to allogeneic, pre-made regulatory T cells that are cryopreserved and ready to be used as an intravenous infusion for the treatment of ARDS.

[0298] Sources and Pharmacology Tregs are isolated from allogeneic, unrelated umbilical cord blood (CB) units obtained from eligible public, licensed, or unlicensed US CB banks based on predetermined 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 wash solutions. CB cells are resuspended in a MgCl2, rHuDNAse, and 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 VIVO15 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 1 × 10 cells. 6 CD25+ cells are suspended at a concentration of 1.0 x 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 of IL-2 (Proleukin, Chiron Corporation, Emeryville, CA). Cells are maintained at a density of 1.0 x 106 viable nucleated cells / mL and cultured at 37°C in 5% CO2 for 14 days.

[0299] On day 14 of culture, cells were harvested, Dynabeads were removed by magnetic separation, and Treg cells were resuspended in Plasmalyte + 0.5% I buffer. The Treg product (CK0802) must pass the release criteria for infusion, including: 7AAD viability of ≥ 70%, purity of ≥ 60% CD4 + CD25+ cells, <10% CD4 - / CD8 + cells, 3 x 10 6 anti-CD3 / anti-CD28 mAB bead count of <100 per cell, Gram stain of "no organisms," and endotoxin of <5EU / kg.

[0300] The harvested cells are then aliquoted into clinical cryobags, cryopreserved using a controlled rate freezer, and labeled as a CK0802 product containing cell dose.

[0301] CK0802 injection In this study, CK0802 dose level 1 = 1.0 × 10 8 cells and dose level 2: 3.0 × 10 8 Infusion of CK0802 cells is considered. Patients are premedicated 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.

[0302] Placebo injection Cryopreserved excipients are infused into 30 ml cryobags. Patients are premedicated with Benadryl® 50 mg via IVPB (IV piggyback) 30 minutes prior to the placebo infusion. The placebo should be infused by gravity within 30 minutes, preferably within 1 hour of thawing. The placebo is compatible with standard blood product tubing and filters.

[0303] Study population The study will recruit subjects who meet all of the inclusion / exclusion criteria detailed below.

[0304] 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]: a ratio of arterial oxygen partial pressure (PaO2) to fraction of inspired oxygen (FiO2) of 200 mmHg or less assessed with a positive end-expiratory pressure (PEEP) of >5 cmH2O. 3. Intubation for less than 120 hours 4. ≥ 18 years old 5. Ability to provide informed consent or a duly appointed health care representative with the authority to provide informed consent.

[0305] 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 history of illness that, in the opinion of the study investigator, may confound the results of the study or pose additional risk to the patient through 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 concurrently active moderate to severe infections at the start of treatment enrollment that, in the investigator's opinion, may affect the safety of CK0802 treatment. 6. Patients who have been 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 above stress doses is excluded: hydrocortisone >50 mg every 6 hours or other systemic steroids >0.5 mg / kg / day intravenous methylprednisolone or >60 mg oral methylprednisolone daily. 10.Administration of investigational cell therapy drugs

[0306] Evaluation during research Clinical evaluation Baseline assessment on the day of infusion of the first dose of assigned treatment group: CK0802 or placebo, then subsequent daily assessments for 14 days after infusion of the first dose of assigned treatment group: CK0802 or placebo.

[0307] Ventilation parameters: Daily average record with range (min and max values) Plateau pressure ·Tidal volume Mean airway pressure FiO2 PEEP ·PaO2 / FiO2 ratio C-STAT / Compliance (static lung compliance)

[0308] Arterial blood gases: Daily average record with range (min and max values) Arterial pH Oxygen partial pressure (PaO2) Partial pressure of carbon dioxide (PaCO2) Bicarbonate (HCO3) Oxygen saturation (O2Sat)

[0309] Vital signs: Daily average record with range (min and max values) Body temperature Blood Pressure (BP): Systolic and diastolic BP measurements ·Respiration rate Heart rate

[0310] SOFA Score The Sequential Organ Failure Assessment (SOFA) score predicts ICU mortality based on laboratory and clinical data.

[0311] SOFA Score [Table 35]

[0312] Example 11: Effect of cryopreservation on the cytosuppressive activity of regulatory T cells derived from umbilical cord blood Cryopreserved umbilical cord blood (CB) Treg cells (CK0802) were shown to have comparable suppressive function compared to fresh CB Treg cells. Tcon cells showed a high proliferation rate 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 observed 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). A similar degree of suppression of the expanding Tcon cells was demonstrated by the lack of dilution of CellTrace™ Violet dye.

[0313] 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 adverse effects of ruxolitinib on Treg cells in patients.

[0314] 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 were placed into secondary culture, the Treg cells lost their suppressive function over time. The addition of ruxolitinib restored the suppressive function.

[0315] Ruxolitinib and CB Treg cells synergistically suppress 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 proinflammatory cytokine IFNγ. IFNγ levels 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 IFNγ release from SLE-PBMC (Figure 32). Camptothecin is used as a control to demonstrate that nonspecific inflammatory stimuli do not increase IFNγ secretion from CB Treg cells.

[0316] A xenogeneic murine 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 1 × 10 donor peripheral blood (PB) mononuclear cells (MNC) were injected. 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 body weight, GVHD score, and survival. Serial blood samples were taken for analysis of cellular compartments and cytokine assays.

[0317] Combination treatment reduced GVHD scores (Figure 34A) and improved survival in mouse models (Figure 34B). Ruxolitinib improved the persistence of CB Tregs in mouse models (Figures 35A-C). Ruxolitinib, both 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).

[0318] Ruxolitinib enhanced the IL-7 and IL-15 survival signaling pathways and attenuated the IL-4 inhibitory signaling pathway 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 IL-15 (Figure 36B) levels increased. Increased IL-7 availability enhanced Treg survival, stabilized Treg molecular signatures, enhanced surface IL-2Rα expression, and improved IL-2 binding (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).

[0319] 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 the administration of CB Treg cells alone, ruxolitinib alone, and the combination of ruxolitinib and CB Treg cells.

[0320] The combination of ruxolitinib and CB Treg cells increased the secretion of anti-inflammatory cytokines in a xenogeneic mouse GVHD model. Plasma levels of IL-1RA (Figure 38A), IL-1a3 (Figure 38B), and IL-12p70 (Figure 38C) increased. The combination of ruxolitinib and CB Treg cells improved hematological parameters in a xenogeneic mouse GVHD model. Administration of both ruxolitinib and CB Treg cells increased platelet levels (Figure 39B). On day 14, a significant decrease in hemoglobin levels was evident in the ruxolitinib alone group compared to the increase in hemoglobin levels in the CB Treg + ruxolitinib group (Figure 39A).

[0321] Example 13: Effect of cord blood-derived regulatory T cells on chimeric antigen receptor T cells The xenogeneic lymphoma model was performed using 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 NSG mice were 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 the CD19-CAR T group, with three mice per group. Mice were followed for weight, GVHD score, and survival. Serial imaging was performed to assess tumor burden using noninvasive bioluminescence. Blood was collected serially for cellular analysis and cytokine assays.

[0322] As shown in Figure 59A, in vivo expansion of GFP-labeled Raji cells was evident in all mice by day +4. CD19-CAR T, but not mock-CAR T cells, reduced tumor burden on day +11. However, by 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-CAR T 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 on 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).

[0323] 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 the CAR T cells.

[0324] Numbered Embodiments Without limiting the scope of the appended claims, the present disclosure describes the following numbered embodiments.

[0325] 1. At least about 1 x 108 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are CD4 + CD25 + and (ii) ≤10% CD4 - CD8 + a population of human Treg cells, wherein the human Treg cells are immunosuppressive.

[0326] 2. At least about 1 x 10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are CD4 + CD25 + and (ii) ≥ 60% CD4 + CD25 + CXCR4 + and (iii) ≤10% CD4 - CD8 + and A population of human Treg cells, wherein the human Treg cells are immunosuppressive.

[0327] 3. At least about 1 x 10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are CD4 + CD25 + and (ii) ≥ 60% CD4 + CD25 + α4β7 + and (iii) ≤10% CD4 - CD8 + and A population of human Treg cells, wherein the human Treg cells are immunosuppressive.

[0328] 4. At least about 1 x 10 8 A population of human Treg cells comprising human Treg cells, (i) ≥ 60% of which are CD4 + CD25 + and (ii) ≥ 60% CD4 + CD25 + CD11a + and (iii) ≤10% CD4 - CD8 +a population of human Treg cells, wherein the human Treg cells are immunosuppressive.

[0329] 5. At least about 1 x 10 9 5. The population of any one of embodiments 1 to 4, comprising human Treg cells.

[0330] 6. The population of any one of embodiments 1-5, wherein the human Treg cells are determined to be immunosuppressive by an assay using carboxyfluorescein succinimidyl ester intracellular staining dye or CellTrace™ Violet intracellular staining dye.

[0331] 7. A method for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit, 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 endogenous Treg cells using a dual selection method based on cell surface expression; d) culturing isolated CD25 in a gas-permeable incubator in a 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 10 days, up to 12 days, or up to 14 days; + Treg cells are expanded ex vivo, with the medium replaced approximately every 48 hours, and activated CD25 + generating and expanding a population of Treg cells; e) extracting the activated CD25 + harvesting the cells to produce an expanded population of activated human Treg cells.

[0332] 8. The method of embodiment 7, wherein a single cord blood unit is used.

[0333] 9. The method of embodiment 7, wherein two to four pooled cord blood units are used.

[0334] 10. The method of any one of embodiments 7 to 9, wherein said reagent that specifically binds to CD25 is an anti-CD25 antibody or an antigen-binding fragment thereof.

[0335] 11. The method of any one of embodiments 7 to 10, wherein the reagent that specifically binds to CD25 is conjugated to a solid support.

[0336] 12. The method of embodiment 11, wherein the solid support is a magnetic microbead.

[0337] 13. The method of any one of embodiments 7 to 11, wherein the reagents that specifically bind to CD3 and CD28 comprise an anti-CD3 antibody or antigen-binding fragment thereof, and an anti-CD28 antibody or antigen-binding fragment thereof.

[0338] 14. The method of any one of embodiments 7 to 12, wherein the reagents that specifically bind to CD3 and CD28 comprise anti-CD3-coated beads and anti-CD28-coated beads.

[0339] 15. The method of embodiment 14, wherein the anti-CD3-coated beads and anti-CD28-coated beads are in a 1:1 ratio.

[0340] 16.CD25 + 16. The method of embodiment 14 or 15, and wherein the anti-CD3 and anti-CD28 coated beads are in a 1:1 ratio.

[0341] 17. The method of any one of embodiments 7-16, wherein the effective amount of IL-2 is up to about 1000 IU / ml.

[0342] 18. The method of any one of embodiments 7 to 17, wherein the effective amount of IL-2 is about 1000 IU / ml.

[0343] 19. CD25 isolated in step c) + 19. The method according to any one of embodiments 7 to 18, wherein the Treg cells are suspended in a medium comprising IL-2 immediately before step d).

[0344] 20. In step e), approximately 1 × 10 6 CD25 + 20. The method of any one of embodiments 7 to 19, wherein cells / ml are cultured.

[0345] 21. In step e), the cells are first cultured in a 10 cm 2 21. The method of any one of embodiments 7 to 20, wherein the cells are cultured in a gas-permeable incubator having a membrane surface area of ​​1000 μg / cm 2 .

[0346] 22.The culture is then transferred to a 100cm 2 22. The method of embodiment 21, wherein the cells are transferred to a gas permeable incubator having a membrane surface area of ​​1000 nm.

[0347] 23. The method according to any one of embodiments 7 to 22, wherein in step d) the culture is not mixed and resuspended.

[0348] 24. Approximately 1×10 9 pieces ~ approx. 2×10 9 Activated CD25 + 24. The method of any one of embodiments 7 to 23, wherein the cells are harvested after 14 days of culture.

[0349] 25. The method according to any one of embodiments 7 to 24, wherein in step a), the cryopreserved human umbilical cord blood unit is thawed in a single step in a water bath.

[0350] 26. The method of any one of embodiments 7 to 25, wherein step b) does not include manual washing.

[0351] 27. The method of any one of embodiments 7 to 26, wherein step b) is carried out in a solution comprising PBS, EDTA, and about 0.5% human serum albumin.

[0352] 28. A double ferromagnetic column method is used in step c) to isolate CD25 + 28. The method of any one of embodiments 7 to 27, wherein Treg cells are isolated.

[0353] 29. A method for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit, said method comprising: a) thawing a cryopreserved human umbilical cord blood unit in a single step in a water bath; b) diluting and washing the thawed cord blood units in a solution containing PBS, EDTA, and 0.5% human serum albumin in a functionally closed system without manual washing; c) CD25 using a dual ferromagnetic column method + Isolating endogenous Treg cells using a dual selection method based on cell surface expression; d) isolated activated CD25 in the presence of approximately 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. + Treg cells are expanded ex vivo, with the medium replaced approximately every 48 hours, and activated CD25 + Generate a population of Treg cells, CD25 + Treg cells and anti-CD3 and anti-CD28 coated beads at a ratio of 1:1; Allowing the culture to grow without mixing and resuspending; e) extracting the activated CD25 + harvesting the cells to produce an expanded population of activated human Treg cells.

[0354] 30. The method of any one of embodiments 7-29, wherein the method further comprises cryopreserving the expanded population of activated human Treg cells.

[0355] 31. A population of activated human Treg cells produced by the method according to any one of embodiments 7 to 30, wherein the Treg cells are at least 90% CXCR4 + That is, a group.

[0356] 32. Treg cells are at least 90% CXCR4 + 7. The population according to any one of embodiments 1 to 6, wherein

[0357] 33. Treg cells are at least 95% CXCR4 + , at least 95% CD45RA + and at least 80% CD45RO + 33. The population of any one of embodiments 1 to 6, 31 and 32, wherein

[0358] 34. Treg cells are also at least 95% CD95 + , at least 95% HLADR + , at least 95% alpha4beta7 + , at least 15% are CXCR3hi + , at least 95% CCR6 + , at least 95% CD54 + , at least 95% CD11A + , at least 85% CD45RARO + , at least 80% CTLA4 + , at least 80% are GPR83 + , and at least 80% CD62L + 34. The population according to any one of embodiments 1 to 6 and 31 to 33, wherein

[0359] 35. Treg cells are at least 95% CXCR4 + , at least 95% CD45RA + , at least 80% CD45RO + , at least 95% CD95 + , at least 95% HLADR + , at least 95% alpha4beta7 +, at least 15% are CXCR3hi + , at least 95% CCR6 + , at least 95% CD54 + , at least 95% CD11A + , at least 85% CD45RARO + , at least 80% CTLA4 + , at least 80% are GPR83 + and at least 80% CD62L + 35. The population according to any one of embodiments 1 to 6 and 31 to 34, wherein

[0360] 36. The population according to any one of embodiments 1-6 and 31-35, wherein the Treg cells exhibit high expression of FOXP3 and low expression of RORγt.

[0361] 37. The population according to any one of embodiments 1-6 and 31-36, wherein the Treg cells maintain their polyclonal T cell receptor Vβ (TCR Vβ) repertoire.

[0362] 38. The population according to any one of embodiments 1 to 6 and 31 to 37, wherein the Treg cells are cryopreserved before use.

[0363] 39. A method for cryopreserving an expanded population of activated human regulatory T (Treg) cells produced from at least one cryopreserved human umbilical cord blood unit, said 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 endogenous Treg cells using a dual selection method based on cell surface expression; d) culturing isolated CD25 in a gas-permeable incubator in a 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 10 days, up to 12 days, or up to 14 days; +Treg cells were expanded ex vivo, with the medium replaced approximately every 48 hours, and the cells were then incubated with activated CD25 + generating and expanding a population of Treg cells; e) Activated CD25 from the culture medium + harvesting the cells to produce an expanded population of activated human Treg cells; f) cryopreserving the expanded population of activated human Treg cells.

[0364] 40. A method for treating or preventing graft-versus-host disease 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 5-28 and 39, or the population of any one of embodiments 1-6 and 31-38.

[0365] 41. A method for treating or preventing graft-versus-host disease in a subject, the method comprising administering to the subject (i) an effective amount of a population of activated human Treg cells produced by the method of any one of embodiments 7-30 and 39, or the population of any one of embodiments 1-6 and 31-38, and (ii) ruxolitinib.

[0366] 42. 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 a method according to any one of embodiments 7 to 30 and 39, or a population according to any one of embodiments 1 to 6 and 31 to 38.

[0367] 43. The method of embodiment 42, wherein the bone marrow failure syndrome is aplastic anemia, primary myelofibrosis, or myelodysplastic syndrome.

[0368] 44. A method for treating or preventing primary myelofibrosis in a subject, the method comprising administering to the subject (i) an effective amount of a population of activated human Treg cells produced by the method of any one of embodiments 7-30 and 39, or the population of any one of embodiments 1-6 and 31-38, and (ii) ruxolitinib.

[0369] 45. 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 a method described in any one of embodiments 7 to 30 and 39, or a population described in any one of embodiments 1 to 6 and 31 to 38.

[0370] 46. ​​A method for treating or preventing multiple myeloma 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 described in any one of embodiments 7 to 30 and 39, or a population described in any one of embodiments 1 to 6 and 31 to 38.

[0371] 47. 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 described in any one of embodiments 7 to 30 and 39, or a population described in any one of embodiments 1 to 6 and 31 to 38.

[0372] 48. The method of embodiment 47, wherein the neuroinflammatory disorder is Guillain-Barré syndrome, amyotrophic lateral sclerosis, multiple sclerosis or a demyelinating neuropathy.

[0373] 49. A method for treating or preventing a respiratory disease, disorder, or condition associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection 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 7-30 and 39, or a population of any one of embodiments 1-6 and 31-38.

[0374] 50. The method of embodiment 49, wherein the respiratory disease, disorder or condition is COVID-19 (coronavirus disease)-mediated acute respiratory distress syndrome (CoV-ARDS).

[0375] 51. A method for treating or preventing cytokine release syndrome (CRS) 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 described in any one of embodiments 7 to 30 and 39, or a population described in any one of embodiments 1 to 6 and 31 to 38.

[0376] 52. The method of embodiment 51, wherein CRS is associated with chimeric antigen receptor T cell therapy.

[0377] 53. The method of any one of embodiments 40-52, wherein an effective amount of the population of activated human Treg cells is administered intravenously to the subject.

[0378] 54. An effective amount of a population of activated human Treg cells is administered to a subject in an amount of about 1 x 10 6 ~Approx. 1×10 7 54. The method of any one of embodiments 40 to 53, wherein the total number of Treg cells per kg is 100.

[0379] 55. An effective amount of activated human Treg cells comprises a population of approximately 1 x 10 8 Treg cells ~ approx. 3 x 10 8 The method of any one of embodiments 40 to 53, wherein the cell is a Treg cell.

[0380] 56. The method of any one of embodiments 40-55, wherein multiple doses of an effective amount of the population of activated human Treg cells are administered to the subject.

[0381] 57. The method of embodiment 56, wherein three doses or four doses are administered to the subject.

[0382] 58. The method of embodiment 56 or 57, wherein the dose is administered to the subject approximately every 4 to 6 weeks.

[0383] 59. The method of any one of embodiments 40-58, wherein following administration of an effective amount of the population of activated human Treg cells, circulating inflammatory cytokine levels in the subject are reduced compared to circulating inflammatory cytokine levels in the subject prior to administration.

[0384] 60. The method of any one of embodiments 40-59, wherein prior to treatment, the subject's serum biomarkers are examined to determine whether the subject will respond to an effective amount of a population of activated human Treg cells.

[0385] 61. The method of any one of embodiments 40-60, wherein following treatment, the subject's serum biomarkers are examined to determine correlations with clinical response.

[0386] 62. The method of embodiment 61, in which serum biomarkers are serially tested to determine whether subsequent retreatment with Treg cells is required.

[0387] 63. The method of any one of embodiments 40-62, wherein the population of activated human Treg cells is prepared from one or more umbilical cord blood units of blood type compatible with the subject.

[0388] 64. The method of any one of embodiments 40 to 63, wherein the population of activated human Treg cells is prepared from umbilical cord blood units that are at least 3 out of 6 HLA (human leukocyte antigen) matches to the subject.

[0389] 65. The method of any one of embodiments 40-62, wherein the population of activated human Treg cells is prepared from an umbilical cord blood unit that is not HLA-matched to the subject.

[0390] 66. Use of a population according to any one of embodiments 1 to 6 and 31 to 38 in the preparation of a medicament.

Claims

1. At least about 1 x 10 ８ A population of human Treg cells comprising human Treg cells, (i) ≥ 60% CD4 ＋ CD25 ＋ and (ii) ≤10% CD4 － CD8 ＋ and A population of human Treg cells, wherein said human Treg cells are immunosuppressive.

2. At least about 1 x 10 ８ A population of human Treg cells comprising human Treg cells, (i) ≥ 60% CD4 ＋ CD25 ＋ and (ii) ≥ 60% CD4 ＋ CD25 ＋ CXCR4 ＋ and (iii) ≤10% CD4 － CD8 ＋ and A population of human Treg cells, wherein said human Treg cells are immunosuppressive.

3. At least about 1 x 10 ８ A population of human Treg cells comprising human Treg cells, (i) ≥ 60% CD4 ＋ CD25 ＋ and (ii) ≥ 60% CD4 ＋ CD25 ＋ α4β7 ＋ and (iii) ≤10% CD4 － CD8 ＋ and A population of human Treg cells, wherein said human Treg cells are immunosuppressive.

4. At least about 1 x 10 ８ A population of human Treg cells comprising human Treg cells, (i) ≥ 60% CD4 ＋ CD25 ＋ and (ii) ≥ 60% CD4 ＋ CD25 ＋ CD11a ＋ and (iii) ≤10% CD4 － CD8 ＋ and A population of human Treg cells, wherein said human Treg cells are immunosuppressive.

5. At least about 1 x 10 ９ The population of any one of claims 1 to 4, comprising human Treg cells.

6. 6. The population of any one of claims 1-5, wherein the human Treg cells are determined to be immunosuppressive by an assay using a carboxyfluorescein succinimidyl ester intracellular staining dye or CellTrace™ Violet intracellular staining dye.

7. 1. A method for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit, said method 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 endogenous Treg cells using a cell surface expression-based dual selection method; d) culturing the isolated CD25 cells in a gas-permeable incubator in a 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 10 days, up to 12 days, or up to 14 days. ＋ and ex vivo expansion of Treg cells, wherein the medium is changed approximately every 48 hours to induce activation of CD25 ＋ Producing and expanding a population of Treg cells; e) isolating the activated CD25 from the culture medium. ＋ harvesting the cells to produce an expanded population of activated human Treg cells.

8. 8. The method of claim 7, wherein a single cord blood unit is used.

9. 8. The method of claim 7, wherein two to four pooled cord blood units are used.

10. The method of any one of claims 7 to 9, wherein the reagent that specifically binds to CD25 is an anti-CD25 antibody or an antigen-binding fragment thereof.

11. The method of any one of claims 7 to 10, wherein the reagent that specifically binds to CD25 is conjugated to a solid support.

12. The method of claim 11 , wherein the solid support is a magnetic microbead.

13. The method of any one of claims 7 to 11, 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.

14. The method of any one of claims 7 to 12, wherein the reagents that specifically bind to CD3 and CD28 comprise anti-CD3 coated beads and anti-CD28 coated beads.

15. 15. The method of claim 14, wherein the anti-CD3 coated beads and the anti-CD28 coated beads are in a 1:1 ratio.

16. CD25 ＋ 16. The method of claim 14 or 15, wherein the cells and the anti-CD3 and anti-CD28 coated beads are in a 1:1 ratio.

17. The method of any one of claims 7 to 16, wherein the effective amount of IL-2 is up to about 1000 IU / ml.

18. The method of any one of claims 7 to 17, wherein the effective amount of IL-2 is about 1000 IU / ml.

19. The CD25 isolated in step c) ＋ The method of any one of claims 7 to 18, wherein the Treg cells are suspended in a medium containing IL-2 immediately before step d).

20. In step e), about 1×10 ６ CD25 ＋ The method according to any one of claims 7 to 19, wherein cells / ml are cultured.

21. In step e), the cells are first cultured in a 10 cm ２ The method according to any one of claims 7 to 20, wherein the culture is carried out in a gas-permeable culture vessel having a membrane surface area of ​​1000 m / s.

22. The culture was then grown to a depth of 100 cm ２ 22. The method of claim 21, wherein the culture medium is transferred to a gas permeable incubator having a membrane surface area of ​​1000 nm.

23. The method according to any one of claims 7 to 22, wherein in step d) the culture is not mixed and resuspended.

24. Approximately 1×10 ９ pieces ~ approx. 2×10 ９ Activated CD25 ＋ The method of any one of claims 7 to 23, wherein the cells are harvested after 14 days of culture.

25. 25. The method of any one of claims 7 to 24, wherein in step a) the cryopreserved human umbilical cord blood unit is thawed in a single step in a water bath.

26. The method of any one of claims 7 to 25, wherein step b) does not include manual washing.

27. 27. The method of any one of claims 7 to 26, wherein step b) is carried out in a solution comprising PBS, EDTA, and about 0.5% human serum albumin.

28. A double ferromagnetic column method is used in step c) to isolate CD25 ＋ The method according to any one of claims 7 to 27, wherein Treg cells are isolated.

29. 1. A method for producing an expanded population of activated human regulatory T (Treg) cells from at least one cryopreserved human umbilical cord blood unit, said method comprising: a) thawing the cryopreserved human umbilical 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 0.5% human serum albumin in a functionally closed system without manual washing; c) CD25 using a double ferromagnetic column method ＋ Isolating endogenous Treg cells using a cell surface expression-based dual selection method; d) culturing the isolated CD25 cells in the presence of about 1000 IU / ml interleukin-2 (IL-2) 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 in a medium containing the cells. ＋ and ex vivo expansion of Treg cells, wherein the medium is changed approximately every 48 hours to induce activation of CD25 ＋ generating a population of Treg cells; CD25 ＋ Treg cells and the anti-CD3 and anti-CD28 coated beads are in a 1:1 ratio; Allowing the culture to grow without mixing and resuspending; e) isolating the activated CD25 from the culture medium. ＋ harvesting the cells to produce an expanded population of activated human Treg cells.

30. The method of any one of claims 7 to 29, wherein the method further comprises cryopreserving the expanded population of activated human Treg cells.

31. A population of activated human Treg cells produced by the method of any one of claims 7 to 30, wherein the Treg cells are at least 90% CXCR4 ＋ That is, a group.

32. The Treg cells are at least 90% CXCR4 ＋ The population according to any one of claims 1 to 6,

33. The Treg cells are at least 95% CXCR4 ＋ , at least 95% CD45RA ＋ and at least 80% CD45RO ＋ 33. The population according to any one of claims 1 to 6, 31 and 32, wherein

34. The Treg cells further comprise at least 95% CD95 ＋ , at least 95% HLADR ＋ , at least 95% alpha4beta7 ＋ , at least 15% are CXCR3hi ＋ , at least 95% are CCR6 ＋ , at least 95% CD54 ＋ , at least 95% CD11A ＋ , at least 85% CD45RARO ＋ , at least 80% CTLA4 ＋ , at least 80% are GPR83 ＋ , and at least 80% CD62L ＋ 34. The population according to any one of claims 1 to 6 and 31 to 33, wherein

35. The Treg cells are at least 95% CXCR4 ＋ , at least 95% CD45RA ＋ , at least 80% CD45RO ＋ , at least 95% CD95 ＋ , at least 95% HLADR ＋ , at least 95% alpha4beta7 ＋ , at least 15% are CXCR3hi ＋ , at least 95% are CCR6 ＋ , at least 95% CD54 ＋ , at least 95% CD11A ＋ , at least 85% CD45RARO ＋ , at least 80% CTLA4 ＋ , at least 80% are GPR83 ＋ and at least 80% CD62L ＋ The population according to any one of claims 1 to 6 and 31 to 34, wherein

36. 36. The population of any one of claims 1 to 6 and 31 to 35, wherein the Treg cells exhibit high expression of FOXP3 and low expression of RORγt.

37. 37. The population of any one of claims 1-6 and 31-36, wherein the Treg cells maintain their polyclonal T cell receptor Vβ (TCR Vβ) repertoire.

38. 38. The population of any one of claims 1 to 6 and 31 to 37, wherein the Treg cells are cryopreserved prior to use.

39. 1. A method for cryopreserving an expanded population of activated human regulatory T (Treg) cells produced from at least one cryopreserved human umbilical cord blood unit, the method 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 endogenous Treg cells using a cell surface expression-based dual selection method; d) culturing the isolated CD25 cells in a gas-permeable incubator in a 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 10 days, up to 12 days, or up to 14 days. ＋ and ex vivo expansion of Treg cells, wherein the medium is changed approximately every 48 hours to induce activation of CD25 ＋ Producing and expanding a population of Treg cells; e) isolating the activated CD25 from the culture medium. ＋ harvesting the cells to produce an expanded population of activated human Treg cells; f) cryopreserving said expanded population of activated human Treg cells.

40. 40. A method for treating or preventing graft-versus-host disease in a subject, said method comprising administering to said subject an effective amount of the population of activated human Treg cells produced by the method of any one of claims 5 to 28 and 39 or the population of any one of claims 1 to 6 and 31 to 38.

41. 40. A method for treating or preventing graft-versus-host disease in a subject, the method comprising administering to the subject (i) an effective amount of the population of activated human Treg cells produced by the method of any one of claims 7 to 30 and 39 or the population of any one of claims 1 to 6 and 31 to 38, and (ii) ruxolitinib.

42. 40. A method for treating or preventing bone marrow failure syndrome in a subject, said method comprising administering to said subject an effective amount of the population of activated human Treg cells produced by the method of any one of claims 7 to 30 and 39 or the population of any one of claims 1 to 6 and 31 to 38.

43. 43. The method of claim 42, wherein the bone marrow failure syndrome is aplastic anemia, primary myelofibrosis, or myelodysplastic syndrome.

44. 40. A method for treating or preventing primary myelofibrosis in a subject, the method comprising administering to the subject (i) an effective amount of the population of activated human Treg cells produced by the method of any one of claims 7 to 30 and 39 or the population of any one of claims 1 to 6 and 31 to 38, and (ii) ruxolitinib.

45. 40. A method for treating or preventing systemic lupus erythematosus (SLE) in a subject, said method comprising administering to said subject an effective amount of the population of activated human Treg cells produced by the method of any one of claims 7 to 30 and 39, or the population of any one of claims 1 to 6 and 31 to 38.

46. 40. A method for treating or preventing multiple myeloma in a subject, said method comprising administering to said subject an effective amount of the population of activated human Treg cells produced by the method of any one of claims 7 to 30 and 39 or the population of any one of claims 1 to 6 and 31 to 38.

47. 40. A method for treating or preventing a neuroinflammatory disorder in a subject, said method comprising administering to said subject an effective amount of the population of activated human Treg cells produced by the method of any one of claims 7 to 30 and 39 or the population of any one of claims 1 to 6 and 31 to 38.

48. 48. The method of claim 47, wherein the neuroinflammatory disorder is Guillain-Barre syndrome, amyotrophic lateral sclerosis, multiple sclerosis or a demyelinating neuropathy.

49. 40. A method for treating or preventing a respiratory disease, disorder, or condition associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a subject, said method comprising administering to the subject an effective amount of the population of activated human Treg cells produced by the method of any one of claims 7-30 and 39, or the population of any one of claims 1-6 and 31-38.

50. 50. The method of claim 49, wherein the respiratory disease, disorder, or condition is COVID-19 (coronavirus disease)-mediated acute respiratory distress syndrome (CoV-ARDS).

51. 40. A method for treating or preventing cytokine release syndrome (CRS) in a subject, said method comprising administering to said subject an effective amount of the population of activated human Treg cells produced by the method of any one of claims 7 to 30 and 39 or the population of any one of claims 1 to 6 and 31 to 38.

52. 52. The method of claim 51, wherein the CRS is associated with chimeric antigen receptor T cell therapy.

53. 53. The method of any one of claims 40 to 52, wherein the effective amount of the population of activated human Treg cells is administered intravenously to the subject.

54. The effective amount of the population of activated human Treg cells is about 1 x 10 body weight of the subject ６ ~Approx. 1×10 ７ The method according to any one of claims 40 to 53, wherein the total number of Treg cells is 100 or more per kg.

55. The effective amount of the population of activated human Treg cells is about 1 x 10 ８ Treg cells to approximately 3 x 10 ８ The method according to any one of claims 40 to 53, wherein the Treg cells are Treg cells.

56. 56. The method of any one of claims 40 to 55, wherein multiple doses of an effective amount of said population of activated human Treg cells are administered to said subject.

57. 57. The method of claim 56, wherein three or four doses are administered to the subject.

58. 58. The method of claim 56 or 57, wherein the dose is administered to the subject about every 4 to 6 weeks.

59. 59. The method of any one of claims 40-58, wherein following administration of the effective amount of the population of activated human Treg cells, circulating inflammatory cytokine levels in the subject are reduced compared to circulating inflammatory cytokine levels in the subject prior to said administration.

60. 60. The method of any one of claims 40-59, wherein prior to treatment, serum biomarkers of the subject are examined to determine whether the subject will respond to the effective amount of said population of activated human Treg cells.

61. 61. The method of any one of claims 40 to 60, wherein following treatment, the subject's serum biomarkers are examined to determine correlations with clinical response.

62. 62. The method of claim 61, wherein the serum biomarkers are serially tested to determine whether subsequent retreatment with Treg cells is required.

63. 63. The method of any one of claims 40 to 62, wherein the population of activated human Treg cells is prepared from one or more cord blood units of a blood type compatible with the subject.

64. 64. The method of any one of claims 40-63, wherein the population of activated human Treg cells is prepared from an umbilical cord blood unit that is at least 3 out of 6 HLA (human leukocyte antigen) matches to the subject.

65. 63. The method of any one of claims 40 to 62, wherein the population of activated human Treg cells is prepared from an umbilical cord blood unit that is not HLA-matched to the subject.

66. Use of a population according to any one of claims 1 to 6 and 31 to 38 in the preparation of a medicament.

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