Polydonor CD4+ t cells expressing il-10 in combination with hematopoietic stem cells for use in a treatment of hematological malignancies

Abstract

The present disclosure provides a population of polydonor CD4IL-10 cells generated by genetically modifying CD4+ T cells from at least two different T cell donors. Further provided are methods of generating the polydonor CD4IL-10 cells and methods of using the polydonor CD4IL-10 cells for immune tolerization, treating GvHD, cell and organ transplantation, cancer, autoimmune and inflammatory diseases and other immune disorders.

Description

Attorney Docket Ref: TR1X-012WOTREATMENT OF HEMATOLOGICAL MALIGNANCIES WITH HSCT IN COMBINATION WITH POLYDONOR CD4+T CELLS EXPRESSING IL- 101. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U. S. Provisional Application No.63 / 753,800, filed February 4, 2025, which is hereby incorporated in its entirety by reference.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted in XML format and is hereby incorporated herein by reference in its entirety’. Said XML copy, created on February 3, 2026, is named Trlx012 Sequence Listing.xml, and is 31,547 bytes in size.3. BACKGROUND

[0003] Regulatory’ T cells belong to a small but important subset of T cells which maintain immunological tolerance to self and non-pathogenic antigens and maintain immune homeostasis. There are two major populations of regulatory T cells - CD4+, FOXP3+CD25+T cells (FOXP3+cells) and type 1 regulatory T (Tri) cells. Both FOXP3+and Tri cells downregulate pathogenic T-cell responses in various preclinical models for organ and pancreatic islet transplantation, graft-versus-host disease (GvHD) and various autoimmune and inflammatory diseases.

[0004] Tri cells have been shown to be effective in clinical studies. Administration of cloned, antigen-specific, autologous Tri cells to patients with ongoing moderate to severe Crohn’s disease resulted in objective, transient remissions (Desreumaux et al..Gastroenterology!. 2012; 143(5): 1207-1217. e2.). In addition, adoptive transfer of donor-derived allo-specific CD4+T cell populations enriched for Tri cells to leukemia patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT) resulted in a rapid reconstitution of the immune system and protection against microbial and viral infections, without severe GvHD. In the responder patients, long term remissions and tolerance (> 7 years) resulting in cures were achieved (Bacchetta et al., Front Immunol. 2014; 5:16).

[0005] Despite these encouraging results, the production of donor-derived or autologous Tri cells for large scale therapy for patients with high unmet medical needs is not always feasible,1IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOis very cumbersome, and also does not allow for the generation of large quantities of pure Tri cells.

[0006] Recently, Locafaro and colleagues circumvented some of these problems by transducing purified CD4+T cells from a single donor with a bidirectional lentiviral vector containing a human IL-10 gene. The resulting single-donor CD4IL-10populations shared the major functions of naturally occurring Tri cells. Like Tri cells, single-donor CD4IL-10cells produce high levels of IL- 10 and downregulate the proliferation of both allogeneic CD4+T cells and allogeneic CD8+T cells. In addition, they are cytotoxic for both normal myeloid cells (including antigen presenting cells, APC) and myeloid leukemia cells. In a humanized xeno-GvHD model, these single-donor CD4IL-10cells were shown to be effective in reducing GvHD in a humanized xeno-GvHD model while retaining graft-versus-leukemia (GvL) activity. See Locafaro et al. Mol Ther. 2017;25(10):2254-2269 and WO 2016 / 146,542.

[0007] Although it is possible to produce highly purified single-donor CD4IL-10cells for therapeutic use, there are still significant limitations, because of qualitative and quantitative differences between the various individual batches, which most likely are related to intrinsic differences between the various donors in addition to variations in the quality of huffy coats.4. SUMMARY

[0008] The present disclosure provides a new Tri -based therapy using a population of polydonor CD4IL-10cells. Poly donor CD4IL-10cells refer to CD4+T cells obtained from at least two different T cell donors and then genetically modified to comprise an exogenous polynucleotide encoding IL- 10. The T cell donors are third party donors who are neither a host to be treated with the polydonor CD4IL-10cells nor an HSC or organ transplant donor. The polydonor CD4IL-10cells are not alloantigen-specific, i.e., they have not been primed or stimulated with cells from the host before administration.

[0009] Applicant demonstrated that the polydonor CD4IL-10cells have cytokine production profiles, immune suppressive- and cytotoxic capabilities comparable to those of single-donor CD4IL-10cells. In addition, in vivo, they are more effective in preventing xeno GvHD mediated by CD4+ T cells than single-donor CD4IL-10cells, while they do not induce GvHD by themselves. Overall, the functional properties of these polydonor CD4IL-10cells both in vitro and in vivo were comparable to or better than those of single donor CD4IL-10cells.2IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0010] Based on these results, Applicant claims that polydonor allogeneic CD4IL-10cells can be used for therapeutic purposes in GvHD, cell and organ transplantation, autoimmune- and inllammatory diseases.

[0011] Further, by using third party T cells and eliminating the requirement of allo-specificity, polydonor CD4IL-10cells makes the Tri -based cell therapy available to a larger population of patients with various genetic backgrounds.

[0012] Accordingly, in a first aspect, the present disclosure provides a population of CD4+T cells that have been genetically modified to comprise an exogenous polynucleotide encoding IL- 10, wherein the CD4+T cells were obtained from at least two different T cell donors (polydonor CD4IL’10cells).

[0013] In some embodiments, the CD4+T cells were obtained from two, three, four, five, six, seven, eight, nine, or ten different T cell donors. In some embodiments, the CD4+T cells in the population collectively have six, seven, eight, nine, ten, eleven, twelve, or more different HLA haplotypes.

[0014] In some embodiments, all the CD4+T cells in the population have at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to each other. In some embodiments, all the CD4+T cells in the population have at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, all the CD4+T cells in the population have 2 / 2 match at the HLA-A locus to each other. In some embodiments, all the CD4+T cells in the population have 2 / 2 match at the HLA-B locus to each other. In some embodiments, all the CD4+T cells in the population have 2 / 2 match at the HLA-C locus to each other. In some embodiments, all the CD4+T cells in the population have at least 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci with each other. In some embodiments, all the CD4+T cells in the population have an A*02 or A*24 allele.

[0015] In some embodiments, all the CD4+T cells in the population have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match at the HLA-A, HLA-B, HLA-C. HLA-DRB1, and HLA-DQB1 loci to each other. In some embodiments, all the CD4 T cells in the population have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, all the CD4+T cells in the population have less than 2 / 2 match at the HLA-A locus to each other. In some embodiments, all the CD4+T cells3IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOin the population have less than 2 / 2 match at the HLA-B locus to each other. In some embodiments, all the CD4+T cells in the population have less than 2 / 2 match at the HLA-C locus to each other. In some embodiments, all the CD4+T cells in the population have less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci with each other.

[0016] In some embodiments, all the CD4+T cells in the population have no match at the HL A- A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to each other. In some embodiments, all the CD4+T cells in the population have no match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, all the CD4+T cells in the population have no match at the HLA-A locus to each other. In some embodiments, all the CD4+T cells in the population have no match at the HLA-B locus to each other. In some embodiments, all the CD4+T cells in the population have no match at the HLA-C locus to each other. In some embodiments, all the CD4+T cells in the population have no match at the HLA-DRB 1 and HLA-DQB 1 loci with each other.

[0017] In some embodiments, none of the CD4+T cells is immortalized. In some embodiments, the exogenous polynucleotide comprises an IL-10-encoding polynucleotide segment operably linked to expression control elements. In some embodiments, the IL- 10 is a human IL-10. In some embodiments, the IL-10 is a viral IL-10. In some embodiments, the IL- 10 is a variant of human IL- 10 having the sequence of human IL- 10 with one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications. In some embodiments, the one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications are substitution with amino acid(s) of viral IL- 10 at corresponding amino acid position(s). In some embodiment, the variant of human IL-10 has the sequence of SEQ ID NO: 8 or 9.

[0018] In some embodiments, the IL-10-encoding polynucleotide segment encodes a protein having the sequence of SEQ ID NO: 1. In some embodiments, the IL-10-encoding polynucleotide segment has the sequence of SEQ ID NO:2. In some embodiments, the expression control elements drive constitutive expression of the encoded IL-10. In some embodiments, the expression control elements drive expression of IL-10 in activated CD4+T cells. In some embodiments, the expression control elements drive tissue-specific or CD4+T cell-specific expression.

[0019] In some embodiments, the exogenous polynucleotide further comprises a sequence encoding a selection marker. In some embodiments, the selection marker is ΔNGFR. In some embodiments, the ΔNGFR has the sequence of SEQ ID NO: 3. In some embodiments,4IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOthe exogenous polynucleotide comprises a sequence of SEQ ID NO:4. In some embodiments, the exogenous polynucleotide having a sequence of SEQ ID NO: 5.

[0020] In some embodiments, the selection marker is a truncated EGFR polypeptide. In some embodiments, the selection marker is a truncated human EGFR polypeptide.

[0021] In some embodiments, the exogenous polynucleotide is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide is not integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide further comprises lentiviral vector sequences. In some embodiments, the exogenous polynucleotide is not integrated into the T cell nuclear genome.

[0022] In some embodiments, at least 70% of the CD4+T cells within the population express IL- 10. In some embodiments, at least 90% of the CD4+T cells within the population express IL-10. In some embodiments, at least 95%, 98% or 99% of the CD4+T cells within the population express IL- 10. In some embodiments the selection marker is ΔNGFR. In some embodiments, expression level of IL- 10 linearly correlates with expression level of the selection marker. In the case, IL- 10 expression level can be determined by the expression level of the selection marker.

[0023] In some embodiments, the genetically modified CD4+T cells constitutively express at least lOOpg IL- 10 per 106of the CD4+T cells / mL of culture medium. In some embodiments, the genetically modified CD4+T cells constitutively express at least 200pg, 500pg, Ing, 5ng, lOng, or 50ng IL-10 per 106of the CD4+T cells / mL. In some embodiments, the genetically modified CD4+T cells express at least lor 2ng IL-10 per 106of the CD4+T cells / mL after activation with anti-CD3 and anti-CD28 antibodies. In some embodiments, the genetically modified CD4+T cells express at least 2ng, 5ng, lOng, lOOng, 200ng, or 500ng IL- 10 per 106of the CD4+T cells / mL after activation with anti-CD3 and anti-CD28 antibodies. In some embodiments, the genetically modified CD4+T cells express IL-10 at a level at least 5-fold higher than unmodified CD4+T cells. In some embodiments, the genetically modified CD4+T cells express IL- 10 at a level at least 10-fold higher than unmodified CD4+T cells.

[0024] In some embodiments, at least 70% of the CD4+T cells within the population express the selection marker from the exogenous polynucleotide. In some embodiments, at least 90% of the CD4+T cells within the population express the selection marker from the exogenous5IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOpolynucleotide. In some embodiments, at least 95%, 98% or 99% of the CD4+T cells within the population express the selection marker from the exogenous polynucleotide.

[0025] In some embodiments, the genetically modified CD4+T cells express CD49b. In some embodiments, the genetically modified CD4+T cells express LAG-3. In some embodiments, the genetically modified CD4+T cells express TGF-β. In some embodiments, the genetically modified CD4+T cells express IFN-y. In some embodiments, the genetically modified CD4+T cells express granzyme B (GzB). In some embodiments, the genetically modified CD4+T cells express perforin. In some embodiments, the genetically modified CD4+T cells express CD18. In some embodiments, the genetically modified CD4+T cells express CD2. In some embodiments, the genetically modified CD4+T cells express CD226. In some embodiments, the genetically modified CD4+T cells express IL-22.

[0026] In some embodiments, the CD4+T cells have not been anergized in the presence of peripheral blood mononuclear cells (PBMCs) from a host. In some embodiments, the CD4+T cells have not been anergized in the presence of recombinant IL- 10 protein, wherein the recombinant IL-10 protein is not expressed from the CD4+T cells. In some embodiments, the CD4+T cells have not been anergized in the presence of DC 10 cells from a host.

[0027] In some embodiments, the CD4+T cells are in a frozen suspension. In some embodiments, the CD4+T cells are in a liquid suspension. In some embodiments, the liquid suspension has previously been frozen.

[0028] In another aspect of the present disclosure provides a pharmaceutical composition comprising:(i) the population of CD4+T cells described herein; suspended in(ii) a pharmaceutically acceptable carrier.

[0029] In yet another aspect, the present disclosure provides a method of making poly donor CD4IL-10cells, comprising the steps of:(i) pooling primary CD4+T cells obtained from at least two different T cell donors; and(ii) modifying the pooled CD4+T cells by introducing an exogenous polynucleotide encoding IL- 10,thereby obtaining the genetically-modified CD4+T cells.6IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0030] In one aspect, the present disclosure provides a method of making polydonor CD4IL-10cells, comprising the steps of:(i) obtaining primary CD4+T cells from at least two different T cell donors; and (ii) separately modifying each donor's CD41T cells by introducing an exogenous polynucleotide encoding IL-10, and then(iii) pooling the genetically modified CD4+T cells,thereby obtaining the genetically-modified CD4+T cells.

[0031] In some embodiments, the method further comprises the step, after step (i) and before step (ii), after step (ii), after step (ii) and before step (iii), or after step (iii), of:incubating the primary CD4+T cells in the presence of an anti-CD3 antibody, and anti-CD28 antibody or anti-CD3 antibody and CD28 antibody coated beads. In some embodiments, poly donor CD4IL’10T cells have been cultured in the presence of T Cell TransAct™ from Miltenyi Biotec. In some embodiments, poly donor CD4IL‘10T cells have been cultured in the presence of ImmunoCult Human T Cell Activator™ from STEMCELL Technologies.

[0032] In some embodiments, the method comprises incubating the primary CD4+T cells further in the presence of IL-2. In some embodiments, the exogenous polynucleotide is introduced into the primary CD4+T cells using a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a chimeric viral vector. In some embodiments, the viral vector is an adeno-associated viral vector. In some embodiments, the exogenous polynucleotide comprises a segment encoding IL-10 having the sequence of SEQ ID NO:1. In some embodiments, the IL-10-encoding polynucleotide segment has the sequence of SEQ ID NO:2.

[0033] In some embodiments, the exogenous polynucleotide further comprises a segment encoding a selection marker. In some embodiments, the encoded selection marker is ΔNGFR. In some embodiments, the encoded selection marker has the sequence of SEQ ID NO:3. In some embodiments, the encoded selection marker is a truncated EGFR polypeptide. In some embodiments, the encoded selection marker is a truncated human EGFR polypeptide.

[0034] In some embodiments, the method further comprises the step, after step (ii), of:isolating the genetically-modified CD4+T cells expressing the selection marker, thereby generating an enriched population of genetically-modified CD4 T cells.7IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0035] In some embodiments, at least 70% of the genetically-modified CD4+T cells in the enriched population express IL- 10. In some embodiments, at least 90%, 95%, or 98% of the genetically -modified CD4+T cells in the enriched population express IL- 10. In some embodiments, at least 70% of the genetically -modified CD4+ T cells in the enriched population express the selection marker. In some embodiments, at least 90%, 95%, or 98% of the genetically -modified CD4+T cells in the enriched population express the selection marker.

[0036] In some embodiments, the method further comprises the step of incubating the enriched population of genetically -modified CD4+T cells. In some embodiments, the step of incubating the enriched population of genetically-modified CD4+T cells is performed in the presence of anti-CD3 antibody and anti-CD28 antibody or CD3 antibody and CD28 antibody coated beads in the presence of IL-2.

[0037] In some embodiments, the method further comprises the later step of freezing the genetically-modified CD4+T cells. In some embodiments, in step (i). the primary CD4+T cells are obtained from two, three, four, five, six, seven, eight, nine, or ten different T cell donors. In some embodiments, the at least two T cell donors have at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to each other. In some embodiments, the at least two T cell donors have at least 1 / 8. 2 / 8, 3 / 8, 4 / 8, 5 / 8. 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, the at least two T cell donors have 2 / 2 match at the HLA-A locus to each other. In some embodiments, the at least two T cell donors have 2 / 2 match at the HLA-B locus to each other. In some embodiments, the at least two T cell donors have 2 / 2 match at the HLA-C locus to each other. In some embodiments, the at least two T cell donors have at least 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to each other. In some embodiments, each of the at least two T cell donors has an A*02 or A*24 allele.

[0038] In some embodiments, the at least two T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to each other. In some embodiments, the at least two T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, the at least two T cell donors have less than 2 / 2 match at the HLA-A locus to each other. In some embodiments, the at least two T cell donors have less than 2 / 28IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOmatch at the HLA-B locus to each other. In some embodiments, the at least two T cell donors have less than 2 / 2 match at the HLA-C locus to each other. In some embodiments, the at least two T cell donors have less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to each other.

[0039] In some embodiments, in step (i), the primary CD4+T cells are obtained from one or more frozen stocks. In some embodiments, in step (i), the primary CD4+T cells are obtained from unfrozen peripheral blood mononuclear cells of the at least two different T cell donors.

[0040] In some embodiments, the method further comprises the step of isolating CD4+T cells from the peripheral blood mononuclear cells. In some embodiments, the peripheral blood mononuclear cells are obtained from buffy coat or apheresis.

[0041] In another aspect, the present disclosure provides method of treating a patient, comprising the step of:administering the poly donor CD4IL-10cells or the pharmaceutical composition of the present disclosure to a patient in need of immune tolerization.

[0042] In some embodiments, the method further comprises the preceding step of thawing a frozen suspension of polydonor CD4IL-10cells.

[0043] In some embodiments, the poly donor CD4IL-10cells or the pharmaceutical composition prevents or reduces severity of pathogenic T cell response in the patient.

[0044] In some embodiments, the poly donor CD4IL-10cells or the pharmaceutical composition prevents or reduces the severity of an inflammatory or autoimmune response.

[0045] In some embodiments, the method further comprises the step of administering mononuclear cells from a hematopoietic stem cells (HSC) donor to the patient. In some embodiments, the poly donor CD4IL’10cells or the pharmaceutical composition and the mononuclear cells from a HSC donor are administered concurrently. In some embodiments, the mononuclear cells from a HSC donor are administered either prior to or subsequent to administration of the polydonor CD4IL-10cells or the pharmaceutical composition. In some embodiments, the mononuclear cells are in the PBMC. In some embodiments, the mononuclear cells are in the bone marrow. In some embodiments, the mononuclear cells are in the cord blood. In some embodiments, the mononuclear cells have been isolated from the PBMC, bone marrow or cord blood.9IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0046] In some embodiments, the method further comprises the step of:administering hematopoietic stem cells (HSC) of an HSC donor to the patient either prior to or subsequent to administration of the poly donor CD4IL-10cells or pharmaceutical composition.

[0047] In some embodiments, the HSC donor is partially HLA-mismatched to the patient. In some embodiments, the HSC donor has less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 match at the HL A- A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to the patient. In some embodiments, the HSC donor has less than 4 / 8, 5 / 8,6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to the patient. In some embodiments, the HSC donor has less than 2 / 2 match at the HLA-A, HLA-B, or HLA-C locus to the patient. In some embodiments, the HSC donor has less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to the patient.

[0048] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the patient. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to the patient. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to the patient. In some embodiments, one or more of the T cell donors have less than 2 / 2 match at the HLA-A, HLA-B, or HLA-C locus to the patient. In some embodiments, one or more of the T cell donors have less than 2 / 4, 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to the patient. In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched with the HSC donor. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to the HSC donor. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to the HSC donor. In some embodiments, one or more of the T cell donors have less than 2 / 2 match at the HLA-A, HLA-B, or HLA-C locus to the HSC donor. In some embodiments, one or more of the T cell donors have less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to the HSC donor.10IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0049] In some embodiments, the poly donor CD4IL-10cells or the pharmaceutical composition prevents or reduces severity’ of GvHD by the transplanted hematopoietic stem cells.

[0050] In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition prevents or reduces severity of pathogenic response of lymphoid cells present in the transplanted hematopoietic stem cells population.

[0051] In some embodiments, the patient has a cancer. In some embodiments, the patient has neoplastic cells. In some embodiments, the neoplastic cells express CD13, HLA-class I and CD54. In some embodiments, the neoplastic cells express CD112, CD58, or CD155.

[0052] In some embodiments, the patient has a cancer. In some embodiments, the cancer is a solid or hematological neoplasm. In some embodiments, the patient has a cancer selected from the group consisting of: Adrenal Cancer, Anal Cancer. Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain / CNS Tumors In Adults, Brain / CNS Tumors In Children, Breast Cancer, Breast Cancer In Men, Cancer of Unknown Primary’, Castleman Disease, Cervical Cancer, Colon / Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family Of Tumors, Eye Cancer, Gallbladder Cancer. Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Acute Lymphocytic (ALL), Acute Myeloid (AML, including myeloid sarcoma and leukemia cutis), Chronic Lymphocytic (CLL), Chronic Myeloid (CML) Leukemia, Chronic Myelomonocytic (CMML), Leukemia in Children, Liver Cancer, Lung Cancer, Lung Cancer with Non-Small Cell, Lung Cancer with Small Cell, Lung Carcinoid Tumor, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma In Children. Oral Cavity' and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary' Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary' Gland Cancer, Sarcoma - Adult Soft Tissue Cancer, Skin Cancer, Skin Cancer -Basal and Squamous Cell, Skin Cancer - Melanoma, Skin Cancer - Merkel Cell, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.11IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0053] In some embodiments, the cancer is a myeloid cancer. In some embodiments, the cancer is AML or CML.

[0054] In some embodiments, the patient has an inflammatory' or autoimmune disease. In some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of: type-1 diabetes, autoimmune uveitis, autoimmune hepatitis, vitiligo, alopecia areata, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus, inflammatory bowel disease, Addison's disease, Graves’ disease, Sjogren’s syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous diseases, scleroderma, Crohn’s disease, celiac disease and celiac disease.

[0055] In some embodiments, the inflammatory or autoimmune disease is Crohn’s disease, ulcerative colitis, celiac disease, type-1 diabetes, lupus, psoriasis, psoriatic arthritis, or rheumatoid arthritis.

[0056] In some embodiments, the patient has a disease or disorder involving hyperactivity of NLPR3 inflammasome. In some embodiments, the patient has type 2 diabetes, neurodegenerative diseases, cardiovascular - or inflammatory bowel disease.

[0057] In some embodiments, the patient has a disease or disorder involving increased IL- 1(3 production by activated monocytes, macrophages or dendritic cells.

[0058] In some embodiments, the patient has a disease or disorder involving increased IL- 18 production by activated monocytes, macrophages or dendritic cells.

[0059] In some embodiments, the patient has a disease or disorder involving increased mature caspase 1 production by activated monocytes, macrophages or dendritic cells.

[0060] In some embodiments, the patient has an allergic or atopic disease. In some embodiments, the allergic or atopic disease is selected from the group consisting of: asthma, atopic dermatitis, and rhinitis. In some embodiments, the patient has a food allergy'.

[0061] In some embodiments, the method further comprises the step of cell and organ transplantation to the patient, either prior to or subsequent to administration of the population of CD4+T cells or the pharmaceutical composition. In some embodiments, the poly donor CD411 -'0cells or the pharmaceutical composition prevents or reduces severity' of host rejection of the cell and organ transplants.12IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0062] In some embodiments, the method further comprises the step of transplanting iPS cell-derived cells or tissues to the patient, either prior to or subsequent to administration of the population of CD4 T cells or the pharmaceutical composition.

[0063] In some embodiments, polydonor CD4IL-10cells or the pharmaceutical composition prevents or reduces severity of host rejection of the transplantation.

[0064] In some embodiments, the method further comprises the step of administering a recombinant Adenovirus, Adeno-Associated Virus (AAV), Herpes simplex virus (HSV), Retrovirus, Lentivirus, Alphavirus, Flavivirus, Rhabdovirus, Measles virus, Newcastle disease Virus, Poxvirus, or Picomavirus to the patient, either prior to or subsequent to administration of the poly donor CD4IL‘10cells or the pharmaceutical composition. In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition reduces immune responses against the recombinant Adenovirus, Adeno-Associated Virus (AAV), Herpes simplex virus (HSV), Retrovirus, Lentivirus, Alphavirus, Flavivirus, Rhabdovirus, Measles virus, Newcastle disease Virus, Poxvirus, or Picomavirus.

[0065] In some embodiments, the patient has an excessive immune response against viral or bacterial infection. In some embodiments, the patient has a coronavirus infection. In some embodiments, the patient has organ and / or tissue damage.

[0066] In some embodiments, the method further comprises the step of administering an immunogenic therapeutic protein to the patient, either prior to or subsequent to administration of the population of polydonor CD4IL-10cells or the pharmaceutical composition. In some embodiments, the population of poly donor CD4IL-10cells, or the pharmaceutical composition reduces immune responses against the immunogenic therapeutic protein. In some embodiments, the immunogenic therapeutic protein is selected from a therapeutic antibody, a factor VIII replacement, a cytokine, and a cytokine mutein.

[0067] In some embodiments, the method further comprises the step of detecting the selection marker in a biological sample obtained from the patient, thereby detecting presence or absence of polydonor CD4IL-10T cells. In some embodiments, the biological sample is a biopsy or blood from the patient.

[0068] In one aspect, the present disclosure provides a method of treating a patient with a malignancy, comprising: administering an allo-HSCT to the patient, and administering a therapeutically effective amount of polydonor CD4IL-10cells. In some embodiments, the allo-13IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOHSCT is administered prior to administration of the polydonor CD4IL-10cells. In some embodiments, the allo-HSCT is administered after administration of the poly donor CD4IL 10cells.

[0069] In some embodiments, none of the donors of the CD4IL’10cells in the poly donor CD4IL-10cells is the donor of the HSCT.

[0070] In another aspect, the present disclosure provides a method of treating a hematological cancer, comprising: administering to a hematological cancer patient an amount of poly donor CD4IL-10cells sufficient to induce anti-cancer effects, wherein the polydonor CD411’10cells comprise CD4+T cells that have been obtained from at least two different T cell donors and then genetically modified by vector-mediated gene transfer of the coding sequence of human IL-10 under control of a constitutive or inducible promoter.

[0071] In some embodiments, the method of treating a hematological cancer comprises the step wherein the administered poly donor CD4+ T cells that are sufficient to induce anti cancer effects have been obtained from the individual donors and are first separately genetically modified by vector-mediated gene transfer of the coding sequence of human IL-10 under the control of a constitutive or inducible promoter and then pooled.

[0072] In some embodiments, the method of treating a hematological cancer comprises the step wherein the administered poly donor CD4+ T cells that are sufficient to induce anti cancer effects have been obtained from the individual donors are first pooled and then the pool is genetically modified by vector-mediated gene transfer of the coding sequence of human IL- 10 under the control of a constitutive or inducible promoter.

[0073] In some embodiments, the method further comprises the step of administering allo HSCT to the patient prior to or subsequence to administration of the poly donor CD4IL-10cells. In some embodiments, the amount of poly donor CD4IL’10cells is further sufficient to suppress or prevent graft-versus-host disease (GvHD) without suppressing graft-versus-leukemia (GvL) or graft- versus -tumor (GvT) efficacy7of the allo HSCT.

[0074] In some embodiments, the hematological cancer is a myeloid leukemia.

[0075] In some embodiments, the polydonor CD4IL-10cells target and kill cancer cells that express CD13. In some embodiments, the polydonor CD4IL-10cells target and kill cancer cells that express HLA-class I. In some embodiments, the myeloid leukemia is acute myeloid leukemia (AML).14IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0076] In some embodiments, the allo-HSCT is obtained from a related or unrelated donor with respect to the recipient. In some embodiments, the poly donor CD4IL-10cells are non-autologous to the recipient. In some embodiments, the polydonor CD4IL-10cells are allogeneic to the recipient. In some embodiments, the poly donor CD4IL-10cells are not anergized to host allo-antigens prior to administration to the host.

[0077] In some embodiments, the poly donor CDd11'11’ cells are Tri -like cells.

[0078] In some embodiments, the polydonor CD4IL-10cells are polyclonal. In some embodiments, the polydonor CD4IL-10cells are polyclonal and non-autologous to the recipient.

[0079] In some embodiments, the poly donor CD4IL-10cells are isolated from at least two donors prior to being genetically modified. In some embodiments, none of the at least two donors is the same donor as the allo-HSCT donor. In some embodiments, the allo-HSCT is obtained from a matched or mismatched donor with respect to the recipient.

[0080] In some embodiments, the polydonor CD4IL-10cells target and kill cells that express CD54. In some embodiments, the polydonor CD4IL’10cells target and kill cancer cells that express HLA-class I and CD54. In some embodiments, the polydonor CD4IL-10cells target and kill cancer cells that express CD112. In some embodiments, the polydonor CD4IL-10cells target and kill cancer cells that express CD58. In some embodiments, the poly donor CD4IL‘10cells target and kill cancer cells in the host.

[0081] One aspect of the present disclosure provides a method of treating a hematological cancer by allogeneic hematopoietic stem cell transplant (allo-HSCT), comprising:administering allo-HSCT to a subject (host);administering to the host an amount of polydonor CD4IL-10cells sufficient to suppress or prevent graft-versus-host disease (GvHD) without suppressing graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT graft;wherein the polydonor CD4IL-10cells comprise CD4+T cells obtained from at least two different T cell donors and that are genetically modified by vector-mediated gene transfer of the coding sequence of human IL- 10 under control of a constitutive or inducible promoter;wherein the polydonor CD4IL-10cells are non-autologous to the host and non-autologous to the allo-HSCT donor;15IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOwherein the polydonor CD4IL-10cells are not anergized to host allo-antigens prior to administration to the host; andwherein the polydonor CD4IL-10cells are polyclonal and Tri -like.

[0082] In some embodiments, the allo-HSCT is administered prior to administration of the polydonor CD4n’10cells. In some embodiments, the allo-HSCT is administered after administration of the poly donor CD4IL‘10cells.

[0083] Another aspect of the present disclosure provides a method of treating a hematological cancer by allogeneic hematopoietic stem cell transplant (allo-HSCT), comprising:administering allo-HSCT to a subject (host);administering to the host an amount of polydonor CD4IL-10cells sufficient to suppress or prevent graft-versus-host disease (GvHD) without suppressing graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT;wherein the polydonor CD4IL-10cells comprise CD4+T cells obtained from at least two different T cell donors and genetically modified by vector-mediated gene transfer of the coding sequence of human IL- 10 under control of a constitutive promoter;wherein the polydonor CD4IL-10cells target and kill cancer cells in the host; wherein the polydonor CD4IL-10cells are not anergized to host allo-antigens prior to administration to the host; andwherein all of the poly donor CD4IL 10cells are non-autologous to the host, and polyclonal, and are Tri -like.

[0084] Another aspect of the present disclosure provides CD4IL-10cells from a single donor or multiple donors, where the IL-10 is viral IL-10. The viral IL-10 having the sequence of SEQ ID NO: 6, 19, 20, or 21. In some embodiments, the viral IL-10 is encoded by a polynucleotide having the sequence of SEQ ID NO: 7. In some embodiments, the IL-10 is human IL- 10 where one, two, three, four, five, six, seven, eight, nine or ten amino-acid from human IL- 10 are replaced by the corresponding amino-acid sequence from viral IL- 10. In some embodiments, the CD4+ T cells are transduced with exogenous viral IL- 10 under the control of constitutive promoter. In some embodiments, the expression control elements drive expression of viral IL-10 in activated CD4+T cells. In some embodiments, the exogenous polynucleotide encoding viral IL-10 is integrated into the T cell nuclear genome. In some16IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOembodiments, the exogenous polynucleotide encoding viral IL- 10 is not integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding viral IL-10 has the sequence of SEQ ID NO: 7.

[0085] Another aspect of the present disclosure provides CD4IL‘10cells from a single donor or multiple donors, where the IL-10 is IL-10 of a mouse (SEQ ID NO: 10), rat (SEQ ID NO: 11), Macaca mulatta (MACMU) (SEQ ID NO: 12), gorilla (SEQ ID NO: 13), cynomolgus monkey (CYNO) (SEQ ID NO: 14), olive baboon (SEQ ID NO: 15), bonobo (SEQ ID NO: 16), chimpanzee (SEQ ID NO: 17), or EBVB9 (SEQ ID NO: 18). In some embodiments, the IL- 10 is a protein having at least 90%, 95%, 98%, or 99% sequence identity to IL- 10 of a mouse (SEQ ID NO: 10), rat (SEQ ID NO: 11), macaca mulatta (MACMU) (SEQ ID NO: 12), gorilla (SEQ ID NO: 13), cynomolgus monkey (CYNO) (SEQ ID NO: 14), olive baboon (SEQ ID NO: 15), bonobo (SEQ ID NO: 16), chimpanzee (SEQ ID NO: 17), or EBVB9 (SEQ ID NO: 18).

[0086] Another aspect of the present disclosure provides CD4IL-10cells from a single donor or multiple donors, where the IL-10 is a variant of human IL-10. The variant of human IL-10 having the sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the IL-10 is human IL-10 where one, two, three, four, five, six, seven, eight, nine or ten amino-acid from human IL- 10 are replaced by the corresponding amino-acid sequence from IL- 10 of another species (e.g.. IL-10 of a mouse (SEQ ID NO: 10). rat (SEQ ID NO: 11), macaca mulatta (MACMU) (SEQ ID NO: 12), gorilla (SEQ ID NO: 13), cynomolgus monkey (CYNO) (SEQ ID NO: 14), olive baboon (SEQ ID NO: 15), bonobo (SEQ ID NO: 16), chimpanzee (SEQ ID NO: 17), or EBVB9 (SEQ ID NO: 18). In some embodiments, the CD4+T cells are transduced with exogenous the IL-10 variant under the control of constitutive promoter. In some embodiments, the expression control elements drive expression of the IL-10 variant in activated CD4+T cells. In some embodiments, the exogenous polynucleotide encoding the IL- 10 variant is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding the IL- 10 variant is not integrated into the T cell nuclear genome.

[0087] In yet another aspect, the present disclosure provides a method of making viral IL- 10 CD4IL-10. comprising the steps of:(i) obtaining primary CD4+T cells from a single T cell donor; and17IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO(ii) modifying the donor CD4+T cells by introducing an exogenous polynucleotide encoding viral IL- 10.thereby obtaining the genetically-modified CD4+T cells.

[0088] In some embodiments, the method further comprises the step, after step (i), or after step (ii), of: incubating the primary CD4+T cells in the presence of an anti-CD3 antibody, and anti-CD28 antibody or anti-CD3 antibody and CD28 antibody coated beads.

[0089] In some embodiments, the method comprises incubating the primary CD4+T cells further in the presence of IL-2. In some embodiments, the exogenous polynucleotide encoding viral IL- 10 using a vector.

[0090] In some embodiments, the exogenous polynucleotide encoding viral IL-10 comprises a segment encoding a selection marker. In some embodiments, the encoded selection marker is ΔNGFR. In some embodiments, the encoded selection marker has the sequence of SEQ ID NO:3. In some embodiments, the encoded selection marker is a truncated EGFR polypeptide. In some embodiments, the encoded selection marker is a truncated human EGFR polypeptide.

[0091] In some embodiments, the method further comprises the step, after step (ii), of: isolating the genetically -modified CD4+T cells expressing the selection marker, thereby- generating an enriched population of genetically-modified CD4+T cells.

[0092] In some embodiments, the method further comprises the step of incubating the enriched population of genetically-modified CD4+T cells. In some embodiments, the step of incubating the enriched population of genetically-modified CD4+T cells is performed in the presence of anti-CD3 antibody and anti-CD28 antibody or CD3 antibody and CD28 antibody coated beads in the presence of IL-2.

[0093] In some embodiments, in step (i), the primary CD4+T cells are obtained from frozen stock. In some embodiments, in step (i), the primary- CD4+T cells are obtained from unfrozen peripheral blood mononuclear cells of the single T cell donor.

[0094] In some embodiments, the method further comprises the step of isolating CD4+T cells from the peripheral blood mononuclear cells. In some embodiments, the peripheral blood mononuclear cells are obtained from buffy coat or apheresis.

[0095] In some embodiments, the method increases reconstitution of natural Tri cells. In some embodiments, the method increases reconstitution of DC-10 cells. In some18IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOembodiments, the method increases reconstitution of FOXP3+ T cells. In some embodiments, the method increases reconstitution of natural Tri, DC-10 and FOXP3+ T cells.

[0096] In some embodiments, administering a therapeutically effective amount of polydonor CD4IL-10 cells increases the number of natural Tri cells. In some embodiments, the number of natural Tri cells increases by about 15-25%, about 20-30%, about 25-35%, about 30-40%, about 35-45%, about 40-50%, about 45-55%, about 50-60%, about 55-65%, about 60-70%, about 65-75%, about 70-80%, about 75-85%, or about 85-95%. In some embodiments, the number of natural Tri cells increases by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%.

[0097] In some embodiments, administering a therapeutically effective amount of polydonor CD4IL-10 cells increases the number of DC-10 cells. In some embodiments, the number of DC-10 cells increases by about 15-25%, about 20-30%, about 25-35%, about 30-40%, about 35-45%, about 40-50%, about 45-55%, about 50-60%, about 55-65%, about 60-70%, about 65-75%, about 70-80%, about 75-85%, or about 85-95%. In some embodiments, the number of DC-10 cells increases by at least about 10%, at least about 15%, at least about 20%. at least about 25%. at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%.

[0098] In some embodiments, administering a therapeutically effective amount of polydonor CD4IL-10 cells increases the number of FOXP3+T cells. In some embodiments, the number of FOXP3+T cells increases by about 15-25%, about 20-30%, about 25-35%, about 30-40%, about 35-45%, about 40-50%, about 45-55%, about 50-60%, about 55-65%, about 60-70%, about 65-75%, about 70-80%, about 75-85%, or about 85-95%. In some embodiments, the number of FOXP3+T cells increases by at least about 10%, at least about 15%, at least about 20%. at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%.19IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0099] In some embodiments, administering a therapeutically effective amount of polydonor CD4IL-10 cells increases the number of at least one of DC-10 cells, natural Tri cells, or FOXP3+T cells. In some embodiments, the number of at least one of natural Tri cells, DC-10 cells, and FOXP3+ T cells increases by about 15-25%, about 20-30%, about 25-35%, about 30-40%, about 35-45%, about 40-50%, about 45-55%, about 50-60%, about 55-65%, about 60-70%, about 65-75%, about 70-80%, about 75-85%, or about 85-95%. In some embodiments, the number of at least one of natural Tri cells, DC-10 cells, and FOXP3+T cells increases by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%. at least about 85%. or at least about 90%.

[0100] In some embodiments, administering a therapeutically effective amount of poly donor CD4IL-10 cells does not result in nonspecific cytotoxicity. In some embodiments, administering a therapeutically effective amount of poly donor CD4IL-10 cells does not substantially increase nonspecific cytotoxicity. In some embodiments, administering a therapeutically effective amount of poly donor CD4IL-10 cells increases nonspecific cytotoxicity by less than about 1%, less than about 2%, less than about 3%. less than about 4%, less than about 5%, less than about 10%, less than about 15%, or less than about 20%.

[0101] In some embodiments, the method does not result in dose-limiting toxicity.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIG. l is a non-limiting illustration of the structure of a bidirectional lentiviral vector for delivering human IL-10 and ANGFR coding sequences into CD4+T cells from multiple donors to produce polydonor CD4IL-10cells.

[0103] FIG. 2 illustrates the complete and circular structure of a bidirectional lentiviral vector for generating the lentiviral vector to deliver human IL-10 and ANGFR coding sequences into CD4+T cells from multiple donors to produce polydonor CD4IL-10cells.

[0104] FIG. 3 illustrates an exemplary protocol for generating CD4IL-10cells.

[0105] FIG. 4A shows percentages of CD4+ΔNGFR+cells (mean± SD, n=10 left grey bar) and vector copy numbers (VCN, mean + SD. n=10 right grey bar) in human CD4+20IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOT cells transduced with LV-IL- 10 / ANGFR (a bidirectional lentiviral vector encoding for human IL- 10 and a truncated form the human NGF receptor). FIG. 4B shows FACS analysis of expression of CD4 and ANGFR in human CD4+T cells from two representative donors (Donor B and Donor C) transduced with LV-IL- 10 / ANGFR and purified using anti-CD271 Microbeads.

[0106] FIG. 5 shows cytokine production profile of single donor CD4IL-10cells after the second (TF2) and third (TF3) restimulation. The TF2 (left panel) and TF3 (right panel) CD4IL-10cells were left unstimulated (as indicated by arrow) or stimulated with immobilized CD3 (1 Opg / mL) and soluble CD28 mAb ( 1 pg / mL) for 48 hours. Culture supernatants were collected and levels of IL- 10, IL -4, IL-5, IFN-y and IL-22 were determined by ELISA. All samples were tested in triplicate. Mean± SD, n=8 donors tested are presented.

[0107] FIG. 6A shows the percentage of CD4IL-10cells expressing granzyme B (GzB) after 2ndround of stimulation (TF2) analyzed by FACS. Box and whiskers of n=7 different single donors are presented. FIG. 6B shows % dead cells when CD4IL-10cells ( 105 / well) were co-cultured with K562 and ALL-CM cells (lO’ / well) at 1:1 ratio for 3 days. Box and whiskers represent data from n=4 donors and dots represent data from single donors.

[0108] FIGs. 7A and 7B show that single donor CD4IL'10cells can suppress the proliferation of allogeneic CD4+T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (5x104cells / well) and stimulated with allogeneic mature dendritic (DC) cells (5x103cells / well) and soluble anti-CD3 mAbs in the absence or presence of CD4IL-10cells (5x104cells / well) at a 1: 1 Responder: Suppressor ratio. After 3 days of culture, the percentages of proliferating responder cells were determined by eFluor® 670 dilution with flow7cytometry after gating on CD4+ANGFR' T cells. FIG. 7A show results from Donor-C, Donor-E, and Donor-F and FIG. 7B show results from Donor-H, Donor-I and Donor-L. Percentages of proliferation and suppression are indicated. The suppression mediated by CD4IL'10cells was calculated as follows: 100-([proliferation of responders in the presence of CD4IL-10cells / proliferation of responders alone]x 100).

[0109] FIGs. 8A and 8B show that single donor CD4IL-10cells can suppress the proliferation of allogeneic CD8+T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (5x104cells / well) and stimulated with allogeneic mature dendritic (DC) cells (5x103cells / well) and soluble anti-CD3 mAbs in the absence or presence of CD4IL-10cells (5x10421IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOcells / well) at a 1:1 Responder: Suppressor ratio. After 3 days of culture, the percentages of proliferating responder cells were determined by eFluor® 670 dilution with flow cytometry after gating on CD8 ANGFR" T cells. FIG. 8A show results from Donor-C, Donor-E, and Donor-F and FIG. 8B show results from Donor-H, Donor-I and Donor-L. Percentages of proliferation and suppression are indicated. The suppression mediated by CD4IL-10cells was calculated as follows: 100-([proliferation of responders in the presence of CD4IL-10cells / proliferation of responders alone]x 100).

[0110] FIG. 9 shows cytokine production profile of polydonor CD4IL-10cells after third (TF3) restimulation, compared to mean levels (+ / - SD) produced by CD4IL’10cells from 8 individual donors. The TF3 CD4IL-10cells from three donors were pooled at a 1: 1: 1 ratio and stimulated with immobilized CD3 (10μg / mL) and soluble CD28 mAb (1μg / mL) for 48 hours. Culture supernatants were collected and levels of IL-10. IL-4, IL-5, IFN-y and IL-22 were determined by ELISA. Dots are results of poly donor CD4IL'10cells; gray bars represent mean+ SD, n=8 single donors.

[0111] FIG. 10A shows the percentage of poly donor CD4IL‘10cells expressing granzyme B (GzB) compared to mean % levels (+ / - SD) of granzyme B expression by CD4IL'10cells of n=3 single donors used to generate the pool. Cells were analyzed by FACS after the 3rdround of stimulation (TF3). FIG. 10B shows % dead cells when polydonor CD4n‘10cells (105 / well) were co-cultured with K562 and ALL-CM cells (105 / well) at 1:1 ratio for 3 days. Residual leukemic cells (CD45+, CD3 -) were counted by FACS for each target cell. Dots are results of polydonor CD4IL-10and gray bars represent mean+ SD of n=3 single donors used to generate the pool.

[0112] FIGs. 11A and 11B show that polydonor CD4IL-10cells can suppress the proliferation of allogeneic CD4+T cells and CD8+T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (5x104cells / well) and stimulated with allogeneic mature dendritic (DC) cells (1x104cells / well) and soluble anti-CD3 mAbs in the absence or presence of polydonor CD4IL-10cells (5x104cells / well) at a 1: 1 Responder: Suppressor ratio. After 3 days of culture, the percentages of proliferating responder cells were determined by eFluor® 670 dilution with flow cytometry after gating on CD4ANGFR T cells and CD8 ANGFR T cells. FIG. 11A shows results from poly donor CD4n‘10cells containing CD4+cells pooled from Donor-C, Donor-E, and Donor-F. FIG. 1 IB shows results from polydonor CD4IL-10cells containing CD4+cells pooled from Donor-H, Donor-I, and Donor-L. The suppression 22IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOmediated by CD411’1" cells was calculated as follows: 100-([proliferation of responders in the presence of CDd11'10cells / proliferation of responders alone]x 100).

[0113] FIG. 12 illustrates a protocol for testing induction of GvHD by human PBMC and / or polydonor CD4n‘10(BC-C / E / F) cells injected on day 0 post-irradiation.

[0114] FIG. 13 shows % of NSG mice free of GvHD on each day after injection of PBMC (5x106cells / mouse), polydonor (three donors; BC-C / E / F) CD4IL-10cells (5x106cells / mouse), or PBMC (5x106cells / mouse) in combination with poly donor CD4IL’10cells (three donors; BC-C / E / F) (5x106cells / mouse).

[0115] FIG. 14 shows migration of CD4IL-10cells to spleen (left panel) and bone marrow (right panel) in NSG mice injected with PBMC (5x106cells / mouse), poly donor (three donors; (BC-C / E / F)) CD4IL’10cells (5x106cells / mouse), or PBMC (5x106cells / mouse) in combination with polydonor CD4IL-10cells (three donors; (BC-C / E / F)) (5x106cells / mouse). Box and whiskers on n=8 tested animals are presented.

[0116] FIG. 15 illustrates a protocol for testing induction of GvHD by CD4+ T cells and polydonor (BC-H / I / L) or single-donor (BC-H) CD4IL-10cells injected on day 3 postirradiation.

[0117] FIG. 16 shows % of NSG mice free of GvHD on each day after injection.

[0118] FIGs. 17A-17C shows graft-versus-leukemia (GvL) effect tested based on reduction of circulating leukemia cells and long-term leukemia free survival. Leukemia was measured as previously described (Locafaro G. et al Molecular Therapy 2017). NSG mice were sub-lethally irradiated and intravenously injected with myeloid leukemia cells (ALLCM) (2.5x106) on day 0. FIG. 17A is an illustration of the experiment. FIG. 17B shows leukemia free survival rate in the animals injected w ith PBMC (2.5x106) or single donor (from donor BC-I and donor BC-H) CD4IL-10cells (2.5x106) on day 3. FIG. 17C shows leukemia free survival rate in the animals injected with PBMC (2.5x106) or poly donor CD4IL-10cells (from donor BC-I and donor BC-H) (2.5x106) on day 3.

[0119] FIGs. 18A-18C show long-term leukemia free survival rate measured in NSG mice sub-lethally irradiated and intravenously injected with ALL-CM cells (2.5x106) at day 0. FIG. 18A is an illustration of the experiment. FIG. 18B shows data from animals inj ected with mononuclear cells (PBMC) (2.5x106) alone or mononuclear cells (PBMC) (2.5x106) + single donor (from donor BC-H and donor BC-I) CD4IL’10cells (2.5x106) at day 3. FIG. 18C 23IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOshows data from animals injected with mononuclear cells (PBMC) (2.5x106) alone or mononuclear cells (PBMC) (2.5x106) + polydonor CD4IL 10cells (BC-I / H) (2.5x106) at day 3.

[0120] FIGs. 19A-19G show inhibition of NLPR3 inflammasome activation by CD4n‘'° cells. FIG. 19A shows the effect of CD4IL’10cell supernatant from a single donor (#1) on the production of IL-10 by LPS activated monocytes. FIG. 19B shows the effect of CD4IL-10cell supernatant from another single donor (#2) on the production of IL-1 by LPS activated monocytes. FIG. 19C shows the effect of CD4IL-10cell supernatant from a single donor (#1) on the inhibition of LPS induced IL-10 production enhanced by NLPR3 inflammasome activator nigericine (NIG). FIG. 19D shows the effect of CD4IL‘10cell supernatant from a single donor (#2) on the inhibition of LPS induced IL-10 production enhanced by NLPR3 inflammasome activator nigericine (NIG). FIG. 19E is a bar graph that shows the effect of CD4IL-10cell supernatant from a single donor (BC-E) and pooled cells from 2 different donors (BC-C / E) on LPS induced IL- 10 production by monocytes in the presence or absence of anti-IL-10 receptor (anti-IL-1 OR) mAb. FIG. 19F shows the effect of polydonor CD4IL-10cell (BC-T / U / V) supernatants on IL-10 production by monocytes in the presence or absence of anti-IL-1 OR mAb. FIG. 19G shows the effects of polydonor CD4IL-10cell (BC-T / U / V) supernatants on IL-18 production induced by LPS in combination with nigericin in the presence or absence of anti-IL-lOR mAb.

[0121] FIG. 20 illustrates an experimental protocol for testing graft versus myeloid leukemia and xeno-GvHD effects. NSG-mice were intravenously injected with ALL-CM cells (2.5x106) on day 0. At day 3 the mice were divided into five groups and each group was treated with (i) none as a control, (ii) allogeneic mononuclear cells (PBMC); (iii) allogeneic PBMC and poly donor CD4IL-10cells (BC-V / T / E, pooled 1:1:1); (iv) allogeneic PBMC and single-donor CD4IL-10cells (BC-E); or (v) polydonor CD4IL‘10cells (BC-V / T / E) at concentrations as indicated in FIG. 20.

[0122] FIG. 21 is a bar graph depicting the cytokine secretion profiles of singledonor (BC-V, BC-T, BC-V, and BC-E) and polydonor CD4IL-10cells (POOL: BC-E, BC-V and BC-T pooled 1:1:1).

[0123] FIG. 22 show suppressive effects of single-donor (BC-V and BC-E) and poly donor CD411’10cells (pool of BC-V / T / E) on in vitro proliferation of allogeneic CD4+ and CD8+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (5x104cells / w ell) and stimulated with allogenic mature dendritic (DC) cells (1x104cells / well) and soluble anti-CD324IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOmAbs in the absence or presence of CD4IL-10cells (5x104cells / well) at a 1: 1 Responder: Suppressor ratio. After 3 days of culture, the percentages of proliferating responder cells were determined by eFluor® 670 dilution with flow cytometry after gating on CD4+ANGFR" (top) or CD8+ ANGFR" (bottom) T cells. FIG.22 shows results from single donors BC-V and BC-E and pooled cells from donors BC-V / T / E. Percentages of proliferation and suppression are indicated. The suppression mediated by CD4IL’10cells was calculated as follows: 100-([proliferation of responders in the presence of CD4IL’10cells / proliferation of responders alone]x 100).

[0124] FIG. 23 shows % of alive cells in a co-culture of single (BC-E and BC-V) or polydonor CD4IL'-10cells (BC-V / T / E) with ALL-CM myeloid tumor cells or K562 cells. The results show selective cytotoxic effect of single-donor and polydonor CD4IL-10cells on ALLCM myeloid tumor cells, but not on K562 cells which lack Class I MHC expression.

[0125] FIG. 24 shows leukemia-free survival rate measured in NSG-mice intravenously injected with ALL-CM cells (2.5x106) on day 0. At day 3 the mice were divided into five groups and each group was treated with (i) none as a control; (ii) allogeneic mononuclear cells (PBMC); (iii) allogeneic PBMC and polydonor CD4IL-10cells (BC-V / T / ET); (iv) allogeneic PBMC and single-donor CD4IL-10cells (BC-E) or (v) polydonor CD4IL-10cells (BC-V / T / E). The graph shows leukemia-free survival rate of animals in each group.

[0126] FIG. 25 shows % of NSG mice free of GvHD on each day following injection with ALL-CM cells (2.5x106) and subsequent administration of (i) none as a control; (ii) allogeneic mononuclear cells (PBMC); (iii) allogeneic PBMC and polydonor CD4IL-10cells (BC-V / T / E); (iv) allogeneic PBMC and single-donor CD4IL-10cells (BC-E) or (v) polydonor CD4IL-10cells were administered at day 3.

[0127] FIG. 26 shows that NSG mice dosed with 2.5E+06 of PBMC (allogeneic to the donors C, E, F and H) all succumbed to acute, and lethal xeno-GvHD at day 22.Administration of single-donor CD4IL-10cells (lot C) or polydonor CD4IL-10cells (lot CEFH) in combination with the PBMCs prevented the development of lethal xeno-GvHD in 75% (3 / 4 mice) and 80% (4 / 5 mice) of the mice, respectively. In contrast, transfer of 2.5E+06 polydonor CD411’10cells did not induce any sign of GvHD. Taken together these results indicate that polydonor CD4n‘10cells from 4 different donors suppress pathogenic human T25IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOcell responses as potently, or slightly more potently than single- donor CD4IL-10cells.PBMC: peripheral mononuclear cells; GvHD: graft vs. host disease.

[0128] FIG. 27 A shows alignment of IL-10 protein sequences of various species, including human (SEQ ID NO: 1). Mus musculus. “MOUSE” (SEQ ID NO: 10); Rattus norvegicus, “RAT” (SEQ ID NO: 11); Macaco mulatto, “MACMU” (SEQ ID NO: 12); Gorilla gorilla, “GORILLA” (SEQ ID NO: 13); Macaca fascicularis, “CYNO” (SEQ ID NO: 14); Papio Anubis, “OLIVE BABOON” (SEQ ID NO: 15); Pan paniscus, “BONOBO” (SEQ ID NO: 16); Pan troglodytes, “CHIMP” (SEQ ID NO: 17); or EBVB9 (SEQ ID NO: 18).

[0129] FIG. 27B provides sequences of IL- 10 variants generated by substituting one or more amino acids of human IL- 10 by amino acids of viral IL- 10 (EBVB9) at the corresponding positions. Also provided are sequences of the exemplary variants, possible huIL-10 hybrid #1 (SEQ ID NO: 19) and possible huIL-10 hybrid #2 (SEQ ID NO: 20). indicates the one or more amino acid positions that are substituted.indicates the preferred 1105 to A105 amino acid substitution for IL-10 hybrid #2 (SEQ ID NO: 20).

[0130] FIG. 27C shows alignment of human IL-10 (SEQ ID NO: 1) with IL10 EBVB9 (SEQ ID NO: 18). “^indicates the one or more amino acid positions that are substituted in IL-10 hybrid #1. " Vindicates the preferred 1105 to A 105 amino acid substitution for IL- 10 hybrid #2.

[0131] FIG. 28 is a study design of the phase I clinical study described in Example 10. In the study design, Sirolimus is administered starting Day +5, tapered from Day +60 to +90 and discontinued on Day +100 to +150. Mycophenolate mofetil (MMF) is administered starting Day +5 and discontinued on Day + 28 to +35. PTCy refers to post-transplant cyclophosphamide. DLT refers to dose limiting toxicities.

[0132] FIGs. 29A and 29B show pharmacokinetics and CD4+ T cell reconstitution in the subjects treated with Dose 1 or Dose 2 in the phase I clinical study. FIG. 29A shows poly donor CD4IL'-10cells (counts per mL) in the subjects; and FIG. 29B shows stem cell donor derived CD4+ T cells (counts per mL) in the subjects.

[0133] FIG. 30 illustrates an exemplary protocol for testing induction of GvHD by human PBMC and / or poly donor CD4n’10cells in a dose-dependent manner, injected on day 0 post-irradiation.26IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0134] FIG. 31 shows percent of NSG mice free of GvHD on each day after injection of PBS, PBMC (5x106cells / mouse). PBMC (5x106cells / mouse) in combination with polydonor (three donors; BC-C / E / F) CD4"'‘10cells ( 1.0x107cells / mouse), PBMC (5x106cells / mouse) in combination with poly donor CD4IL-10cells (three donors; BC-C / E / F) (5x106cells / mouse), or PBMC (5x106cells / mouse) in combination with poly donor CD4IL’10cells (three donors; BC-C / E / F) (2.5x106cells / mouse).

[0135] FIGs. 32A-32B show' polydonor CD4IL-10cell count in the subjects. FIG. 32A shows polydonor CD4IL’10cells (counts per mL) in the subjects of Dose Level 1 (DL1), Dose Level 2 (DL2), Dose Level 3 (DL3), and Dose Level 4 (DL4). FIG. 32B shows the area under the curve (PK AUC) in the subjects of DL 1, DL2, LD3 and DL4.

[0136] FIGs. 33A-33B show FOXP3 Treg cell count in the subjects. FIG. 33A shows FOXP3 Treg cells (counts per mL) in the subjects of DL1, DL2, DL3 and DL4. FIG. 33B shows FOXP3 Tregs (counts per mL) at Day 28 for subjects in DL1, DL2, DL3 and DL4.

[0137] FIG. 34A shows CD14+CD16+DC-10 cells (counts per mL) in the subjects of DL1, DL2, DL3 and DL4.

[0138] FIG. 34B shows Natural Tri cells (counts per mL) in the subjects of DL1, DL2, DL3 and DL4.

[0139] The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.6. DETAILED DESCRIPTION6.1. Definitions

[0140] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0141] G raft-vers us-leukemia effect” or GvL refers to an effect that appears after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). T lymphocytes in the allogeneic graft eliminate malignant residual host leukemia cells.27IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0142] “Graft versus tumor effect’' or “GvT refers to an effect that appears after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). T lymphocytes in the allogeneic graft eliminate malignant residual host cancer cells, e.g., cells of myeloma and lymphoid and myeloid leukemias, lymphoma, multiple myeloma and possibly breast cancer. The term GvT is generic to GvL.

[0143] The terms “treatment”, “treating”, and the like are used herein in the broadest sense understood in the medical arts. In particular, the terms generally mean obtaining a desired pharmacologic and / or physiologic effect. “Treatment” as used herein covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms). Improvements in any conditions can be readily assessed according to standard methods and techniques known in the art. The population of subjects treated by the method of the disease includes subjects suffering from the undesirable condition or disease, as well as subjects at risk for development of the condition or disease.

[0144] “HLA-matched” as used herein refers to a pair of individuals having a matching HLA allele in the HLA class I (HLA-A. HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci that allow the individuals to be immunologically compatible with each other. HLA compatibility can be determined using any of the methods available in the art, for example, as described in Tiervy, Haematol ogica 2016 Volume 101(6):680-687, which is incorporated by reference herein.

[0145] For a given locus, a pair of individuals have 2 / 2 match when each of two alleles of one individual match with the tw o alleles of the other individual. A pair of individuals have ½ match when only one of two alleles of one individual match with one of two alleles of the other individual. A pair of individuals have 10 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci when all of the ten alleles (two for each of the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci) of one individual match with all ten alleles of the other individual.

[0146] In preferred embodiments, allele level typing is used for determination of HLA compatibility. Allele level typing corresponds to a unique nucleotide sequence for an 28IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOHLA gene, as defined by using all digits in the first, second, third and fourth fields, e.g. A*02:01:01:01. Functionally, the third and fourth fields which characterize alleles that differ, respectively, by silent substitutions in the coding sequence and by substitutions in the non-coding sequence, are irrelevant, except when substitutions prevent the expression of HLA alleles (e.g. the null allele B* 15:01: 01:02N). Missing a null allele will lead to a mismatch that is very likely to be recognized by alloreactive T cells and have a deleterious clinical impact. Substitutions in non-coding sequences may influence the level of expression (e.g. the A241ow allele A*24:02:01:02L). Such variability may also have an impact on anti-HLA allorecognition.

[0147] The term “HLA-mismatched” as used herein refers to a pair of individuals having a mis-matching HLA allele in the HLA class I (HLA- A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci that make the individuals to be immunologically incompatible with each other.

[0148] The term “partially HLA-mismatched" as used herein refers to a pair of individuals having a mis-matching HLA allele in the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci that make the individuals to be immunologically incompatible with each other in a permissible degree. Some studies have identified permissive mismatches. Some HLA class I incompatibilities are considered to be more permissive.

[0149] “HLA haplotype” refers to a series of HLA loci-alleles by chromosome, one passed from the mother and one from the father. Genotypes for HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci can be used to determine the HLA haplotype.

[0150] The term “therapeutically effective amount” is an amount that is effective to treat, and thus ameliorate a symptom of a disease.

[0151] The term prophylactically effective amount is an amount that is effect in terms of completely or partially preventing a disease, condition, or symptoms thereof The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a neurodegen erative disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.29IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO6.2. Other interpretational conventions

[0152] Ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36. 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.6.3. Polydonor CD4IL 10cells

[0153] In a first aspect, a population of CD4+T cells that have been genetically modified to comprise an exogenous polynucleotide encoding IL- 10 is provided (CD4IL‘10cells). The population comprises CD4+T cells obtained from at least two different T cell donors (polydonor CD4IL'10cells).6.3.1. CD4+T cells and T cell donors

[0154] CD4+T cells used in polydonor CD4IL-10populations can be isolated from peripheral blood, cord blood, or other blood samples from a donor, using methods available in the art. In ty pical embodiments, CD4+T cells are isolated from peripheral blood, preferably a human donor. In certain embodiments, CD4+T cells are isolated from peripheral blood by leukapheresis. In certain embodiments the CD4+T cells are obtained from third party-blood banks. In certain embodiments the CD4+ T cells are obtained from buffy coats from centrifugation of whole blood.

[0155] In some embodiments, CD4+T cells are isolated from a prior-frozen stock of blood or a prior-frozen stock of peripheral blood mononuclear cells (PBMCs). In some embodiments, CD4+T cells are isolated from peripheral blood or from PBMCs that have not previously been frozen. In some embodiments, the CD4+ T cells are separately isolated from blood or PBMCs obtained from a plurality of donors, and then pooled. In some embodiments, the CD4+ T cells are isolated from blood or PBMCs that have first been pooled from a plurality of donors.

[0156] In some embodiments, the CD4+T cells are obtained from three, four, five, six, seven, eight, nine, or ten different T cell donors.30IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0157] In some embodiments, the at least two different T cell donors are selected without regard to genotype. In some embodiments, the at least two different T cell donors are selected based on genotype.

[0158] In certain embodiments, the at least two different T cell donors are selected based on their HLA haplotypes.

[0159] In some embodiments, some or all of the at least two different T cell donors have matching HLA haplotypes. In some embodiments, some or all of the at least two different T cell donors have a mis-matched HLA haplotype.

[0160] In some embodiments, all of the CD4+T cells in the population have at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to each other. In some embodiments, all of the CD4+T cells in the population have at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, all the CD4+T cells in the population have 2 / 2 match at the HLA-A locus to each other. In some embodiments, all the CD4+T cells in the population have 2 / 2 match at the HLA-B locus to each other. In some embodiments, all the CD4+T cells in the population have 2 / 2 match at the HLA-C locus to each other. In some embodiments, all the CD4+T cells in the population have at least 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA DQB1 loci with each other. In some embodiments, all the CD4 T cells in the population have an A*02 or A*24 allele.

[0161] In some embodiments, all of the CD4+T cells in the population have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci to each other. In some embodiments, all of the CD4+T cells in the population have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, all the CD4+T cells in the population have less than 2 / 2 match at the HLA-A locus to each other. In some embodiments, all the CD4+T cells in the population have less than 2 / 2 match at the HLA-B locus to each other. In some embodiments, all the CD4+T cells in the population have less than 2 / 2 match at the HLA-C locus to each other. In some embodiments, all the CD4+T cells in the population have less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA DQB1 loci with each other.

[0162] In some embodiments, all of the CD4+T cells in the population have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRBL and HLA-31IPTS / 200307516.4Attorney Docket Ref: TR1X-012WODQB1 loci to each other. In some embodiments, all of the CD4+T cells in the population have less than 4 / 8, 5 / 8. 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, all the CD4+T cells in the population have less than 2 / 2 match at the HLA-A locus to each other. In some embodiments, all the CD4+T cells in the population have less than 2 / 2 match at the HLA-B locus to each other. In some embodiments, all the CD4+T cells in the population have less than 2 / 2 match at the HLA-C locus to each other. In some embodiments, all the CD4+T cells in the population have less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HL A DQB1 loci with each other.

[0163] In preferred embodiments, none of the at least two different T cell donors is a host to be treated with the CD4IL-10cells. In preferred embodiments, none of the at least two different T cell donors is a donor of stem cells (e.g., HSC), tissue or organ that will be used together with the CD4IL-10cells in the methods of treatment described herein.

[0164] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the patient to be treated (host). In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to the patient. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to the patient. In some embodiments, one or more of the T cell donors have less than 2 / 2 match at the HLA-A, HLA-B, or HLA-C locus to the patient. In some embodiments, one or more of the T cell donors have less than 2 / 4, 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to the patient.

[0165] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched with the HSC donor. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 match at the HLA-A, HLA-B, HLA-C. HLA-DRB1. and HLA-DQB1 loci to the HSC donor. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to the HSC donor. In some embodiments, one or more of the T cell donors have less than 2 / 2 match at the HLA-A, HLA-B, or HLA-C locus to the HSC donor. In some embodiments, one or more of the T cell donors have less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to the HSC donor.

[0166] In the preferred embodiments, none of the CD4+T cells is immortalized.32IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO6.3.2. Exogenous polynucleotide encoding IL-10

[0167] Poly donor CD4IL-10cells of the present disclosure are CD4+T cells that have been genetically modified to comprise an exogenous polynucleotide encoding IL-10. The exogenous polynucleotide comprises an IL-10-encoding polynucleotide segment operably linked to expression control elements.

[0168] The IL- 10-encoding polynucleotide segment can encode IL- 10 of a human, bonobo or rhesus. In some embodiments, the IL- 10-encoding polynucleotide segment encodes human IL-10 having the sequence of SEQ ID NO: 1. In some embodiments, the IL- 10-encoding polynucleotide segment encodes a variant of human IL-10 having at least 90%. 95%. 98%. or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-10-encoding polynucleotide segment has the nucleotide sequence of SEQ ID NO:2. In some embodiments, the IL- 10-encoding polynucleotide segment has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2.

[0169] In some embodiments, the IL- 10-encoding polynucleotide segment encodes IL-10 of aMus musculus, ‘‘MOUSE'’ (SEQ ID NO: 10); RciUus norvegicus, “RAT"’ (SEQ ID NO: 11); Macaca mulatta. “MACMU” (SEQ ID NO: 12); Gorilla gorilla, •■GORILLA” (SEQ ID NO: 13); Macaca fascicularis, “CYNO” (SEQ ID NO: 14); Papio Anubis, “OLIVE BABOON” (SEQ ID NO: 15); Pan paniscus, “BONOBO” (SEQ ID NO: 16); Pan troglodytes, “CHIMP” (SEQ ID NO: 17); and EBVB9 (SEQ ID NO: 18). In some embodiments, the IL- 10-encoding polynucleotide segment encodes a protein having at least 90%, 95%, 98%, or 99% sequence identity to IL-10 of a Mus musculus, “MOUSE” (SEQ ID NO: 10); Rattus norvegicus, “RAT” (SEQ ID NO: 11); Macaca mulatta, “MACMU” (SEQ ID NO: 12); Gorilla gorilla, “GORILLA” (SEQ ID NO: 13); Macaca fascicularis, “CYNO” (SEQ ID NO: 14); Papio Anubis, “OLIVE BABOON” (SEQ ID NO: 15); Pan paniscus, “BONOBO” (SEQ ID NO: 16); Pan troglodytes, “CHIMP” (SEQ ID NO: 17); and EBVB9 (SEQ ID NO: 18).

[0170] In some embodiments, the exogenous polynucleotide encodes viral-IL-10. In various embodiments, the exogenous polypeptide encodes IL- 10 from HCMV, GMCMV, RhCMV, BaCMV, MOCMV, SMCMV, EBV, Bonobo-HV, BaLCV, OvHV-2, EHV-2, CyHV-3, AngHV-1, ORFV, BPSV, PCPV, LSDV, SPV, GPV, or CNPV. In some embodiments, the exogenous polypeptide encodes viral IL-10 from EBV or ORFV.33IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0171] In some embodiments, the IL-10-encoding polynucleotide segment encodes a variant of human IL- 10 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions compared to human IL-10 (e.g, SEQ ID NO: 1). In some embodiments, the one, two, three, four, five, six, seven, eight, nine or ten amino acid substitution are substitution(s) with amino acid(s) of viral IL-10 at corresponding amino acid position(s). In some embodiments, the IL-10-encoding polynucleotide segment encodes a variant of human IL- 10 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid insertion, deletion or modification compared to human IL-10 (e.g, SEQ ID NO: 1). In some embodiments, the variant of human IL-10 has the sequence of SEQ ID NO: 8 or 9.

[0172] In some embodiments, the IL-10-encoding polynucleotide segment encodes a variant of human IL-10 having one, tw o, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, insertions, and / or deletions compared to human IL- 10 (e.g. SEQ ID NO: 1). In some embodiments, the modifications are substitutions, insertions, and / or deletions with amino acids of Mus musculus, “MOUSE’’ (SEQ ID NO: 10); Rattus norvegicus, “RAT” (SEQ ID NO: 11); Macaca mulatto. “MACMU” (SEQ ID NO: 12); Gorilla gorilla, “GORILLA” (SEQ ID NO: 13); Macaca fascicularis. “CYNO” (SEQ ID NO: 14); Papio Anubis, “OLIVE BABOON” (SEQ ID NO: 15); Pan paniscus, “BONOBO” (SEQ ID NO: 16); Pan troglodytes, “CHIMP” (SEQ ID NO: 17); and EBVB9 (SEQ ID NO: 18), at the corresponding positions. In some embodiments, the variant of human IL- 10 has the sequence of SEQ ID NO: 19 or SEQ ID NO: 20.

[0173] In some embodiments, the IL-10-encoding polynucleotide segment encodes a variant of human IL- 10 having reduced immunostimulatory activity compared to human IL- 10. In some embodiments, the variant of human IL-10 includes I105A substitution. In some embodiments, a variant of human IL-10 is made using the method described in A Single Amino Acid Determines the Immunostimulatory Activity of Interleukin 10, J Exp Med, 191, 2, 2000, p.213-223.

[0174] The exogenous polynucleotide further comprises expression control elements that direct expression of the encoded IL- 10 in transduced CD4+T cells.

[0175] In some embodiments, the expression control elements comprise a promoter capable of directing expression of IL-10 in CD4 T cells. In some embodiments, the34IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOpromoter drives constitutive expression of IL-10 in CD4+T cells. In some embodiments, the promoter drives expression of IL-10 in activated CD4+T cells.

[0176] In some embodiments, an inducible promoter is used to induce expression of IL-10 when therapeutically appropriate. In some embodiments, the IL-10 promoter is used. In some embodiments a tissue-specific promoter is used. In some embodiments, a lineagespecific promoter is used. In some embodiments, a ubiquitously expressed promoter is used.

[0177] In some embodiments, a native human promoter is used. In some embodiments, a human elongation factor (EF) la promoter is used. In some embodiments, a human phosphoglycerate kinase promoter (PGK) is used. In some embodiments, a human ubiquitin C promoter (UBI-C) is used.

[0178] In some embodiments, a synthetic promoter is used. In certain embodiments, a minimal CMV core promoter is used. In particular embodiments, an inducible or constitutive bidirectional promoter is used. In specific embodiments, the synthetic bidirectional promoter disclosed in Amendola et al.. Nature Biotechnology, 23(1): 108-116 (2005) is used. This promoter can mediate coordinated transcription of two mRNAs in a ubiquitous or a tissue-specific manner. In certain embodiments, the bidirectional promoter induces expression of IL-10 and a selection marker.

[0179] In some embodiments, the exogenous polynucleotide further comprises a segment encoding a selection marker that permits selection of successfully transduced CD4+T cells. In some embodiments, the selection marker is ANGFR. In certain embodiments, the selection marker is a polypeptide having the sequence of SEQ ID NO: 3. In certain embodiments, the selection marker is a polypeptide having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3. In particular embodiments, the nucleotide sequence encoding the ANGFR selection marker has the sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the ANGFR selection marker has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 4.

[0180] In some embodiments, expression of the selection marker correlates with expression of IL- 10 from the exogenous polynucleotide. In some embodiments, expression of the selection marker linearly correlates with expression of IL-10 from the exogenous polynucleotide. Accordingly, in some embodiments, expression of the selection marker is measured to infer expression of IL-10 from the exogenous polynucleotide.35IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0181] In some embodiments, the selection marker is a truncated form of EGFR polypeptide. In some embodiments, the selection marker is a truncated form of the human EGFR polypeptide, optionally huEGFR disclosed in Wang et al. “A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells”, Blood, v. 118, n. 5 (2011), incorporated by reference in its entirety herein.

[0182] In some embodiments, the exogenous polynucleotide further comprises a sequence encoding an antibiotic resistance gene. In some embodiments, the exogenous polynucleotide comprises a sequence encoding an ampicillin resistance gene.

[0183] In typical embodiments, the exogenous polynucleotide is delivered into CD4+ T cells using a vector. In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector.

[0184] In certain embodiments, the exogenous polynucleotide is delivered into CD4+ T cells using a lentiviral vector and the exogenous polynucleotide comprises lentiviral vector sequences. In certain embodiments, a lentiviral vector disclosed in Matrai et al., Molecular Therapy 18(3):477-490 (2010) (“Matrai”), incorporated by reference herein, is used.

[0185] In some embodiments, the lentiviral vector is capable of integrating into the T cell nuclear genome. In some embodiments, the lentiviral vector is not capable of integrating into T cell nuclear genome. In some embodiments, an integration-deficient lentiviral vector is used. For example, in some embodiments, an integration-deficient or other lentiviral vector disclosed in Matrai is used. In some embodiments, an integrase-defective lentivirus is used. For example, an integrase-defective lentivirus containing an inactivating mutation in the integrase (D64V) can be used as described in Matrai et al., Hepatology 53:1696-1707 (2011), which is incorporated by reference herein, is used.

[0186] In some embodiments, the exogenous polynucleotide is integrated in the T cell nuclear genome. In some embodiments, the exogenous polynucleotide is not integrated in the nuclear genome. In some embodiments, the exogenous polynucleotide exists in the T cell cytoplasm.

[0187] In particular embodiments, the exogenous polynucleotide has the sequence of SEQ ID NO:5. In some embodiments, the exogenous polynucleotide has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 5.36IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO6.3.3. Gene expression of polydonor CD4IL-10T cells

[0188] Polydonor CD4IL-10T cells express IL-10. In some embodiments, polydonor CD4IL-10T cells constitutively express IL-10. In some embodiments, polydonor CD4IL-10T cells express IL- 10 when activated.

[0189] In some embodiments, polydonor CD4IL-10T cells constitutively express at least 100 pg of IL- 10 per 106of the CD4+T cells / mL of culture. In some embodiments, polydonor CD4IL-10T cells constitutively express at least 200pg, 500pg, Ing, 5ng, lOng, or 50ng of IL- 10 per 106of the CD4+T cells / mL of culture.

[0190] In some embodiments, polydonor CD4IL-10T cells express at least Ing or 2ng IL-10 per 106of the CD4+T cells / mL of culture after activation with a combination of anti-CD3 and anti-CD28 antibodies, or anti-CD3 antibody and anti-CD28 antibody coated beads. In some embodiments, poly donor CD4IL’10T cells express at least 5ng, lOng, lOOng, 200ng, or 500ng IL-10 per 106of the CD4+T cells / mL of culture after activation with anti-CD3 and anti-CD28 antibodies or CD3 antibody and CD28 antibody coated beads.

[0191] In various embodiments, the amount of IL-10 production is determined 12 hours, 24 hours, or 48 hours after activation using various methods for protein detection and measurement, such as ELISA, spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, western blotting, etc. In preferred embodiments, the amount of IL- 10 production is determined by ELISA 48 hours after activation with anti-CD3 and anti-CD28 antibodies.

[0192] In some embodiments, polydonor CD4IL-10T cells express IL-10 at a level at least 5-fold higher than unmodified CD4+T cells. In some embodiments, polydonor CD4IL-10T cells express IL-10 at a level at least 6, 7, 8, 9, 10, 11. 12. 13, 14, 15, 20, 30, 40, or 50-fold higher than unmodified CD4+T cells.

[0193] In some embodiments, polydonor CD4IL’10T cells further express a selection marker. In some embodiments, polydonor CD4IL-10T cells express a protein typically expressed in Tr1 cells. In some embodiments, polydonor CD4IL-10T cells express a marker protein characteristic of Tr1 cells.

[0194] In some embodiments, polydonor CDd11'11’ T cells express CD49b. In some embodiments, polydonor CD4IL-10T cells express LAG-3. In some embodiments, polydonor CD4n~'° T cells express TGF-. In some embodiments, polydonor CDd11'10T cells express 37IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOIFNy. In some embodiments, polydonor CD4IL-10T cells express granzyme B (GzB). In some embodiments, polydonor CD4IL 10T cells release granzyme B (GzB) when activated with myeloid antigen-presenting cells or myeloid tumor cells In some embodiments, polydonor CD4IL-10T cells express perforin. In some embodiments, polydonor CD4IL-10T cells release perforin when activated with myeloid antigen-presenting cells or myeloid tumor cells In some embodiments, polydonor CD4IL-10T cells express CD18. In some embodiments, polydonor CD4IL’10T cells express CD2. In some embodiments, polydonor CD4IL-10T cells express CD226. In some embodiments, polydonor CD4IL-10T cells express IL-22. In some embodiments, polydonor CD4IL’10T cells express IL-10.

[0195] In some embodiments, polydonor CD4IL-10T cells exhibit at least one phenoty pic function of Tri cells. In various embodiments, the function is secretion of IL-10, secretion of TGF-, and by the specific killing of myeloid antigen-presenting cells through the release of Granzyme B (GzB) and perforin.6.3.4. Product by process

[0196] In typical embodiments, polydonor CD4IL’10T cells are obtained by modifying CD41T cells w ith an exogenous polynucleotide encoding IL- 10.

[0197] In some embodiments, the exogenous polynucleotide is introduced to CD4+T cells by a viral vector or a plasmid vector. In particular embodiments, CD4+T cells are transduced with a lenti virus containing a coding sequence of IL- 10.

[0198] In some embodiments, polydonor CD4IL-10T cells are generated by (i) pooling primary- CD4+T cells obtained from at least two different T cell donors; and (ii) modifying the pooled CD4+T cells by introducing an exogenous polynucleotide encoding IL-10. In some embodiments, polydonor CD4IL’10T cells are generated by (i) obtaining primary CD4+T cells from at least two different T cell donors; (ii) separately modifying each donor’s CD4+T cells by introducing an exogenous polynucleotide encoding IL- 10, and then (iii) pooling the genetically modified CD4+T cells.

[0199] In some embodiments, polydonor CD4IL-10T cells have been cultured in the presence of proteins capable of activating CD4+T cells. In some embodiments, poly donor CD4IL-10T cells have been cultured in the presence of anti-CD3 antibody and anti-CD28 antibody, or anti-CD3 antibody and anti-CD28 antibody coated beads. In some embodiments, polydonor CDd11'"10T cells have been cultured in the presence of anti-CD3 antibodies, anti-38IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOCD28 antibodies, and IL-2, or anti-CD3 antibody and anti-CD28 antibody coated beads and IL-2. In some embodiments, polydonor CD4IL-10T cells have been cultured in the presence of T Cell TransAct™ from Miltenyi Biotec. In some embodiments, polydonor CD4IL’10T cells have been cultured in the presence of ImmunoCult Human T Cell Activator™ from STEMCELL Technologies.

[0200] In some embodiments, polydonor CD4IL-10T cells are in a frozen stock.6.4. Pharmaceutical compositions

[0201] In another aspect, pharmaceutical compositions are provided. The pharmaceutical comprises the poly donor CD4IL’10T cells disclosed herein and a pharmaceutically acceptable carrier or diluent.

[0202] The pharmaceutical composition can be formulated for administration by any route of administration appropriate for human or veterinary medicine. In typical embodiments, the composition is formulated for intravenous (IV) administration. In some embodiments, the composition is formulated for intravenous (IV) infusion. In embodiments formulated for IV administration, the pharmaceutical composition will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.

[0203] In some embodiments, the pharmaceutically acceptable carrier or diluent is saline, lactated Ringer’s solution, or other physiologically compatible solution. In various embodiments, the pharmaceutical composition solution comprises 2-20%, preferably 5 %, human serum albumin.

[0204] In some embodiments, unit dosage forms of the pharmaceutical composition are provided that are adapted for administration of the pharmaceutical composition by systemic administration, in particular, for intravenous administration.

[0205] In some embodiments, the unit dosage form contains 104to 1011poly donor CDd1™0T cells, 104to 1010polydonor CD4IL’10T cells, 104to 109polydonor CD4IL’10T cells, 105to IO10polydonor CD4IL-10T cells, 105to 109polydonor CD4IL-10T cells, 105to 108polydonor CD4IL-10T cells, or 105to 107polydonor CD4I, _|° T cells.

[0206] In typical embodiments, the pharmaceutical composition in the unit dosage form is in liquid form.39IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO6.5. Methods of making polydonor CD4IL-10cells

[0207] In another aspect, the present disclosure provides a method of making polydonor CD4IL’10cells.

[0208] In some embodiments, the method comprises the steps of: (i) pooling primary CD4+T cells obtained from at least two different T cell donors; and (ii) modifying the pooled CD4+T cells by introducing an exogenous polynucleotide encoding IL- 10. In other embodiments, the method comprises the steps of: (i) obtaining primary CD4+T cells from at least two different T cell donors; (ii) separately modifying each donor's CD4+T cells by introducing an exogenous polynucleotide encoding IL- 10; and then (iii) pooling the genetically modified CD4+T cells, thereby obtaining the polydonor CD4IL-10cells. Various methods known in the art can be used to introduce an exogenous polynucleotide encoding IL-10 to primary CD4+T cells.

[0209] In some embodiments, the method further comprises the step of incubating the primary CD4+T cells or genetically -modified CD4+T cells in the presence of an anti-CD3 antibody and anti-CD28 antibody, or anti-CD3 antibody and anti-CD28 antibody coated beads. In some embodiments, the method further comprises the step of incubating the primary CD4+T cells or genetically -modified CD4+T cells in the presence of anti-CD3 antibody, anti-CD28 antibody and IL-2 or anti-CD3 antibody and anti-CD28 antibody coated beads and IL-2. In some embodiments, the method further comprises the step of incubating the primary CD4+T cells or genetically-modified CD4+T cells in the presence of a mixture of feeder cells. In some embodiments, the method further comprises the step of incubating the primary CD4+T cells or genetically -modified CD4+T cells in the presence of nanopreparations of anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the incubation is done in the presence of T Cell TransAct™ from Miltenyi Biotec. In some embodiments, the incubation is done in the presence of ImmunoCult Human T Cell Activator™ from STEMCELL Technologies.

[0210] In some embodiments, the incubation step is performed before introducing an exogenous polynucleotide encoding IL- 10. In some embodiments, the incubation step is performed after (i) pooling primary CD4+T cells obtained from at least two different T cell donors; but before (ii) modifying the pooled CD4+T cells by introducing an exogenous polynucleotide encoding IL- 10. In some embodiments, the incubation step is performed after (i) obtaining primary CD4+T cells from at least tw o different T cell donors; but before (ii)40IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOseparately modifying each donor’s CD4+T cells by introducing an exogenous polynucleotide encoding IL- 10.

[0211] In some embodiments, the incubation step is performed after step (ii). In other words, in some embodiments, the incubation step is performed after (ii) modifying the pooled CD4+T cells by introducing an exogenous polynucleotide encoding IL-10. In some embodiments, the incubation step is performed after (ii) separately modifying each donor’s CD4+T cells by introducing an exogenous polynucleotide encoding IL- 10, but before (iii) pooling the genetically modified CD4+T cells, thereby obtaining the genetically-modified CD4+T cells. In some embodiments, the incubation step is performed after (iii) pooling the genetically modified CD4+T cells, thereby obtaining the polydonor CD4IL-10cells.

[0212] In some embodiments, the incubation step is performed more than once. In some embodiments, the incubation step is performed both before and after genetic modification of CD4+T cells.

[0213] In some embodiments, the exogenous polynucleotide is introduced into the primary CD4+T cells using a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the exogenous polynucleotide comprises a segment encoding IL-10 having the sequence of SEQ ID NO: 1. In some embodiments, the exogenous polynucleotide comprises a segment encoding IL-10 having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-10-encoding polynucleotide segment has the sequence of SEQ ID NO: 2. In some embodiments, the IL- 10-en coding polynucleotide segment has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the exogenous polynucleotide further comprises a segment encoding a marker permitting selection of successfully transduced CD4+ T cells. In some embodiments, the encoded selection marker is ANGFR. In certain embodiments, the encoded selection marker has the sequence of SEQ ID NO:3. In particular embodiments, the exogenous polynucleotide comprises a sequence of SEQ ID NO:4. In some embodiments, the encoded selection marker is a truncated form of human EGFR polypeptide.

[0214] In some embodiments, the method further comprises the step of isolating the genetically -modified CD4+T cells expressing the selection marker, thereby generating an enriched population of genetically -modified CD4IL’10cells.41IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0215] In some embodiments, at least 70% of the genetically-modified CD4+T cells in the enriched population express a selection marker. In some embodiments, at least 95% of the genetically-modified CD4+T cells in the enriched population express a selection marker. In some embodiments, at least 96, 97, 98, or 99% of the genetically-modified CD4+T cells in the enriched population express a selection marker.

[0216] In some embodiments, the method further comprises the step of incubating the enriched population of the genetically -modified CD4+T cells. In some embodiments, the incubation is performed in the presence of anti-CD3 antibody and anti-CD28 antibody, or anti-CD3 antibody and anti-CD28 antibody coated beads. In some embodiments, the incubation is performed further in presence of IL-2. In some embodiments, the incubation is performed in the presence of feeder cells. In some embodiments, the incubation is performed in the presence of nanopreparations of anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the incubation is performed in the presence of T Cell TransAct™ from Miltenyi Biotec. In some embodiments, the incubation is performed in the presence of ImmunoCult Human T Cell Activator™ from STEMCELL Technologies.

[0217] In some embodiments, the method further comprises the step of freezing the genetically -modified CD4+T cells.

[0218] In some embodiments, the primary CD4+T cells are from donors selected based on their HLA haplotypes. In some embodiments, the method further comprises the step of selecting T cell donors by analyzing their genetic information. In some embodiments, the method comprises the step of analyzing genetic information or HLA haplotype of potential T cell donors.

[0219] In some embodiments, the primary CD4+T cells are from donors having at least a partial HLA match with a host to be treated w ith the primary CD4+T cells or a modification thereof. In some embodiments, the primary CD4+T cells are from donors having at least a partial HLA match with a stem cell (HSC). tissue or organ donor. In some embodiments, the primary CD4+T cells are obtained from third party donors who are not biologically related with a host. In some embodiments, the primary CD4+T cells are obtained from third party donors who are not biologically related with a stem cell, tissue or organ donor.42IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0220] In some embodiments, in step (i), the primary CD4+T cells are obtained from two, three, four, five, six, seven, eight, nine, or ten different T cell donors. In some embodiments, the at least two T cell donors have at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to each other. In some embodiments, the at least two T cell donors have at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8. or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, the at least two T cell donors have 2 / 2 match at the HLA-A locus to each other. In some embodiments, the at least two T cell donors have 2 / 2 match at the HLA-B locus to each other. In some embodiments, the at least two T cell donors have 2 / 2 match at the HLA-C locus to each other. In some embodiments, the at least two T cell donors have at least 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to each other. In some embodiments, each of the at least two T cell donors has an A*02 or A*24 allele.

[0221] In some embodiments, the at least two T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci to each other. In some embodiments, the at least two T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci to each other. In some embodiments, the at least two T cell donors have less than 2 / 2 match at the HLA-A locus to each other. In some embodiments, the at least two T cell donors have less than 2 / 2 match at the HLA-B locus to each other. In some embodiments, the at least two T cell donors have less than 2 / 2 match at the HLA-C locus to each other. In some embodiments, the at least two T cell donors have less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to each other.

[0222] In some embodiments, in step (i), the primary CD4+T cells are obtained from one or more frozen stocks. In some embodiments, in step (i), the primary CD4+T cells are obtained from unfrozen peripheral blood mononuclear cells of the at least two different T cell donors. In some embodiments, the method further comprises the step of isolating CD4 T cells from the peripheral blood mononuclear cells. In some embodiments, in step (i), the primary CD4+T cells are obtained from a liquid suspension. In some embodiments, the liquid suspension is obtained from a previously frozen stock.

[0223] In some embodiments, CD4+T cells from donors are contacted with patient antigen-presenting cells (monocytes, dendritic cells, or DC-10 cells), generating allo-specific CD4+T cells that are then modified to produce high levels of IL-10 (allo-CD4IL‘10cell).43IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0224] In some embodiments, the method does not comprise the step of anergizing the CD4+T cells in the presence of peripheral blood mononuclear cells (PBMCs) from a host. In some embodiments, the method does not comprise the step of anergizing the CD4+T cells in the presence of recombinant IL- 10 protein, wherein the recombinant IL- 10 protein is not expressed from the CD4+T cells. In some embodiments, the method does not comprise the step of anergizing the CD4+T cells in the presence of DC 10 cells from a host.6.6. Methods of using polydonor CD4IL-10cells

[0225] In yet another aspect, the present disclosure provides a method of treating a patient, comprising the step of administering the polydonor CD4IL-10cells or the pharmaceutical composition provided herein to a patient in need of immune tolerization.

[0226] In some embodiments, the method further comprises the preceding step of thawing a frozen suspension of polydonor CD4IL-10cells.

[0227] In some embodiments, the polydonor CD4n‘10cells or the pharmaceutical composition prevents or reduces severity of pathogenic T cell response in the patient. In some embodiments, the polydonor CD4IL’10cells or the pharmaceutical composition reduces inflammation. In some embodiments, the polydonor CD4IL’10cells or the pharmaceutical composition enhances tissue repair. In some embodiments, the polydonor CD4IL‘10cells or the pharmaceutical composition enhances immunological tolerance to self and non-pathogenic antigens and maintain immune homeostasis. In some embodiments, the polydonor CD4IL’10cells or the pharmaceutical composition downregulates pathogenic T-cell responses associated with organ transplantation, GvHD and various autoimmune and inflammatory diseases. In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition treats autoimmune disease. In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition reduces hyperactivity of NLPR3 inflammasome or reduces symptoms associated with hyperactivity of NLPR3 inflammasome. In some embodiments, the poly donor CD4IL-10cells or the pharmaceutical composition induces death of tumor cells or reduces tumor growth. In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition increases disease free survival (e.g., absence of minimal residual disease). In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition induces wound healing or tissue repair.

[0228] In some embodiments, the poly donor CD4I,‘I" cells or the pharmaceutical composition are administered at an amount effective to prevent or reduce severity of44IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOpathogenic T cell response in the patient. In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition are administered at an amount effective to reduce inflammation. In some embodiments, the polydonor CD4IL’10cells or the pharmaceutical composition are administered at an amount effective to enhance tissue repair. In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition are administered at an amount effective to enhance immunological tolerance to self and pathogenic antigens and maintain immune homeostasis. In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition are administered at an amount effective to down regulate pathogenic T-cell responses associated with organ transplantation, GvHD and various autoimmune or inflammatory diseases. In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition are administered at an amount effective to treat autoimmune disease. In some embodiments, the polydonor CD4IL‘10cells or the pharmaceutical composition are administered at an amount effective to reduce hyperactivity of NLPR3 inflammasome or reduces symptoms associated with hyperactivity of NLPR3 inflammasome. In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition are administered at an amount effective to induce death of tumor cells or reduces tumor growth. In some embodiments, the polydonor CD4IL’10cells or the pharmaceutical composition are administered at an amount effective to increase disease free survival (e.g., absence of minimal residual disease).

[0229] In some embodiments, the treatment method further comprises monitoring polydonor CD4n’10cells in a patient after administration. In some embodiments, the method comprises the step of detecting a selection marker in a biological sample obtained from the patient, thereby detecting presence or absence of polydonor CD4IL-10T cells. In some embodiments, the selection marker is detected at multiple time points to trace changes in presence of poly donor CD4IL-10cells in a patient. In some embodiments, the biological sample is a biopsy or blood sample from the patient.

[0230] The polydonor CD4n’"' T cells are administered in a therapeutically effective amount. The amount can be determined based on the body weight and other clinical factors. In some embodiments, 103to 109cells / kg are administered. In some embodiments, 103to 108cells / kg are administered. In some embodiments, 103to 107cells / kg are administered. In some embodiments, 103to 106cells / kg are administered. In some embodiments, 103to 105cells / kg are administered. In some embodiments, 103to 104cells / kg are administered.45IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0231] In various embodiments, polydonor CD4IL-10T cells are administered on a therapeutically effective schedule. In some embodiments, polydonor CD4IL-10T cells are administered once. In some embodiments, polydonor CD4IL-10cells are administered every day, every 3 days, every 7 days, every 14 days, every 21 days, or every month.

[0232] The polydonor CD4IL-10T cells can be administered according to different administration routes, such as systemically, subcutaneously, or intraperitoneally. In some embodiments, the cells are administered within a saline or physiological solution which may contain 2-20%. preferably 5 % human serum albumin.

[0233] In some embodiments, administering the polydonor CD4IL-10is prophylactic, in terms of completely or partially preventing a disease, condition, or symptoms thereof.6.6.1. Methods of reducing or preventing GvHD

[0234] In some embodiments, the poly donor CD4n’10cells or the pharmaceutical composition comprising polydonor CD4IL-10cells is used to treat a patient before a hematopoietic stem cell (HSC) transplant (HSCT), concurrently with an HSCT, or following an HSCT.

[0235] In various embodiments, the HSCT is a matched related HSCT. In various embodiments, the HSCT is a haploidentical HSCT, a mismatched related HSCT, or a mismatched unrelated HSCT.

[0236] In some embodiments, the patient has a hematological malignancy which requires treatment with allo-HSCT. In some embodiments, the hematological malignancy is mediated by aberrant myeloid cells.

[0237] In some embodiments, T cell donors are selected based on genetic information of a patient to be treated with polydonor CD411'"’ cells and HSC, and / or genetic information of the HSC donor. In some embodiments, T cell donors are selected based on HLA haplotype of a patient to be treated with polydonor CD4IL-10cells and HSC, and / or HLA haplotype of the HSC donor. In some embodiments, the method further comprises the step, prior to administering CD4IL-10cells, of analyzing genetic information or HLA haplotype of T cell donors. In some embodiments, the method further comprises the step of analyzing genetic information or HLA haplotype of a host. In some embodiments, the method further comprises the step of analyzing genetic information or HLA haplotype of an HSC donor.46IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0238] In some embodiments, T cell donors, a host and an HSC donor are not biologically related. In some embodiments, T cell donors, a host and an HSC donor have different HLA haplotypes. In some embodiments, T cell donors, a host and an HSC donor have at least partial mismatch in HLA haplotype. In some embodiments, T cell donors are selected when they have HLA haplotype with an HLA match over a threshold value.

[0239] In some embodiments, the HSC donor is partially HLA mismatched to the patient. In some embodiments, the HSC donor has less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 match at the HLA- A, HLA-B. HLA-C, HLA-DRB1, and HLA-DQB1 loci to the patient. In some embodiments, the HSC donor has less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA- A, HLA-B, HLA-C, and HLA-DRB1 loci to the patient. In some embodiments, the HSC donor has less than 2 / 2 match at the HLA- A, HLA-B, or HLA-C locus to the patient. In some embodiments, the HSC donor has less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB 1 loci to the patient.

[0240] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the patient. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 match at the HL A- A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB 1 loci to the patient. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA- A, HLA-B, HLA-C, and HLA-DRB1 loci to the patient. In some embodiments, one or more of the T cell donors have less than 2 / 2 match at the HLA-A, HLA-B, or HLA-C locus to the patient. In some embodiments, one or more of the T cell donors have less than 2 / 4, 3 / 4 or 4 / 4 match at the HLA-DRB 1 and HLA-DQB 1 loci to the patient.

[0241] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched with the HSC donor. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB 1, and HLA-DQB1 loci to the HSC donor. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match at the HLA-A, HLA-B, HLA-C, and HLA-DRB 1 loci to the HSC donor. In some embodiments, one or more of the T cell donors have less than 2 / 2 match at the HLA-A. HLA-B, or HLA-C locus to the HSC donor. In some embodiments, one or more of the T cell donors have less than 3 / 4 or 4 / 4 match at the HLA-DRB1 and HLA-DQB1 loci to the HSC donor. In some embodiments, the47IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOpolydonor CD4n'’10cells or the pharmaceutical composition prevents or reduces severity of GvHD by the transplanted hematopoietic stem cells.

[0242] In some embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition prevents or reduces severity of pathological T cell response by the transplanted hematopoietic cells. In specific embodiments, the polydonor CD4IL-10cells prevents or reduces GvHD.

[0243] In some embodiments, the polydonor CD4n‘10cells or the pharmaceutical composition prevents or reduces severity of tissue damage induced by the pathogenic T cells or the inflammation.6.6.2. Methods of treating cancer

[0244] In some embodiments, polydonor CD4IL-10cells are used for treatment of cancer. In preferred embodiments, the polydonor CD4IL-10cells directly mediate anti-tumor effects and in particular embodiments, an anti-leukemic effect.

[0245] In some embodiments, polydonor CD4IL-10cells are administered in combination with allogeneic mononuclear cells or PBMC for treatment of cancer. In some embodiments, polydonor CD4IL‘10cells are administered prior to or subsequence to administration of PBMC. In some embodiments, polydonor CD4IL-10cells and allogeneic mononuclear cells or PBMC are administered concurrently.

[0246] In some embodiments, polydonor CD4IL-10cells and allogeneic mononuclear cells or PBMC are administered at 1:3, 1:2, 1:1, 2:1 or 3:1 ratio.

[0247] In some embodiments, the neoplastic cells express CD13. In some embodiments, the neoplastic cells express HLA-class I. In some embodiments, the neoplastic cells express CD54. In some embodiments, the neoplastic cells express CD13, HLA-class I and CD54. In some embodiments, the neoplastic cells express CD112. In some embodiments, the neoplastic cells express CD58. In some embodiments, the neoplastic cells express CD155. In some embodiments, the tumor expresses CD112, CD58. or CD155. In various embodiments, the tumor is a solid or hematological tumor.

[0248] In some embodiments, the patient has a cancer selected from the group consisting of: Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain / CNS Tumors In Adults, Brain / CNS Tumors In Children, Breast Cancer, Breast48IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOCancer In Men, Cancer of Unknown Primary, Castleman Disease, Cervical Cancer, Colon / Rectum Cancer. Endometrial Cancer. Esophagus Cancer, Ew ing Family Of Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Acute Lymphocytic (ALL), Acute Myeloid (AML, including myeloid sarcoma and leukemia cutis), Chronic Lymphocytic (CLL). Chronic Myeloid (CML) Leukemia. Chronic Myelomonocytic (CMML), Leukemia in Children, Liver Cancer, Lung Cancer, Lung Cancer with Non-Small Cell, Lung Cancer with Small Cell, Lung Carcinoid Tumor, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma In Children, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma - Adult Soft Tissue Cancer, Skin Cancer, Skin Cancer - Basal and Squamous Cell. Skin Cancer -Melanoma, Skin Cancer - Merkel Cell, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.

[0249] In some embodiments, the cancer is a myeloid tumor. In particular embodiments, the cancer is AML or CML. In some embodiments, the cancer is a myeloid tumor.

[0250] In some embodiments, the method is used to treat a hematological cancer affecting blood, bone marrow, and lymph nodes. In various embodiments, the hematological cancer is a lymphoma (e.g. Hodgkin's Lymphoma), lymphocytic leukemias, myeloma. In various embodiments, the hematological cancer is acute or chronic myelogenous (myeloid) leukemia (AML, CML), or a myelodysplastic syndrome.

[0251] In some embodiments, the cancer is refractory or resistant to a therapeutic intervention.

[0252] In some embodiments, the polydonor CD4IL-10cells are used in combination with a therapeutic intervention. The combination may be simultaneous or performed at different times. Preferably the therapeutic intervention is selected from the group consisting49IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOof: chemotherapy, radiotherapy, allo-HSCT, immune suppression, blood transfusion, bone marrow transplant, growth factors, biologicals.

[0253] In some embodiments, the polydonor CD4IL-10cells induce cell death of tumor infiltrating and tumor growth promoting myeloid lineage cells (e.g., monocytes, macrophages, neutrophils).6.6.3. Methods of treating inflammatory or autoimmune disease

[0254] In some embodiments, polydonor CD4IL’10cells are administered to treat inflammatory or autoimmune disease. In some embodiments, polydonor CD4IL‘10cells are administered to treat a disease or disorder involving hyperactivity of NLPR3 inflammasome.

[0255] The NOD-like receptor family (NLR) protein NLRP3 is an intracellular signaling molecule that senses danger signals from pathogenic, environmental or endogenous source. Following activation, NLPR3 interacts with caspase-1, forming a complex termed the inflammasome. This results in the activation of caspase- 1, which cleaves the proinflammatory cytokines IL-1β and IL-18 to their active forms and mediates a type of inflammatory cell death known as pyroptosis.

[0256] In some embodiments, polydonor CD411'11’ cells are administered to treat an inflammatory disease selected from Muckle-Wells syndrome (MWS), familial cold auto-inflammatory syndrome (FCAS) and neonatal onset multi-system inflammatory disease (NOMID). In some embodiments, polydonor CD4IL-10cells are administered to treat a chronic disease selected from metabolic syndrome, type 2 diabetes, atherosclerosis, Alzheimer, Parkinson, ALS, non-alcoholic steatohepatitis, osteoarthritis, silicosis, asbestosis, gout, and lung fibrosis. In some embodiments, poly donor CD4IL’10cells are administered to treat Crohn’s disease, Ulcerative colitis, Multiple sclerosis and systemic lupus erythromytosis or inflammatory eye diseases such as diabetic retinopathy, acute glaucoma and age related macular degeneration.

[0257] In some embodiments, polydonor CD411'"’ cells are administered to treat a disease associated with NLRP3. The disease can be selected from the group consisting of: CAPS, NASH, Alzheimer, Parkinson, cardiovascular disease, osteoarthritis, gout, pseudogout, nephrocalcinosis, type II diabetes, Sjogren syndrome, sickle cell disease (SCD). AMD, infections, cerebral malaria, asbestosis, contact hypersensitivity, sunbum, silicosis,50IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOcystic fibrosis, inflammatory bowel disease, nephrocalcitosis, ALS, myelodysplastic syndrome, and uveitis.

[0258] In some embodiments, the disease is a brain disorder selected from Parkinson, Alzheimer, age-related cognitive impairment, frontotemporal dementia, traumatic brain injury, intracerebral hemorrhage, sepsis-associated encephalopathy, cerebral ischemia, subarachnoid hemorrhage, epilepsy, acrylamide poisoning, opioid-induced neuroinflammation, chronic migraine, perioperative neurocognitive disorder, poststroke cognitive impairment, post-cardiac arrest cognitive impairment, social isolation-induced cognitive impairment, anxiety and post-traumatic stress disorder.

[0259] In some embodiments, the disease is a lung disorder selected from asthma, IR lung injury, ARDS / COPD, particulate matter-induced lung injury, radiation pneumonitis, pulmonary hypertension, sarcoidosis, cystic fibrosis, and allergic rhinitis.

[0260] In some embodiments, the disease is a heart disorder selected from atherosclerosis, heart failure, hypertension, myocardial infarction, atrial fibrillation, cardiac injury induced by metabolic dysfunction,. and endothelial dysfunction.

[0261] In some embodiments, the disease is a gastrointestinal disease, such as colitis. In some embodiments, the disease is a liver disorder selected from acute liver failure, circadian regulation of immunity, NASH, cognitive dysfunction in diabetes, IR liver injury, idiosyncratic drug-induced liver injury and liver fibrosis. In some embodiments, the disease is a pancreas or kidney disorder selected from diabetic encephalopathy, diabetes-associated atherosclerosis, insulin resistance, islet transplantation rejection, chronic crystal nephropathy, renal fibrosis, I / R kidney injury, obesity-associated renal disease, and renal hypertension. In some embodiments, the disease is a skin or eye disorder selected from psoriasis and retinal neovascularization. In some embodiments, the disease is a reproductive disorder such as preterm birth. In some embodiments, the disease is an immune disorder selected from primary dysmenorrhea, innate immunity, innate to adaptive immunity, systemic lupus erythematosus-lupus nephritis, and multiple sclerosis. In some embodiments, the disease is an inheritable disorder selected from Muckle-Wells syndrome, rheumatoid arthritis, sickle cell disease and VCP-associated disease. In some embodiments, the disease is a pain disorder selected from multiple sclerosis-associated neuropathic pain, chronic prostatitis / chronic pelvic pain, cancer-induced bone pain, and hyperalgesia. In some embodiments, the disease is cancer, such as human squamous cell carcinoma of head and neck cancer. In some51IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOembodiments, the disease is an infective disorder, such as bacterial, viral or parasitic infection.

[0262] In some embodiments, polydonor CD4IL-10cells are used in combination with a currently available treatments for NLRP3 related diseases, such as a biologic agent that target IL-1. The biologic agent includes the recombinant IL-1 receptor antagonist Anakinra, the neutralizing IL- 13 antibody Canakinumab and the soluble decoy IL-1 receptor Rilonacept.

[0263] In some embodiments, polydonor CD4IL-10cells are administered to treat a disease selected from Type 2 diabetes, metabolic syndrome, cardiovascular diseases, SLE, MS, CD, Ulcerative colitis (UC), osteoarthritis, Nonalcoholic steatohepatitis (Nash), Parkinson, ALS, lung fibrosis, silicosis, asbestosis, diabetic retinopathy, and age-related macular degeneration.

[0264] In some embodiments, polydonor CD4IL-10cells are administered to treat inflammation. The inflammation can be related to coronary' artery' disease (CAD), Type 2 diabetes, neurodegenerative diseases, or inflammatory bowel disease, but is not limited thereto.

[0265] In some embodiments, polydonor CD4IL’10cells are administered to treat a disease or disorder involving increased IL-1 [3 production by activated monocytes, macrophages or dendritic cells. In some embodiments, polydonor CD4IL-10cells are administered to treat a disease or disorder involving increased IL-18 production by activated monocytes, macrophages or dendritic cells. In some embodiments, polydonor CD4IL’10cells are administered to treat a disease or disorder involving increased mature caspase 1 production by activated monocytes, macrophages or dendritic cells.

[0266] In some embodiments, polydonor CD4IL’10cells are administered to reduce IL-ip production by7activated monocytes, macrophages or dendritic cells. In some embodiments, polydonor CD4IL-10cells are administered to reduce IL-18 production by activated monocytes, macrophages or dendritic cells. In some embodiments, polydonor CD4IL-10cells are administered to reduce mature caspase 1 production by activated monocytes, macrophages or dendritic cells.52IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO6.6.4. Methods of treating other disorders

[0267] In some embodiments, polydonor CD4IL-10cells are administered to treat autoimmune disease.

[0268] In some embodiments, the autoimmune disease is selected from the group consisting of: type-1 diabetes, autoimmune uveitis, autoimmune hepatitis, vitiligo, alopecia areata, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus, inflammatory bowel disease, Addison’s disease, Graves’ disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous diseases, scleroderma, and celiac disease. In some embodiments, the autoimmune disease is Crohn’s disease, ulcerative colitis, celiac disease, type-1 diabetes, lupus, psoriasis, psoriatic arthritis, or rheumatoid arthritis. In some embodiments, the patient has an allergic or atopic disease. The allergic or atopic disease can be selected from the group consisting of: asthma, atopic dermatitis, and rhinitis. In some embodiments, the patient has a food allergy.

[0269] In some embodiments, polydonor CD4IL-10cells are administered to prevent or reduce severity of pathogenic T cell response to cell and organ transplantation other than HSCT. In some embodiments, the method comprises the step of organ transplantation to the patient, either prior to or subsequent to administration of polydonor CD4IL-10T cells or the pharmaceutical composition. In certain embodiments, the organ is a kidney, a heart, or pancreatic islet cells. In preferred embodiments, the polydonor CD4IL-10cells or the pharmaceutical composition prevents or reduces severity of host rejection of the organ transplantation.

[0270] In some embodiments, polydonor CD4IL-10cells are administered to prevent or reduce immune response associated w ith gene therapy, e.g., administration of recombinant AAV (rAAV). In these embodiments, the method further comprises the step of administering a recombinant AAV to the patient, either prior to or subsequent to administration of the polydonor CD4IL-10cells or the pharmaceutical composition.

[0271] In some embodiments, polydonor CD4IL-10cells are administered to prevent or reduce immune response associated with transplantation of iPS-derived tissues or cells. The iPS-derived tissues and cells include, but are not limited to cardiomyocytes, hepatocytes, epithelial cells, cartilage, bone and muscle cells, neurons.53IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0272] In some embodiments, polydonor CD4IL-10cells are administered to reduce patient hyperactive immune response to viral infection. In some embodiments, the virus is SARS-coV-2. In some embodiments, poly donor CD4" ‘10cells are administered to reduce hyperactive immune responses to bacterial infections, such as toxic shock and cytokine storm.

[0273] In some embodiments, the method further comprises the step of administering an immunogenic therapeutic protein to the patient, either prior to or subsequent to administration of the population of polydonor CD4IL-10cells or the pharmaceutical composition. In some embodiments, the population of polydonor CD4IL-10cells, or the pharmaceutical composition reduces immune responses against the immunogenic therapeutic protein. In some embodiments, the immunogenic therapeutic protein is selected from a therapeutic antibody, a factor VIII replacement, a cytokine, and a cytokine mutein.6.7. Examples

[0274] The following examples are provided by way of illustration not limitation.6.7.1. Summary of experimental observations

[0275] The present disclosure provides the methods for production and use of highly purified, allogeneic CD4+T cells that have been transduced with a bidirectional lentiviral vector containing the human IL- 10 gene and a truncated, non-signaling form of the human NGFR. The successfully transduced CD4+T cells were purified utilizing aNGFR specific monoclonal antibody resulting in > 95% pure IL- 10 producing and NGFR expressing CD4+T cells (designated CD4IL-10cells). CD4n'"’ cells from 3 different allogeneic HLA mismatched donors were pooled at 1:1:1 ratios.

[0276] These pooled populations, also referred to herein as polydonor CD4IL-10cells, had cytokine production profiles comparable to those of single-donor CD4IL-10cells and naturally derived type 1 regulatory T (Tri) cells. They produce high levels of IL-10 and IL-22, variable levels of IFN-y and IL-5 and low levels of IL-4. The poly donor CD4IL’10cells were polyclonal (has multiple antigen specificities) and suppressed proliferation of both allogeneic CD4+and CD8+T cells in vitro. In addition, they specifically killed myeloid leukemia cells in vitro. Additionally, the polydonor CD4IL-10cells inhibited NLPR3 inflammasome activation and the pro inflammatory IL- 1 and IL- 18 production by human monocytes in vitro.54IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0277] Adoptive transfer of poly donor CD4IL’10cells in a humanized mouse model for Graft versus Host Disease (GvHD) indicated that these cells efficiently home to the spleen and bone marrow Adoptive transfer of poly donor CD4IL‘10cells in a humanized mouse model of GvHD inhibited severe xeno-GvHD induced by human CD4+T cells or PBMC Importantly, even at high concentrations, polydonor CD4IL-10 cells did not induce GvHD by themselves. Additionally, polydonor CD4IL-10cells had cy totoxic effects on cancer cells in an NSG mouse intravenously injected with ALL-CM cells. Injection of single- and poly donor CD41, _|0cells 3 days after administration of the ALL-CM cells (when already massive expansion of these cells is ongoing) resulted in inhibition of tumor growth. These results indicate that polydonor CD4IL-10cells have direct therapeutic anti myeloid leukemia effects in vivo. When the single- and polydonor CD4IL-10cells were administered with PBMC, the Dd11"10cells further down regulated xeno-GvHD induced by allogeneic PBMC.

[0278] These results demonstrate that polydonor CD4IL-10cells can be used for the treatment and / or prevention of GvHD; can be used as an adjunct to allogeneic hematopoietic stem cell transplant (HSCT) for treatment of leukemias and other malignancies to reduce GvHD while preserving GvL or GvT therapeutic effects of the HSCT; and for treating cell and organ rejection and autoimmune and inflammatory diseases.6.7.2. Example 1: Generation of poly donor CD4IL 10cellsVector production

[0279] Poly donor CD4IL-10cells were produced by transduction with a lentiviral vector (LV-IL-10 / ANGFR) containing coding sequences of both the human IL-10 and a truncated form of the NGFR (ANGFR) (FIGs. 1 and 2), as described in WO2016 / 146542, incorporated by reference in its entirety herein. The sequence of the plasmid encoding for human IL-10 and ANGFR (pLVIL-10) used to manufacture LV-IL- 10 / ANGFR is provided as SEQ ID NO:5. In short, pLVIL-10 was generating by ligating the coding sequence of human IL-10 from 549 bp fragment of pH15C (ATCC 68192)) into plasmid #1074.1071.hPGK. GFP. WPRE.mhCMV.dNGFR. SV40PA. The presence ofthe bidirectional promoter (human PGK promoter plus minimal core element of the CMV promoter in the opposite direction) allows co-expression of the two transgenes. The plasmid further contains a coding sequence of an antibiotic resistance gene (e.g., ampicillin or kanamycin).55IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0280] The lentiviral vectors were produced by Ca3PO4transient four-plasmid co-transfection into 293T cells and concentrated by ultracentrifugation: 1 pM sodium butyrate was added to the cultures for vector collection. Titer was estimated on 293T cells by limiting dilution, and vector particles were measured by HIV-1 Gag p24 antigen immune capture (NEN Life Science Products; Waltham, MA). Vector infectivity was calculated as the ratio between titer and particle. For concentrated vectors, titers ranged from 5x108to 6x109transducing units / mL, and infectivity from 5x104to 5x105transducing units / ng.Production of CD4IL 10cells

[0281] FIG. 3 is a schematic representation of the production process of CD4IL-10cells. CD4 T cells from healthy donors were purified. Human CD4+T cells were activated with soluble anti-CD3, soluble anti-CD28 mAbs, and rhIL-2 (50 U / mL) for up to 48 hours before transduction with a bidirectional lentiviral vector encoding for human IL-10 and a truncated form the human NGF receptor (LV-IL-10 / ANGFR) at multiplicity of infection (MOI) of 20.

[0282] After 9-11 days, transduced cells were analyzed by FACS for the expression of ANGFR, and the vector copy number (VCN) was quantified by digital droplet PCR (ddPCR).

[0283] The mean transduction efficiency of CD4+T cells from 10 different donors was 45 ± 17% with VCN of 2.7 ± 0.6%. FIG. 4A shows percentages of CD4+ΔNGFR+cells (mean± SD, n=10 left bar) and vector copy numbers (VCN, mean + SD, n=10 right bar) in human CD4+T cells transduced with LV-IL-10 / ANGFR (a bidirectional lentiviral vector encoding for human IL- 10 and a truncated form the human NGF receptor). The frequency of CD4+ANGFR+cells and the vector copy numbers were quantified by digital droplet PCR (ddPCR) in CD4IL-10cells.

[0284] ANGFR+T cells were purified using anti-CD271 mAb-coated microbeads and resulted in > 95% pure CD4IL-10cells populations. After purification, cells were stained with markers for CD4 and ANGFR and analyzed by FACS. The data showed purity resulting from the purification step was over 98%. FIG. 4B shows FACS data from two representative donors (Donor B and Donor C) out of 10 donors tested. The purity of the CD4IL-10cells for these two donors was respectively 98.3% and 99.2%. The purified CD4IL-10cells wereIPTS / 200307516.4Attorney Docket Ref: TR1X-012WOrestimulated 3 times at 14 day intervals and their in vitro and in vivo functions were tested after the second (TF2) and or third restimulation (TF3) functions.

[0285] Resting CD4IL-10cells produced IL- 10 constitutively. Upon activation, the level of IL- 10 produced was strongly enhanced.CD41L 10cells have a cytokine production profile which is comparable to that of naturally derived Tri cells.

[0286] Cytokine production profiles of single donor CD4IL 10cells were analyzed after the second (TF2) and third (TF3) restimulation and the results are provided in FIG. 5. Specifically, CD4IL-10cells (2x105cells in 200 pl) were restimulated as previously described (Andolfi et al. Mol Then 2012;20(9): 1778-1790 and Locafaro et al. Mol Ther.2017;25(10):2254-2269). At day 14, after the 2ndround (TF2) and 3rdround (TF3) of restimulation, CD4IL-10cells were left unstimulated or were activated with with immobilized CD3 (10μg / mL) and soluble CD28 mAb (1μg / mL) for 48 hours. Culture supernatants were collected and levels of IL-10, IL-4, IL-5, IFN-y and IL-22 were determined by ELISA. All samples were tested in triplicate. Mean± SD, n=8 donors tested are presented. The results provided in FIG. 5 show that CD4IL’10cells stimulated with immobilized anti-CD3 and soluble anti-CD28 mAbs show a Tri cell cytokine production profile.

[0287] Although variations between the different donors were observed, the overall cytokine production profiles after the second (TF2) (FIG. 5 left panel) or the third (TF3) (FIG. 5 right panel) restimulation were comparable and reflected those of Tri cells (Roncarolo et al., Immunity. 2018). Like Tri cells, the CD4IL-10cells produced high levels of IL- 10 and IL-22, variable levels of IL-5 and IFN-y, but relatively low levels of IL-4 and undetectable levels of IL -2 (not shown).CD4IL-10cells express high levels of Granzyme B and selectively kill myeloid leukemia cells

[0288] The CD4IL-10cells were further analyzed after the 2ndround (TF2) of restimulation for expression of granzyme B (GzB). The data in FIG. 6A show that more than 95 % of all CD4IL-10cells derived from 7 different donors expressed high levels of Granzyme B.57IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0289] The CD4IL-10cells from the 2ndround (TF2) of restimulation were further analyzed for their cytotoxic effects against a human myeloid leukemia cell line (ALL-CM) and an erythroid leukemia cell line (K562). CD4IL-10cells (105 / well) were co-cultured with K562 and ALL-CM cells (105 / well) at 1:1 ratio for 3 days. Residual leukemic cell lines (CD45low, CD3-) were counted by FACS for each target cell.

[0290] The CD4IL-10cells selectively killed the myeloid leukemia cells (ALL-CM) as shown in FIG. 6B. The % of killed ALL-CM cells varied between 62% and 100%. whereas the killing of the erythroid leukemia cell line K562 (which are highly sensitive for nonspecific cytotoxic and natural killer (NK) cell activities) varied between 0 and 27% (4 different donors tested). Taken together, these data confirm that CD4IL-10cells express Granzyme B and efficiently kill myeloid leukemia cells. As expected, some variations in the killing capacity of the CD4IL-10cells derived from individual donors was observed.CD4IL-10cells suppress the proliferative responses of both allogeneic CD4+and CD8+T cells

[0291] The CD4IL’10cells were also analyzed for their effects on allogeneic CD4+T cells or CD8+T cells. Specifically, allogeneic PBMC cells were labeled with eFluor® 670 (5x104cells / well) and stimulated with allogeneic mature dendritic (mDC) cells (5x103cells / well) and soluble anti-CD3 mAbs in the absence or presence of CD4IL-10cells (5x104cells / well) at a 1:1 Responder: Suppressor ratio. After 3 days of culture, the percentages of proliferating responder cells were determined by eFluor® 670 dilution with flow cytometry after gating on CD4 ANGFR T cells or CD8 ANGFR T cells. FIGs. 7A and 7B show effects of CD4IL-10cells from six different, unpooled, donors (Donor-C, Donor-E, and Donor-F in FIG. 7 A and Donor-H, Donor-I, and Donor-L in FIG. 7B) on CD4+T cells with percentages of proliferation and suppression. FIGs. 8A and 8B show effects of CD4n’10cells from six different single donors (Donor -C, Donor-E, and Donor-F in FIG. 8A and Donor-H. Donor-I, and Donor-L in FIG. 8B) on CD8+T cell proliferation.

[0292] The results demonstrated that CD4IL’10cells from 6 different single donors, unpooled and tested separately, downregulated the proliferative responses of both allogeneic CD4+and CD8+T cells. The suppressive effects on the CD4+T cells varied between 51% and 96%, while the suppressive effects on the CD8+T cells varied between 62% and 73 %.58IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOProduction and characterization of polydonor CD4IL-10cells

[0293] CD4IL-10cells were generated as described above and FIG. 3 using CD4+cells from multiple donors. CD4IL-10cells from each donor were stimulated by the second (TF2) and third (TF3) restimulation. After the third stimulation. CD4IL’10cells from the three donors were pooled at a 1: 1: 1 ratio and stimulated with with immobilized CD3 (10μg / mL) and soluble CD28 mAb (1μg / mL) for 48hrs.Polydonor CD4IL-10cells have a cytokine production profile which is comparable to that of CD4IL-10cells of individual donors and Tri cells.

[0294] Culture supernatants were collected and levels of IL-10, IL-4, IL-5, IFN-y and IL-22 were determined by ELISA. The results provided in FIG. 9 show that the cytokine production of polydonor CD4IL-10cells pooled from 3 different allogeneic donors (pooled 1:1:1) (black dot) was comparable to that of CD4IL-10cells from individual donor (n=8) derived CD4IL-10cells (gray bars). The polydonor CD4IL-10cells produced high levels of IL-10 and IL-22, variable levels of IL-5. IFN-y and low levels of IL-4 and undetectable levels of IL-2 (not shown). These data indicate that it is feasible to pool CD4IL-10cells and that these polydonor CD4IL-10cells maintain the cytokine production signature of single donor derived CD4IL-10cells and Tri cells. Importantly, the pooled allogeneic cell populations contained > 95% viable cells indicating that they did not kill each other.Polydonor CD4IL 10cells express high levels of Granzyme B and kill myeloid leukemia cell lines.

[0295] The polydonor CD411’1" cells were further analyzed after 3rdround (TF3) of restimulation for expression of granzyme B (GzB). The data in FIG. 10A show that most of the polydonor CD4IL-10cells express GzB. Over 95 % of the polydonor CD4IL’10cells expressed Granzyme B, comparable to the GzB expression of single donor derived CD4IL-10cells (FIG. 10A).

[0296] The CD4IL-10cells from 3rdround (TF3) of restimulation were further analyzed for their cytotoxic effects on myeloid leukemia cells (ALL-CM cell line) or K562. The polydonor CD4IL’10cells (105 / well) were co-cultured with K562 and ALL-CM cells (105 / well) at 1:1 ratio for 3 days. Residual leukemic cell lines (CD45low, CD3 ) were counted by FACS for each target cell. The results provided in FIG. 10B show that some level of cytotoxicity' against K562 cells, which are highly sensitive for nonspecific cytotoxicity.59IPTS / 200307516.4Attorney Docket Ref: TR1X-012WONevertheless, a level of selective killing of the polydonor CD4IL-10cells (black dot) towards myeloid leukemia cells (ALL-CM) was obtained which is comparable to that of single donor derived CD4IL’10cells (open bar).Polydonor CD41L 10cells suppress the proliferative responses of both allogeneic CD4+ and CD8+ T cells.

[0297] The polydonor CD4IL-10cells were also analyzed for their effects on allogeneic CD4+T cells or CD8+T cells. Specifically, allogeneic PBMC cells were labeled with eFluor® 670 (5x104cells / well) and stimulated with allogenic mature dendritic (DC) cells (5x103cells / well) and soluble anti-CD3 mAbs in the absence or presence of polydonor CD4n‘10cells (5x104cells / well) at a 1:1 Responder: Suppressor ratio. After 3 days of culture, the percentages of proliferating responder cells were determined by eFluor® 670 dilution with flow' cytometry after gating on CD4+ΔNGFR-T cells or CD8+ΔNGFR-T cells.FIG. 11A shows results from polydonor CD4IL-10cells containing CD4IL-10cells from Donor-C, Donor-E, and Donor-F (C-E-F). FIG. 1 IB shows results from polydonor CD4IL-10cells containing CD4IL‘10cells from Donor-H, Donor-I, and Donor-L (H-I-L), which had been frozen, stored and thawed prior to testing.

[0298] FIG. 11 A shows that the polydonor CD4IL-10cells (from 3 different donors) suppress CD4+and CD8+T-cell responses by 97% and 74%, respectively. Comparable results were obtained with a second, different batch of polydonor CD4IL-10cells which was tested after the cells had been frozen, stored and thawed prior to testing (FIG. 1 IB).Suppression of CD4+and CD8+T cell proliferation was 68% and 75 %, respectively. These data indicate that polydonor CD4IL-10cells can be frozen, stored, and thawed without loss of function.

[0299] Collectively the data obtained with polydonor CD4IL10cells indicate that these cell preparations can be pooled without any problems. They contain > 95 % viable cells and maintain all the relevant functions (cytokine production, cytotoxic capacity, and suppression of allogeneic T cell responses) of single donor CD4IL’10cells. The use of larger pools of polydonor CD4IL-10cells should reduce the natural variations observed between CD4IL-10cell lots originating from different individual donors, and should provide a large quantity of off-the-shelf CD4IL-10cells for human therapy.60IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0300] A polydonor CD4n'’10cell product will have significant advantages in terms of a more homogeneous product which will allow the determination of well defined, less lot-to-lot variation, potency, and release criteria. In addition, it will enable the development of a continuous large-scale cell production process.Other methods for production of polydonor CD41L 10cells

[0301] Before the lentiviral transduction, huffy coats from minimally 3-5 different donors are pooled. CD4+cells are isolated from huffy coats by positive selection using anti-CD4 antibody. Purify of the pooled CD4+cells is checked by FACS. Alternatively, frozen human CD4+cells are obtained from minimally 3-5 normal healthy donors. The frozen human CD41cells are thawed before use. CD41cells from buffy coats or frozen stocks are activated for 24-48 hours by a combination of CD3 and CD28 antibodies or CD3- and CD28 antibody coated beads in the presence of IL-2. In some cases, CD4+cells from buffy coats or frozen stocks are activated with soluble anti-CD3, soluble anti-CD28 mAbs, and rhIL-2 (50 U / mL) for 48 hours and transduced with a bidirectional lentiviral vector encoding for human IL-10 as described above for production of CD4IL-1° cells.

[0302] In some cases, the HLA haplotype of the T cell donors (or CD41cells isolated from the donors) are first determined and CD4+cells having desired HLA haplotypes are selectively pooled and used.

[0303] Polydonor CD4n -|(' cells are generated by transducing the activated CD4+cells described above with the lentiviral vector containing human IL- 10 and ANGFR coding sequences described above.

[0304] On Day 7-11, which is 5-9 days after the transduction, the cells are harvested and successfully transduced T cells purified utilizing an anti-NGFR antibody. This process generally results in 95% pure populations of polydonor CD4IL‘10cells.

[0305] The purified polydonor CD4IL 10cells are counted and re-stimulated by a mixture of CD3- and CD28 antibodies, CD3- and CD28 antibody coated beads, optionally in the presence of feeder cells for another 8-10 days in the presence of IL-2. In some cases, the purified polydonor CD4IL-10cells are re-stimulated in the presence of feeder cells.

[0306] After a total culture period of 5 weeks, CD4IL-10cells are harvested, counted and tested for their capacity to produce IL- 10 spontaneously or following activation with CD3 and CD28 antibodies or CD3 and CD28 antibody coated beads. Additionally, the levels 61IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOof GrzB and perforin are measured. Their capacity to suppress human T cell (PBMC) and purified CD4+and CD8+T cell proliferation are also tested.

[0307] In addition, the production of IL-22 is measured both constitutively and following activation of 200,000 CD4IL‘10cells in a volume of 200 microliter using a combination of CD3 and CD28 antibodies as described previously for the production of other cytokines such as IFNy, IL-10, IL-4 and IL-5. IL-22 production levels are measured in IL-22 specific ELISA as described for the other cytokines in WO2016 / 146542. The pooled CD4IL-10cells are frozen before storage.6.7.3. Example 2: Treatment or prevention of GvHD using polydonor CD4IL 10cellsEffects of polydonor CD4IL-10cells in vivo.

[0308] A population of polydonor CD4IL-10cells were tested in a humanized xeno GvHD disease model, an NSG mouse model, for their effect on xeno-GvHD induced by human PBMC as illustrated in FIG. 12. NSG mice were sub-lethally irradiated and intravenously injected with (i) human PBMC (5x106cells / mouse), (ii) polydonor (three donors; BC-C / E / F) CD4IL’10cells (5x106cells / mouse), or (iii) with human PBMC (5x106cells / mouse) in combination with polydonor CD4IL-10cells (BC-C / E / F) (5x106cells / mouse). Xeno-GvHD was evaluated as previously described (Bondanza et al. Blood 2006) based on survival, weight loss (>20% weight loss), skin lesions, fur condition, activity, and hunch.

[0309] FIG. 13 shows % of NSG mice free of xeno-GvHD on each day after injection. Administration of 5x 106human PBMC to irradiated NSG mice resulted unexpectedly in an unusually fulminant xeno-GvHD. All mice died at day 10 which reflects very lethal xeno-GvHD. Co-administration of 5x106polydonor CD4IL-10cells delayed this fulminant xeno-GvHD, but the mice were sacrificed at day 14 because they reached the prespecified humane 20 % body weight loss criterion for sacrifice (FIG. 13). Nevertheless, these results indicate that polydonor CD4n -,° can delay extremely severe xeno-GvHD.Importantly, polydonor CD4IL-10cells administered alone at the same dose as the PBMC (5x106cells) failed to induce any sign of xeno-GvHD.

[0310] The presence of human CD4IL-10cells was also tested in the spleen (FIG. 14, left panels) and bone marrow (FIG. 14, right panels) of the NSG mice injected with human PBMC (5x106cells / mouse), polydonor (three donors; BC-C / E / F) CD4IL-10cells (5x106cells / mouse), or human PBMC (5x106cells / mouse) in combination with polydonor CD4IL’1062IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOcells (three donors; BC-C / E / F)) (5x106cells / mouse) at 14 days post injection. The results provided in FIG. 14 show that polydonor CD4IL’10cells migrated to spleen and bone marrow. Low percentages of these cells were found to be present 14 days after infusion of the cells. These results indicate that polydonor CD4n''0cells delayed fulminant xeno-GvHD induced by human PBMC and that they do not themselves induce any xeno-GvHD.Polydonor CD4IL~10cells inhibit severe xeno-GvHD by purified CD4+cells.

[0311] Polydonor CD4IL-10cells were tested in a humanized xeno-GvHD model in which GvHD disease was induced by administration of 2.5 x 106purified human CD4+T cells as illustrated in FIG. 15. NSG mice were sub-lethally irradiated at day 0 and on day 3 were intravenously injected with human CD41T cells (2.5x106cells / mouse) alone or in combination with polydonor CD4IL-10cells (three different donors; BC-H / T / L) (2.5x106cells / mouse) or with CD4IL-10cells from a single donor (BC-H) from the pool (2.5x106cells / mouse). Xeno-GvHD was evaluated as previously described (Bondanza et al. Blood 2006) based on survival, weight loss (>20% weight loss), skin lesions, fur condition, activity, and hunch.

[0312] FIG. 16 shows % of NSG mice free of GvHD on each day after injection. The results show that polydonor CD4IL’10(BC-H / I / L) cells can inhibit the xeno-GvHD mediated by human allogeneic CD4+T cells. In this experiment, xeno-GvHD was very7severe, because all mice in the control group which received CD4+ T cells were dead at day 20. In contrast, co-administration of 2.5 x 106polydonor CD4IL-10inhibited GvHD by 75 %. Single-donor CD4IL-10cells were also protective but the effects were less potent.Other experiments

[0313] Therapeutic effects of the polydonor CD4n’10cells are tested in four different groups of mice: (i) mice receiving human PBMC from a donor unrelated to the CD4IL’10cells (xeno-GvHD positive control); (ii) mice receiving the polydonor CD4IL-10cells (negative control); (iii) mice receiving a combination of PBMC and the polydonor CDd117'10cells at 1: 1 ratio; and (iv) mice receiving a combination of PBMC and the polydonor CD4IL’10cells at 2: 1 ratio or at different ratios. Among animals receiving combination of PBMC and the polydonor CD411"10cells, some animals receive PBMC and the polydonor CD4" ‘l0cells concurrently, some animals receive polydonor CD4IL-10cells several days (e.g., 5 days) after63IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOreceiving PBMC, and some animals receive poly donor CD4n'’10cells several days (e.g., 5 days) before receiving PBMC.

[0314] The mice are monitored for development of GvHD by measuring weight at weeks 1, 2. 3, 4, and if necessary week 5, after administration of PBMC and / or the poly donor CD4IL-10cells. In addition to weight loss, the mice are inspected for skin lesions, fur condition and activity7. The mice in the treatment groups are monitored for additional periods to determine effects of the poly donor CD4IL-10cells on long term survival.

[0315] The amount and localization of the poly donor CD4IL-10cells are also monitored in peripheral blood and tissues after administration. Specifically, presence of poly donor CD4IL-10cells are monitored in peripheral blood and at sites of inflammation: spleen and bone marrow. Other sites presence of poly donor CD4IL-10cells are monitored include lymph nodes and gut. The mice in the treatment group(s) are monitored for an additional 3 weeks to determine long-term survival.

[0316] The results demonstrate that poly donor CD4IL’10cells are effective in reducing and preventing xeno-GvHD.6.7.4. Example 3: Inhibition of GvHD and treatment of cancer

[0317] A population of poly donor CD4IL-10cells are tested in an NSG mouse model transplanted with human PBMC and AML tumor cells for their effect on xeno-GvHD induced by human PBMC and anti -tumor effects. AML cells (ALL-CM) are administered i.v. as described previously in WO 2016 / 146542. PBMC or polydonor CD4IL-10cells or combinations thereof are administered 3 days later.

[0318] Poly donor CD4IL-10cells are obtained as described in Example 1. Therapeutic effects of the poly donor CD4n -1" cells are tested in four different groups of mice, each having received irradiation and 5x106ALL-CM cells (AML mice) at day 0: (i) AML mice without additional treatment; (ii) AML mice receiving 5x106human PBMC from a donor unrelated to the poly donor CDd11'1" cells - the PBMCs cause severe xeno-GvHD; (iii) AML mice receiving 2.5x106polydonor CD4ir -10cells; and (iv) AML mice receiving combinations of PBMC and the poly donor CD4IL’10cells at 1: 1 or 2: 1 ratio or at different ratios. One additional group of mice do not receive ALL-CML cells but receive 5x106human PBMC at day 3 after irradiation.64IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0319] Effects of the poly donor CD4n’10cells on xeno-GvHD induced by human PBMC are tested based on weight loss, skin lesions, fur condition, activity, death rate and long-term survival. Anti-tumor or graft versus leukemia (GvL) effects of the poly donor CD4IL-10cells are tested based on reduction of tumor cells in the circulation and long-term tumor free survival.

[0320] Some mice are monitored for up to 7 weeks in order to monitor long-term survival and complete tumor remissions.

[0321] Results demonstrate that polydonor CD4IL-10cells are effective in both inhibition of xeno-GvHD and treatment of cancer.6.7.5. Example 4: Treatment of cancer using polydonor CD4IL 10cells

[0322] A population of polydonor CD4IL-10cells were tested in an ALL-CM leukemia model of T cell therapy in NSG mice.

[0323] NSG mice were sub-lethally irradiated and intravenously injected with myeloid leukemia cells (ALL-CM) (2.5x106) at day 0. In the first group of animals, no additional cells were administered. In the second group of animals PBMC (2.5x106) were injected at day 3. In the third group of animals, polydonor CD4IL-10cells (2.5x106) were injected at day 3. In the fourth group of animals, single donor (from donor BC-I) CD4IL-10cells (2.5x106) were injected at day 3. In the fifth group of animals, single donor (from donor BC-H) CD4IL 10cells (2.5x106) were injected at day 3. Graft-versus-leukemia (GvL) effect was tested in the animals based on reduction of circulating leukemia cells and long-term leukemia free survival. Leukemia was measured as previously described (Locafaro G. et al Molecular Therapy 2017). See FIG 17A.

[0324] As provided in FIG. 17B and FIG. 17C, all of the mice injected with ALL-CM myeloid leukemia cells alone had extensive leukemia progression at day 17. Administration of 2.5x106PBMC resulted in a strong inhibition of leukemia progression. Interestingly, a comparable level of inhibition of leukemia progression was obtained by both single-donor CD ^10(Figl7B) or polydonor CD4IL10(Figl7C) cells. These data indicate that single donor and polydonor CD4IL1° have strong direct anti leukemia effects.

[0325] Graft-versus-leukemia (GvL) effects of single-donor CD4IL1° and polydonor CD4n 10were further tested in combination with PBMC in mice injected with ALL-CM myeloid leukemia cells (FIG. 18A). Administration of 2.5x106PBMC resulted in a strong 65IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOinhibition of leukemia progression. Administration of 2.5x106PBMC combined with single donor CD4IL10(2.5x106) cells resulted in a stronger inhibition of leukemia progression (FIG.18B). Administration of 2.5x106PBMC combined with 2.5x106polydonor CD4IL10cells had a comparable synergistic inhibition of leukemia effect to the single donor CD4IL1° (2.5x106) cells (FIG.18C). These data indicate that poly donor CD4IL1° cells do not interfere with the protective GVL effects of the PBMC, but act in synergy with the PBMC to mediate strong GvL effects.6.7.6. Example 5: Treatment of chronic inflammatory and autoimmune diseases using polydonor CD4IL 10cells

[0326] Activation of the NLPR3 inflammasome has been implicated in many chronic inflammatory and autoimmune diseases. The NLPR3 inflammasome can be activated by “danger signals” which lead to caspase 1 -mediated production of the pro-inflammatory cytokines IL-1 p and IL- 18 by monocytes / macrophages. A series of in vitro experiments are performed to investigate the effects of poly donor CD4IL’10cells on the NLPR3 inflammasome and IL-1 p / IL-18 production by human monocytes.

[0327] First, human PBMC are isolated from peripheral blood by standard density centrifugation on Ficoll / Paque (Sigma- Aldrich). Monocytes are isolated from the human PBMC by negative selection using monocyte isolation kit II (Miltenyi) according to the manufacturer’s instructions. Negative selection is preferred because positive selection or adherence can lead to undesired activation of the cells. Isolated monocytes are plated at 5x104cells / 200 pl in the presence of 2xl05or IxlO5polydonor CD4IL-10cells / 200 pl per well in 96-well microtiter plates in culture medium containing 3% toxin free human AB serum.

[0328] Table 1 summarizes treatment conditions applied to 9 sets of monocytes, each set including 6 wells of cells.Table 1Group # Monocytes CD4 Inhibitors* LPS** Other*** cells / supernatantMedium controlIncubation time: 1 hour; followed by activation by LPS + / - Nigericin as indicated in FIG 19A-G1 Monocytes No No No LPS2 Monocytes No No LPS3 Monocytes No Z-YVADfmk LPS4 Monocytes No MCC950 LPS66IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOCulture of monocytes in the presence of supernatants**** obtained from single donor or polydonor CD4IL 10cell- or from CD4GFFcell-culture 5 Monocytes 50% supernatant No LPSof CD4IL-10cells6 Monocytes 25% supernatant No LPSof CD4IL-10cells7 Monocytes 12.5%supematant No LPSof CD41L'10cells8 Monocytes 50% supernatant No LPS Anti-IL-10 ofCD41L'10cells receptor antibody 9 Monocytes 50% supernatant No LPSof CD4GFPcells* Z-YVADfmk is an inhibitor specific to caspase 1. 20 uM Z-YVADfmk (Biovision. Enzo Life Sciences, or Axxora Life Sciences) dissolved in DMSO is used as indicated. MCC950 is an NLRP3 inhibitor. 10 uM MCC950 (Invivogen) is used as indicated.** LPS (Sigma- Aldrich) lOOng / mL plus nigericin as indicated (Nig, Invivogen) 10 uM added during the last 30m of LPS incubation.***Anti-IL-l OR antibody (Biolegend) 30ug / mL.**** Supernatants of CD4IL’10and CD4 GFP cultures are obtained by incubating CD4IL’10or CD4 GFP cells at lX106 / mL for 3days and collecting the supernatants. IL- 10 production levels are measured by IL-10 specific ELISA.

[0329] After treatments outlined in Table 1, supernatants are collected from 6 wells for each group and IL-1 [3 / IL-18 production is measured by ELISA specific for mature IL- 1 P or IL-18 (Biolegend). Cells collected from 6 wells for select groups are analyzed by Western Blot to determine levels of activated caspase 1.

[0330] Data from the experiments show that polydonor CD4IL-10cells down-regulate IL- i and IL- 18 production by activated monocytes. They further show that poly donor CD4IL-10cells down-regulate mature caspase- 1 production in activated monocytes.Additionally, poly donor CD4IL‘10and IL-10 produced by the poly donor CD4IL’10down-regulate inflammasome.

[0331] Similar experiments are performed with human macrophages or dendritic cells instead of monocytes. Results from the experiments demonstrate that polydonor CD4IL-10cells further down-regulate IL-ip, IL-18, and mature caspase-1 production from activated macrophages and dendritic cells.

[0332] These data suggest that polydonor CD4IL-10cells can be used to treat diseases or disorders involving hyperactivation of NLPR3 inflammasome. In particular, polydonor CD4n _'0cells can be used to treat chronic inflammatory and autoimmune diseases. The67IPTS / 200307516.4Attorney Docket Ref: TR1X-012WONLPR3 inflammasome can be activated by exogenous or endogenous “danger signals’", such as Pathogen Associated Molecular Patterns (PAMPs). silica, asbestos, Danger Associated Molecular Patterns (DAMPs) like products from damaged mitochondria, necrotic and stressed cells, and uremic acid crystals.6.7.7. Example 6: Supernatant of polydonor CD41L 10cells inhibit NLPR3 Inflammasome activation and IL-ip and IL-18 production by human monocytes

[0333] CD14+monocytes were isolated from PBMC using a pan monocyte isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and plated in 96 flat microtiter wells at 2x I O5 / 2OOpL per well and cultured in the presence of LPS. The cells were cultured further in the presence of Z-YVADfmk (20 microMol), MMC950 (lOmicroMol), IL-10 (lOng / mL) or various concentrations of single- or pooled donor CD4IL-10cell supernatants as summarized in Table 1.

[0334] The supernatants were obtained from single- or pooled donor CD4IL-10cells activated for 72 hours with a combination of CD3 and CD28 antibodies as described previously. (Andolfi et al. 2012, Mol. Therapy Vol. 20, 1778-1790, Locafaro et al. Mol Ther 2017, 25, 2254) In some cases (FIG. 19C and 19D), the monocytes were incubated with LPS in combination with the NLPR3 inflammasome activator nigericin (“NIG”) which was added during the last 30 minutes of the LPS activation.

[0335] The NLPR3 inflammasome was activated by LPS. resulting in the production of mature caspase 1 and the biologically active forms of IL-10 and IL-18. Monocytes plated in the absence of LPS activation did not produce detectable levels of IL- 10 during the incubation period (not shown).

[0336] Addition of the supernatant of single donor derived CD4IL-10cells (containing 1769 pg IL-10 / mL) inhibited IL-10 production by LPS activated monocytes from donor #1 and #2 at concentrations of 50%, 25% and 12.5 % respectively, in a dose dependent fashion (FIG. 19A and FIG. 19B). Supernatant of CD4+ cells transduced with GFP was used as a control. Complete inhibition of IL-10 production is observed at concentrations of 50% and 25%, whereas supernatants of GFP transduced control CD4+ cells at concentrations of 50% were ineffective.

[0337] Various concentrations of CD4IL-10T cell supernatant (50%, 25% or 12.5%), Z-YVADfmk or MCC950 were further tested on monocytes activated with LPS and nigericin 68IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO(“NIG’'). Supernatants from single donor (BC-E) or pooled donor CD4IL’10cells contained 5295 or 3532 pg IL-10 / mL respectively. Supernatants of single donor CD4IL 10cells were also very effective in inhibiting LPS induced IL-1 [3 production enhanced by the NLPR3 inflammasome activator nigericin (FIG. 19C and FIG. 19D).

[0338] The data demonstrate that the supernatants from CD41| _|" cells at concentrations of 50% were as effective as the irreversible caspase 1 inhibitor Z-YVADfmk (Guo et al. 2015, Nature Med 21, 677), the selective NLPR3 inflammasome inhibitor MCC950 (Coll et al. 2019. Nature Chem. Biol 15.556) and recombinant IL-10, indicating that IL-10 containing supernatants inhibit NLPR3 inflammasome activation and mature caspasel production resulting in strong inhibition of the production of the proinflammatory cytokine IL-ip (FIG. 19A-19D).

[0339] Comparable results were obtained in second series of experiments with supernatants of single donor (BC-E) and pooled CD4IL-10cells from 2 different donors (BC-C / E). The CD4IL 10cells were activated by a combination of CD3 and CD28 antibodies as described (Andolfi et al. 2012). After 3 days the supernatants from the CD4IL‘10cells were collected. These supernatants contained 5295 and 3532 pg IL-10 / mL respectively, and inhibited LPS induced IL-ip production by monocytes from donor #3 in a dose dependent fashion (FIG. 19E). Supernatants at concentrations of 50% were as effective as Z-YVADfmk and MCC950. The inhibitory effects of the supernatants of the pooled CD4IL-10cells were completely neutralized by an anti-IL-10 receptor antibody. Similarly, supernatants pooled from 3 different donors containing 2589 pgIL-10 / mL, dose dependently inhibited IL-ip production by monocytes from donor #4 (FIG. 19F). The inhibitory effects of the supernatants are completely neutralized by an IL- 10 receptor antibody demonstrating that NLPR3 activation is mediated by IL- 10. The results indicate that production of the pro inflammatory cytokine IL-1 P is strongly inhibited by IL-10 produced by the poly donor CD4IL-1° T cells. As expected, the anti IL-10 receptor antibody had no effect of the inhibition of IL-1 p production mediated by Z-VADfmk and MCC950 (FIG. 19E).

[0340] CD4IL-10T cells were further tested on monocytes from donor #4 activated by LPS and nigericin. Various concentrations of single donor (BC-V) or polydonor (three donors; BC-T / U / V) CD4IL-10cell supernatants containing 2583 or 2589pg IL-10 / mL respectively, ZYVADfmk or MCC950 were tested. Data provided in FIG. 19G show that69IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOpooled supernatants of 3 different donors (BC T-U-V) down regulate IL-18 production induced by LPS in combination with nigericin.

[0341] Collectively, these data indicate that IL-10 produced by single- and polydonor CD4IL-10cells strongly down regulates the NLPR3 inflammasome resulting in strong inhibition of the pro inflammatory cytokines IL-1 [3 and IL-18.6.7.8. Example 7: Single-donor and polydonor CD4IL 10cells inhibit xeno GvHD and myeloid tumor growth in vivo

[0342] Functional properties and quality7of the single donor (BC-T, BC-V, and BC-E) or polydonor (BC-V / T / E) CD4IL-10cells were tested as described in Andolfi et al. Mol Ther 2012. 20, 177 and Locafaro et al. Mol Ther 2017, 25, 2254. Both single donor- and poly donor CD4IL-10cells produced high levels of IL-10, variable levels of IFN-y, very low levels of IL-4 and no detectable IL-2 (the latter not shown), reflecting the characteristic cytokine production profde of Tri cells (FIG. 21).

[0343] Further, the suppressive capacity of the single donor (BC-T, BC-V, and BC-E) or polydonor (BC-V / T / E) CD4IL-10cells on CD4+and CD8+T cell proliferation was measured in vitro on allogeneic PBMC. PBMC were labeled with eFLuor670 (Invitrogen). Labeled PBMC (IxlO5) were activated with immobilized CD3 (lOpg / mL) and soluble CD28 antibodies (Ipg / mL). Single and polydonor CD4IL-10cells were added at a 1: 1 ratio in a final volume of 0.2mL in 96 well round bottom plates. After 4 days of co-culture, their suppressive effects on the proliferation of eFluor670 labeled responder cells was determined by eFluor670 dilution using flow cytometry as described (Locafaro et al. Mol Ther 2017, 25, 2254). FIG. 22 provides results from the flow cytometry7. Single- and poly donor CD4IL’10cells strongly inhibited in vitro proliferation of both allogeneic CD4+ and CD8+ T cells by more than 80% (FIG. 22).

[0344] The CD4IL 10cells were further analyzed for their cytotoxic effects against myeloid leukemia cells (ALL-CM) and an erythroid leukemia cell line (K562). Single (BC-E and BC-V) or poly donor (BC-V / T / E) CD4IL-10cells were co-cultured at a 1: 1 ratio with ALLCM or K562 cells. After 3 days the cells were harvested and surviving CD45lowCD3" target cells were counted and analyzed by FACS as described ((Locafaro et al. Mol Ther 2017, 25, 2254). The single donor and poly donor CD4IL’10cells also mediated strong direct cytotoxic effects on ALL-CM myeloid tumor cells, whereas they failed to kill the sensitive K562 cells,70IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOwhich lack Class I MHC expression required for their cytotoxic activity (FIG. 23). Single (BC-E and BC-V) or poly donor (BC-V / T / E) CD4IL 10cells had comparable cytotoxic activities against these two target cell lines (ALL-CM and K562).

[0345] Cytotoxic effects of single-donor (BC-E) and poly donor (BC-V / T / E) CD4IL-10cells were also tested in vivo, using a humanized xeno GvHD disease model - an NSG mouse intravenously injected with ALL-CM cells (2.5x106). Their effect on GvHD induced by human PBMC from an allogeneic donor as well as their effect on the growth of acute myeloid leukemia in cell line ALL CM in a therapeutic setting were tested as illustrated in FIG. 20.

[0346] Eight to ten-w eek-old female NOD scid gamma, (NSG) mice were obtained from Charles-River Italia (Calco, Italy). The experimental protocol was approved by the internal committee for animal studies of the Ospedale San Raffaele (Institutional Animal Care and Use Committee (IACUC). At day 0, the mice received total body irradiation from a linear accelerator. ALL-CM cells (2.5x106) were injected at day 0. On day 0, different groups of mice were injected with nothing, allogeneic PBMC (2.5 x106), single donor (BC-E, 2.5 x106) or poly donor CD4IL’10cells pooled at 1:1:1 ratio from 3 different donors (BC-V / T / E, 2.5 x106) in combination with allogeneic PBMC (2.5x106) or poly donor CD4IL-10cells (2.5x106) on day 3. All cells were administered i.v. in volumes of 250 pl of Iscove's modified Dulbecco’s medium. Mice were monitored 3-4 times per week.

[0347] The NSG mice were divided into five cohorts of 5 mice and each group was treated on day 0 with (i) none as a control; (ii) allogeneic mononuclear cells (PBMC); (iii) allogeneic PBMC and polydonor CD4IL-10cells (BC-V / T / E); (iv) allogeneic PBMC and single-donor CD4IL-10cells (BC-E); or (v) polydonor CD4IL‘10(BC-V / T / E) cells administered at day 3 Myeloid leukemia progression was measured as previously described ((Locafaro et al. Mol Ther 2017, 25, 2254).

[0348] Administration of ALL-CM cells to NSG mice resulted in a rapid expansion of these cells and all the mice died or had to be sacrificed on day 20. Injection of PBMC prevented leukemia progression as expected. Both single- and polydonor CD4IL10cells given in combination with allogeneic PBMC did not interfere with anti myeloid leukemia effects of the PBMC.

[0349] Injection of polydonor CD4IL-10(BC-V / T / E) cells 3 days after administration of the ALL-CM cells (when already massive expansion of these cells is ongoing) resulted in71IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOinhibition of tumor growth. These results indicate that polydonor CD4IL-10cells have direct therapeutic anti myeloid leukemia effects in vivo (FIG. 24).

[0350] However, despite their beneficial anti myeloid leukemia effects, the PBMC induced a very severe form of xeno-GvHD and all mice died by day 24 (FIG. 25). In the study, single-donor (BC-E) and polydonor (BC-V / T / E) CD4IL-10cells were tested on their capacity to inhibit xeno-GvHD induced by PBMC following administration in NSG mice. On day 0, the NSG mice were injected with ALL-CM cells (2.5x106). The mice were divided into five groups and each group was treated with (i) none as a control; (ii) allogeneic mononuclear cells (PBMC); (iii) allogeneic PBMC and polydonor (BC-V / T / E) CD4IL-10cells; (iv) allogeneic PBMC and single-donor CD4IL-10cells (BC-E) or polydonor (BC-V / T / E) CD4IL-10cells injected at day 3. In the animals, xeno-GvHD was measured by survival and weight loss. In addition, hunching, fur condition and skin integrity were monitored as described (Bondanza et al. Blood, 2006. 107, 1828). If weight loss reached more than 20%, the mice were sacrificed for ethical reasons. FIG. 25 shows % of NSG mice free of GvHD in each day following day 1 injection with ALL-CM cells (2.5x106) and subsequent treatment with PBMC with or without single-donor or polydonor CD4IL-10cells.

[0351] The results show that polydonor CD4IL-10cells did not induce xeno-GvHD, and down regulated xeno-GvHD induced by allogeneic PBMC. Collectively these results indicate that polydonor CD4n‘10cells downregulate severe xeno-GvHD, have direct anti myeloid leukemia effects in a therapeutic setting and do not interfere with the protective anti myeloid leukemia effects of the PBMC.6.7.9. Example 8: Adoptive transfer of polydonor CD4IL-10 cells derived from four different donors

[0352] Adoptive transfer of polydonor CD4IL-10cells derived from four different donors was tested for the transfer’s ability to inhibit PBMC -induced xeno-GvHD.

[0353] In these experiments, single-donor CD4IL-10cells (donor C; lot C) and polydonor CD4IL-10cells derived from 4 different donors(donors C. E, F, and H; lot CEFH) were tested in a humanized mouse model of GvHD induced by allogeneic PBMC. In this model, NSG mice were sub-lethally irradiated at day 0 and injected at day 3 (slow bolus i.v.) with (i) 2.5E+06 allogeneic PBMC, (ii) 2.5E+06 allogeneic PBMC in combination with 2.5E+06 single-donor CD4IL-10cells (lot C), (iii) 2.5E+06 allogeneic PBMC in combination with 2.5E+06 cells polydonor CD4IL‘10cells (lot CEFH), or (iv) 2.5E+06 cells polydonor 72IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOCD4IL-10cells (lot CEFH) alone. Xeno-GvHD was determined using a composite score of weight loss, fur appearance, skin appearance, hunch, and activity (see Bondanza A, et al. Blood 2006; 107: 1828-36 ]. As shown in FIG. 26, only mice administered with the poly donor CD4IL-10cells were 100% free of xeno-GvHD at the end of the study.

[0354] In summary, this data demonstrated that adoptive transfer of poly donor CD4IL-10cells derived from four different donors inhibits PBMC-induced xeno-GvHD and does not induce xeno GvHD.6.7.10. Example 9: Dose escalation treatment or prevention of GvHD using polydonor CD4IL 10cells

[0355] A dose escalation of polydonor CD4IL-10cells were tested in a humanized xeno GvHD disease model, an NSG mouse model, for their effect on xeno-GvHD induced by human PBMC as illustrated in FIG.30. NSG mice received total body irradiation and were intravenously injected with (i) PBS, (ii) human PBMC (5x106cells / mouse), (iii) human PBMC (5x106cells / mouse) in combination polydonor (three donors; BC-C / E / F) CD4IL-10cells (1.0x107cells / mouse), (iv) human PBMC (5x106cells / mouse) in combination with polydonor CD4IL’10cells (BC-C / E / F) (5x106cells / mouse), or with (v) human PBMC (5x106cells / mouse) in combination with polydonor CD4IL-10cells (BC-C / E / F) (2.5x106cells / mouse). Xeno-GvHD was evaluated as previously described (Bondanza et al. Blood 2006) based on survival, weight loss (>20% weight loss), skin lesions, fur condition, activity, and hunch.

[0356] FIG. 31 shows the % of NSG mice free of xeno-GvHD on each day after injection. Administration of 5x 106human PBMC to irradiated NSG mice resulted in fulminant xeno-GvHD. Co-administration of polydonor CD4IL-10cells delayed this fulminant xeno-GvHD, but the mice w ere sacrificed accordingly when they reached the pre-specified humane 20 % body w eight loss criterion for sacrifice. Mice that received co-administration of 1.0x107cells / mouse demonstrated increased survival time compared to mice that received 5x106cells / mouse, which in turn, survived longer than mice that received 2.5x106cells / mouse. These results show that poly donor CD411''1’ can delay extremely severe xeno-GvHD in a dose-dependent manner.6.7.11. Example 10: Generation of a variant of IL-10

[0357] Variants of human IL-10 are generated by introducing amino acid modification(s) (e g., substitution, insertion, deletion) in view of IL-10 sequences of other73IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOspecies. Modification sites are determined by sequence alignment as provided in FIG. 27 A. Amino acid positions having different amino acids among species are identified from the alignment and modified by introducing substitution, insertion, or deletion of amino acids.

[0358] Two examples of the variant of human IL- 10 are provided in FIG. 27B.Possible huIL-10 HYBRID #1 (SEQ ID NO: 19) is generated by substituting three amino acids (D, I and A) of human IL- 10 with three different amino acids (E, A, and D) of viral IL-10 (EBVB9) at the corresponding positions. Possible huIL-10 HYBRID #2 (SEQ ID NO: 20) is generated by substituting one amino acid (1105) of human IL- 10 with another amino acid (A105) of viral IL-10 (EBVB9) at the corresponding position. FIG. 27C shows alignment of human IL- 10 (SEQ ID NO: 1) with IL 10 EBVB9 (SEQ ID NO: 18) with “*’'indicating the one or more amino acid positions that are substituted in IL-10 hybrid #1 and “ / / "’indicating the preferred 1105 to A105 amino acid substitution for IL-10 hybrid #2.

[0359] The variants of human IL- 10 are cloned into an expression vector as described in the above section and tested for the expression and function of the variant proteins.Selected variants of human IL-10 are used to generate CD4" ‘10cells. Efficiency of CD4" ‘10cells are tested as provided herein.6.7.12. Experimental methods and materials

[0360] Cell preparation and cell lines. Peripheral blood mononuclear cells (PBMC) were prepared by centrifugation over Ficoll-Hypaque gradients. CD4+T cells were purified with a CD4 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) with a resulting purity of >95%. Mature dendritic cells (DC) were generated from peripheral blood CD14+monocytes positively selected using CD14+MicroBeads (Miltenyi Biotech. Germany) according to the manufacturer’s instructions and cultured in RPMI 1640 (Lonza, Italy) supplemented with 10% fetal bovine serum (FBS; Lonza, Italy), 100 U / mL penicillin / streptomycin (Lonza, Italy), 2 mM L-glutamine (Lonza, Italy), at 37°C in the presence of 10 ng / mL recombinant human (rh) IL-4 (R& D Sy stems, Minneapolis MN, USA) and 100 ng / mL rhGM-CSF (Genzyme, Seattle, WA, USA) for 5 days and matured with 1 mg / mL of lipopolysaccharide (LPS, Sigma, CA, USA) for an additional two days.

[0361] Plasmid construction. The coding sequence of human IL-10 was excised from pH15C (ATCC n° 68192), and the 549bp fragment was cloned into the multiple cloning site of pBluKSM (Invitrogen) to obtain pBluKSM-hIL-10. A fragment of 555bp was obtained74IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOby excision of hIL-10 from pBluKSM-hIL-10 and ligation to1074.1071.hPGK. GFP. WPRE.mhCMV.dNGFR. SV40PA (here named LV-ANGFR), to obtain pLVIL-10. The presence of the bidirectional promoter (human PGK promoter plus minimal core element of the CMV promoter in opposite direction) allows co-expression of the two transgenes (Locafaro et al. Mol Then 2017;25(10):2254-2269). The sequence of pLVIL-10 was verified by pyrosequencing (Primm).

[0362] Vector production and titration. VSV-G-pseudoty ped third generation bidirectional lentiviral vectors were produced by Ca3PO4transient four-plasmid cotransfection into 293T cells and concentrated by ultracentrifugation as described (Locafaro et al. Mol Then 2017;25(10):2254-2269). Titer was estimated by limiting dilution, vector particles were measured by HIV-1 Gag p24 antigen immune capture (NEN Life Science Products; Waltham, MA), and vector infectivity was calculated as the ratio between titer and particle. Titers ranged from 5x108to 6x109transducing units / mL. and infectivity from 5x104to 105transducing units / ng of p24.

[0363] Generation of CD4IL 10cell lines. Polyclonal CD4-transduced cells were obtained as previously described (Andolfi et al. Mol Ther. 2012;20(9): 1778-1790, Locafaro et al. Mol Ther 2017, 25, 2254).. Briefly, CD4 purified T cells were activated for 48 hours with soluble anti-CD3 monoclonal antibody (mAb, 30 ng / mL, OKT3, Janssen-Cilag, Raritan, NJ. USA), anti-CD28 mAb (1 pg / mL, BD) and rhIL-2 (50 U / mL. PROLEUKIN. Novartis. Italy). T cells were transduced with LV-IL-10 / ANGFR (CD4IL-10) with multiplicity of infection (MOI) of 20. At day 11, CD4+ANGFR+cells were beads-sorted using CD271 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and expanded in X-VIVO15 medium with 5% human serum (BioWhittaker-Lonza. Washington), 100 U / mL penicillinstreptomycin (BioWhittaker), and 50 U / mL rhIL-2 (PROLEUKIN, Novartis, Italy). At day 7 and 10, medium was replaced by fresh medium supplemented with 50U / mL of rhIL-2. At day 14, cells were collected, washed, and restimulated with allogeneic feeder mixture as previously described (Locafaro et al. Mol Ther 2017, 25, 2254). After 14 days, cells were collected and frozen. Thawed CD4IL‘10cells were restimulated and after the 2ndand 3rdrestimulation and expansion were functionally characterized in vitro and used for in vivo experiments.

[0364] Vector Copy Number Analysis. Cells were cultured for at 11 days after transduction in order to get rid of non-integrated vector forms. Genomic DNA was isolated 75IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOwith QIAamp DNA Blood Mini Kit (QIAGEN, 51106), according to the manufacturer’s instructions. Vector integrations were quantified by QX200 Droplet Digital PCR System (Bio-Rad), according to the manufacturer’s instructions.

[0365] Cytokine determination. To measure cytokine production, after 2ndand 3rdre-stimulation single donor and polydonor CD4IL-10cells were left unstimulated or stimulated with immobilized anti-CD3 (10 pg / mL) and soluble anti-CD28 (Ipg / mL) mAbs in a final volume of 200 pL of medium (96 well round-bottom plates, 2xl05 / well). Supernatants were harvested after 48 hours of culture and levels of IL- 10, IL-4, IL-5, IFN-y and IL-22 were determined by ELISA according to the manufacturer's instructions (BD Biosciences).

[0366] Flow cytometry analysis. For the expression of Granzyme B (clone MHGB04, Invitrogen, USA) after surface staining with CD4, CD4IL-10cells were fixed, permeabilized, and stained using the BD Cytofix / Cytoperm™ Kit according to the manufacturer’s instructions (Cat. No. 554714, Biolegend, USA). Stained cells were washed two times with PBS supplemented with 1% FBS and analysed with a BD LSRFortessa analysed utilizing FlowJo 10 software.

[0367] Killing assays. After 2ndand 3rdre-stimulation, cytotoxicity of single-donor and polydonor CD4IL-10cells was analysed in co-culture experiments (Locafaro et al. Mol Ther 2017, 25, 2254). Briefly, non-myeloid leukemia and a myeloid leukemia cell lines, K562 and ALL-CM respectively, were used as target cells and plated with CD4IL-10cells at 1: 1 ratio (105target cells and I05CD4IL 10cells) for 3 days. At the end of co-culture, cells were harvested and K562 and ALL-CM cells were analysed based on CD45+, CD3 -expression and counted by FACS.

[0368] Suppression assays. To measure the suppressive capacity of single donor and polydonor CD4IL-10cells, allogeneic PBMC were labeled with Cell Proliferation Dye eFluor® 670 (Invitrogen, CA, USA), according to manufacturer’s instructions. The labeled cells were activated with allogenic mature dendritic cells (DC) from CD14+cells in the presence of GM-CSF and IL-4 in the presence an anti-CD3 mAb (50 ng / mL). Peripheral blood CD14 monocytes w ere positively selected using CD14 Microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were cultured in RPMI 1640 (Lonza) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin / streptomycin (Lonza), 2 mM L-glutamine (Lonza), at 37°C in the presence of 10 ng / mL recombinant human (rh) IL-4 (R& D Systems) and 100 ng / mL recombinant human granulocyte-macrophage colony-stimulating 76IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOfactor (rhGM-CSF) (Genzyme) for 5 days. To generate mature dendritic cells (mDCs), on day 5 the cells were stimulated with 1 mg / mL lipopolysaccharide (LPS; Sigma) for an additional 2 days. At day 7, DCs were collected, phenotypically analyzed, and used to stimulate T cells. The purity and maturation state of DCs were checked by flow cytometry to determine expression of CDla, CD14, CD86, CD83, and HLA-DR.

[0369] The labeled cells were plated in 96 well round well plates in final volumes of 200 pL and incubated for 3 days as follows: (i) Labeled PBMC alone 5x104cells / well; (ii) Labeled PBMC 5x104cells / well+ mature DC 5x103cells / well + anti-CD3 mAb (50 ng / mL); (iii) Labeled PBMC 5x104cells / well + single or polydonor CD4IL-10 cells 5x104cells / well + mature DC 5xl0Jcells / well +anti-CD3 mAb (50 ng / mL).

[0370] After 3 days of culture, the cells were harvested and transferred to 96 V bottom well plates for immunofluorescence staining. Cells were analyzed by FACS gated on living CD4+ eFluor670+ and CD8+ eFluor670 + cells. Percentages of inhibition were calculated by measuring dilution of the eFluor670 label as described previously (Locafaro et al. Mol Ther 2017, 25, 2254).

[0371] Graft-versus Host Disease models: In all experiments 6 / 8 week-old female NSG mice were used. On day 0 mice received total body irradiation with a single dose of 175-200 cGy from a linear accelerator according to the weight of the mice. In some experiments mice received an single dose irradiation of 350 cGy. The mice were intravenously injected with PBMC cells (5x106or 2.5x106). or CD4IL-10cells (single-donors or polydonor - pool of three donors - 5x106or 2.5x106), or with PBMC (5x106or 2.5x106) in combination with CD4IL-10cells (5x106or 2.5x106). Survival, weight loss, activity, fur, skin, and hunch were monitored at least 3 times per w eek as previously described (Bondanza et al. Blood. 2006; 107(5): 1828- 1836). Mice w ere euthanized for ethical reasons when their loss of bodyweight was 20%.

[0372] Alternatively, on day 0 mice received total body irradiation as above. On day 3 mice were injected with CD4+T cells (2.5x106), single and polydonor (pool of three donors) CD4IL-10cells (2.5x106), or CD4+T cells (2.5x106) in combination with single and polydonor (pool of three donors) CD4IL-10cells (2.5x106). GvHD induction was monitored as indicated above.6.7.13. Example 11: A Phase I, First in Human Open Label Study to Evaluate the Safety and Tolerability of polydonor CD4IL 10cells 77IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOInfusion in Subjects with Hematological Malignancies Undergoing HLA-Mismatched Related or Unrelated Hematopoietic Stem Cell Transplantation (HSCT)

[0373] The use of post-transplant cyclophosphamide (PTCy) for patients undergoing allogeneic HSCT has reduced the incidence of graft versus host disease (GvHD) and expanded donor options for patients to include haploidentical and mismatched unrelated donors. Though PTCy is effective at reducing acute GvHD, 30-40% of patients receiving peripheral blood stem cell (PBSC) grafts are at risk of chronic GvHD, which creates significant long-term consequences. There are several other risks associated w ith PTCy, including a higher rate of serious infections, organ dysfunction and delayed immune reconstitution.

[0374] Consequently, there is an urgent unmet medical need for the development of additional therapies for GvHD prevention and improved immune reconstitution with better protection against severe infections, reduction of relapse events, and better long-term posttransplant outcome in HSCT recipients.

[0375] Poly donor CD4IL-10cells were produced as described above, by transducing CD4+ T cells from healthy donors with a bi-directional lentiviral vector that encodes human IL-10 and a non-signaling form of human truncated Nerve Growth Factor Receptor (ANGFR, CD271). The resulting polydonor CD4IL‘10cells were highly pure, engineered T cell products allogeneic to the subject and to the donor of the HSCT. Poly donor CD4IL-10cells recapitulated all the major regulatory functions of naturally occurring Tri cells.

[0376] The safety and tolerability of polydonor CD4IL-10cells infusion were tested in patients with hematological malignancies undergoing HLA-Mismatched related or unrelated HSC transplantation (HSCT) in this Phase I. First in Human Open Label Study. Subjects are enrolled sequentially in 4 escalating Dose Level cohorts to determine the maximum tolerated dose (MTD). The escalating Dose Level includes Dose 1: 25 xlO6cells (± 10%); Dose 2: 75 xlO6cells (± 10%); Dose 3: 225 xlO6cells (± 10%); and Dose 4: 675 xlO6cells (± 10%).

[0377] The endpoints of the clinical study and the subject’s inclusion and exclusion criteria are provided in the below' table. The study design and protocol are summarized in the flowchart provided in FIG.28.78IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO dlbliiiiiii E Etttnponsg crerayThe safety and tolerability of polydonor CD4IL-10cells, assessed by the incidence and severity of treatment emergent adverse events (TEAE) and treatment emergent serious adverse events (TESAE)Primary The safety of poly donor CD4IL-10cells as determined by stem cell engraftmentand donor chimerism after HSCTThe safety of poly donor CD4IL’10cells determined by negative Replication Competent Lentivirus (RCL) at 3-month, 6-month, and 1-yearThe incidence of Grade I I-I V acute GvHD by Day +100 using the Mount Sinai MAGIC criteriaThe incidence of Grade III-IV acute GvHD by Day +100 using the Mount Sinai MAGIC criteriaSecondaryIncidence and severity of chronic GvHD from Day +100 to Day +365 Overall survival at Day +365Subjects with one of the following hematologic malignancies: Acute Lymphoblastic Leukemia [ALL], Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS), and Chronic Myelomonocytic Leukemia (CMML)InclusionAvailable mismatched related (haploidentical) or unrelated donors for peripheral blood stem cell (PBSC) donationAbsence of uncontrolled bacterial, viral or fungal infection at time of enrollmentPrior allogeneic bone marrow, peripheral blood, or cord blood HSCT History of significant organ dysfunctionExclusion Received another investigational agent for treatment of disease understudy within 28 daysSubjects with a previous history of Thrombotic Thrombocytopenic Purpura (TTP) or Hemolytic Uremic Syndrome (HUS)00378] A total of 6 patients were dosed: 3 in Cohort 1, Dose Level 1 and 3 in Cohort 2, Dose Level 2. No dose limiting toxicities were observed at Day 28 after infusion of polydonor CD4IL-10cells. Thus, 6 more patients were dosed: 3 at Dose Level 3 and 3 at Dose Level 4.

[0379] Conditioning regimens used in this study are as provided below:IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0380] Conditioning Regimen MAC- 1 (FTBI / Flu): On Day -7 Fludarabine is administered [30 mg / m2]; On Day -6 Fludarabine is administered [30 mg / m2]; On Day -5 Fludarabine is administered [30 mg / m2]; On Day -4, Subject undergoes Fractionated Total Body Irradiation (FTBI) [150 cGy x 2 doses]; On Day -3, FTBI [150 cGy x 2 doses]; On Day -2, FTBI [150 cGy x 2 doses]; On Day -1, FTBI [150 cGy x 2 doses],

[0381] Conditioning Regimen MAC -2 (FB4): On Day -6, Busulfan at an initial dose of 0.8 mg / kg (or 130 mg / m2) every 6h IV infusion over two hours + Fludarabine 40 mg / m2; On Day -5. Busulfan administered at a dose of 0.8 mg / kg (or 130 mg / m2) every 6h IV infusion over two hours or per blood level monitoring* + Fludarabine 40 mg / m2; On Day -4, Busulfan administered at a dose of 0.8 mg / kg (or 130 mg / m2) every 6h IV infusion over two hours or per blood level monitoring* + Fludarabine 40 mg / m2; On Day -3, Busulfan administered at a dose of 0.8 mg / kg (or 130 mg / m2) every 6h IV infusion over two hours or per blood level monitoring* + Fludarabine 40 mg / m2(total dose of Fludarabine: 160 mg / m2).* The Busulfan total dose is recommended to be adjusted to achieve a plasma steady state concentration of 800-900 ng / ml (or AUC equivalent).

[0382] Conditioning Regimen RIC-1 (Flu / Cy / TBI) per JHMI regimen: From Day -6 to Day -2, Fludarabine 30 mg / m2IV qd (adjusted for renal function); On Day -6 and Day -5, Cyclophosphamide 14.5 mg / kg IV qd; On Day -1. Total body irradiation. 200 cGy.

[0383] Conditioning Regimen RIC- 2 (FB2): On Day -6 Fludarabine 40 mg / m2; On Day -5 Fludarabine 40 mg / m2; On Day -4. Busulfan at an initial dose of 0.8 mg / kg (or 130 mg / m2) every 6h IV infusion over two hours + Fludarabine 40 mg / m2; On Day -3, Busulfan administered at a dose of 0.8 mg / kg (or 130 mg / m2) every 6h IV infusion over two hours or per blood level monitoring* + Fludarabine 40 mg / m2(total dose of Fludarabine: 160 mg / m2). * The Busulfan total dose is recommended to be adjusted to achieve a plasma steady state concentration of 800-900 ng / ml (or AUC equivalent).

[0384] Conditioning Regimen RIC- 3 (Flu / Mel + / - TBI): Days -6 (or Day -5) through -2, Fludarabine 25, 30 or 40 mg / m2IV qd (adjusted for renal function and age), based on haploidentical or mismatched unrelated transplant; Days -2, Melphalan 140 mg / m2(For subjects > 60 years of age Melphalan should be capped at 100 mg / m2); Day -1 200cGy for subjects receiving haploidentical transplant.80IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO

[0385] In the study design provided in FIG. 28, Day 0 is defined as the day that the stem cell infusion was completed. Based on site standard guidelines, subjects were hospitalized throughout the ANC (absolute neutrophil counts) nadir until myeloid engraftment and / or physician decision. Post-transplant immune suppression regimens are provided below.

[0386] Post Transplant Cyclophosphamide (PTCy): PTCy is dosed according to ideal body weight (IBW) if subject actual body weight (ABW) is between 100-125% of IBW. If subject ABW is < 100% of IBW, the ABW is used to calculate dose. If subject ABW is > than 125% of IBW, then the dose is adjusted to IBW or according to institutional guidelines. Cyclophosphamide (Cy) 50mg / kg IV, over 1-2 hours (depending on volume), is given on Day +3 (ideally between 60 and 72 hours after bone marrow infusion) and on Day +4 (approximately 24 hours after Day +3 Cy). Hydration prior and during Cy administration, management of volume status, and monitoring for hemorrhagic cystitis follows institutional guidelines. Institutional guidelines for Mesna use and dosing may be applied when necessary'. Mesna recommendation: Mesna IV dose > 80% of the total daily dose of Cy and given in divided doses 30 minutes before and at 3, 6, and 8-9 hours after completion of Cy. It is crucial that no systemic immunosuppressive agents are given until at least 24 hours after the completion of the PTCy. This includes corticosteroids and anti-emetics.

[0387] Sirolimus: On Day +5, at least 24 hours after Cy completion, subjects are started on sirolimus using a loading dose of 4-6 mg followed by 1-2 mg daily. In the absence of GvHD or relapse, sirolimus is tapered from Day +60 to Day +90 and discontinued on Day +100 to Day +150 as determined by subject status and PI discretion. Sirolimus levels are checked per institutional practice. Sirolimus blood levels should be between 5-12 ng / mL as measured by HPLC or immunoassay. Dose adjustments may be required if a new medication is initiated or if the subject develops renal insufficiency or hepatic dysfunction.

[0388] Mycophenolate mofetil (MMF): MMF begins on Day +5, at least 24 hours after completion of PTCy. The MMF dose is 10 mg / kg PO, with a total daily dose not to exceed 3 g (i.e., maximum 1 g PO TID). An equivalent IV dose (1:1 conversion) may be given instead. MMF prophylaxis is discontinued after the last dose on Day +30 to Day +35 (per PI discretion) or may be continued if there is GvHD.

[0389] Growth factors: G-CSF (Granulocyte stimulating factor) (filgrastim) or a biosimilar can be administered beginning on Day +5 at a dose of 5 mcg / kg / day (according to 81IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO 11h Diemoerancsactual body weight) IV or SC (rounding to the nearest vial dose is allowed), until the ANC is > l,000 / mm3 over the course of 3 consecutive assessments. Additional G-CSF may be administered as warranted. Pegfilgrastim and granulocyte-macrophage colony stimulating factor (GM-CSF) or their biosimilars are not permitted.

[0390] On Day +5 (re., 24 hours prior to polydonor CD4IL-10cells dose) subjects were confirmed eligible to proceed with polydonor CD4IL-10cells dosing. Eligibility criteria include the following: No clinical signs or symptoms of active infections; No clinically significant vital signs or clinically significant abnormal lab values indicative of active infections.

[0391] On Day +6, the subject was premedicated with: Acetaminophen (15 mg / kg or per institutional guidelines) and Diphenhydramine (1 mg / kg or per institutional guidelines). Cyclophosphamide has an elimination half-life of 3 to 12 hours, allowing sufficient time for its clearance before the poly donor CD4I, _|" cells infusion. Following poly donor CD4IL-10cells infusion, subjects were observed for 24 hours inpatient for infusion related reactions. All adverse reactions, including infusion-related reactions, were recorded.

[0392] The below table show s patient demographics for Cohort 1 and 2, their treatment and outcome. In the table, “engraftment day” represents the 3rdconsecutive assessments of ANC > 500 neutrophils / uL.Cohort 1 Cohort 2Subject 1001-001 1001-003 1003-001 1001-006 1001-008 1001-009Age / Gender 37 / Female 52 / Female 67 / Male 65 / Female 73 / Female 71 / MaleDisease AML ALL B-ALL AML B-ALL AML DiagnosisNot Not Not Ethnicity & Hispanic / L Hispanic / L Hispanic / Latino atino Hispanic / L Hispanic / L atino Hispanic / L race atino atino atino (White) (White) (White) (White) (Asian) (Asian) Donor Haploident Haploident Haploidenti Mismatche Mismatche Haploidenti ical ical cal d Unrelated d Unrelated cal TypeDonor 19 / Female 17 / Female 41 / Male 31 / Male 22 / Female 36 / Female Age / GenderIPTS / 200307516.4Attorney Docket Ref: TR1X-012WO 11 11 11f O HSCT HSCT S (ttomeaeucvConditioningregimen MAC-1 MAC-1 RIC-3 RIC-3 RIC-3 RIC-3 CD34 cellsinfused 4.05 x l064.97 x lO65.01 x 10s8.10 x 10s8.05x l0S5.90 x l0S(cells / kg)Engraftment(>500 Yes Yes Yes Yes Yes Yes neutrophils / (Day +20) (Day +19) (Day +19) (Day +18) (Day +19) (Day +33) pL)100 % Donor Yes Yes Yes Yes Yes Yes Chimerism (Day +34) (Day +32) (Day +34) (Day +33) (Day +34) (Day +29) Acute GvHD Yes: Grade Yes: GradeQ 2 2 No No No No O >ChronicGvHD Yes: mild Yes: ocular No No No No polydonorCD4TT-10None None None None None None relatedSAEs & AEsEarlyFollow up terminatevisit d on Day Day +210 Day +150 Day +120 Day +49 Day +35 post HSCT + 120(relapse)00393] The results from the study of all 12 patients from DL1-DL4 are summarized in the below table. Additionally, FIG. 29A shows poly donor CD4IL-10cells (CD45+CD3+CD4+CD271+) in blood quantified by flow cytometry in Cohort 1 (n=3) and Cohort 2 (n=3). FIG.29B shows total CD4+T cell levels monitored up to Day 98 (Cohort 2 data available up to Day 56 in patient 1001-006).No acute infusion reactions and no DLT at 28d (12 / 12pts)Timely stem cell engraftment by absolute neutrophil count (12 / 12pts) 100% donor chimerism (12 / 12pts)SAFETY No SAE related to polydonor CD4IL 10cellsNo RCL positive results (12 / 12pts)2 grad 1 AEs related to polydonor CD4IL-10cells1 infusion related reaction at Dose Level 4 due to rapid infusionPK studies demonstrate presence of polydonor CD4IL-10cells in the peripheral EARLY blood of the patients >14d post-infusion at Dose Level 1(DL1)PK Increase in exposure and detectability in periphery at Dose Level 2 (DL2) Dose dependent exposure, expansion and persistence at Dose Level 3 and 4 (DL3 and DL4)IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO1 acute GvHD grade IV at DL34 acute GvHD grade II (2 at DL1, 1 at DL3 and 1 at DL4)2 mild chronic GvHD (1 at DL1 and 1 at DL3)1 moderate chronic GvHD at DL31 severe chronic GvHD at DL1EARLY Preliminary PD studies show improved immune reconstitution vs. historical PD dataPreliminary data shows immunomodulatory activity of polydonor CD4IL-10cells, including increase in Treg cellsTolerized environment via enhanced donor-derived DC10, FOXP3 and natural Tri cells

[0394] These results show that all subjects had engrafted, with 100% donor chimerism achieved by all patients by Day 42 post-HSCT. PK demonstrated the presence of polydonor CDT11'"10(CD271+ cells) in peripheral blood for more than 14 days after infusion of poly donor CD4IL-10(FIG. 29 A). PD showed robust CD4+ T cell reconstitution in all subjects (FIG. 29B).

[0395] Due to the dose tolerability of Cohort 1 and 2, 6 more patients were dosed: 3 at Dose Level 3 and 3 at Dose Level 4. A dose dependent persistence of polydonor CD4n‘'° cells (CD45+CD3+CD4+CD271+) in peripheral blood is shown in FIG. 32A, with detectable circulating cells persisting for > 14 days in DL3 and DL4. Additionally, FIG. 32B shows a non-linear exposure in area under the curve (AUC), showing expansion of poly donor CD4n’10cells.

[0396] Enhanced immune reconstitution and a reduced period of immunodeficiency in HSCT patients is shown in FIGs.33A and 33B. Where FIG. 33A shows a 10-20 time increase in FOXP3 Treg cells following administration of polydonor CD41L'10cells, when compared to a historical control (Al Malki et al. Blood Advance, 2021). This increase in FOXP3 Tregs is also dose-dependent, as seen in FIG. 33B. Furthermore, FIG.34A quantifies tolerogenic dendritic cells (CD 14+CD 16+ DC- 10). showing a dose dependent increase in DC-10 cell counts. Additionally, FIG.34B shows that polydonor CD4IL-10cells promote an increase in natural Tri cells.

[0397] Without wishing to be bound by theory, the results from this study show rapid reconstitution of DC-10, natural Tri, and FOXP3+ T cells following polydonor CD4IL-10cell infusion, suggesting evidence of long-term tolerance.IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO7. INCORPORATION BY REFERENCE

[0398] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.8. EQUIVALENTS

[0399] While various specific embodiments have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). Many variations will become apparent to those skilled in the art upon review of this specification.9. SEQUENCESSEQ ID NO: 1 (Human IL-10 amino acid sequence— Protein Sequence: Ref P22301) MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 2 (Human IL-10 exemplary nucleic acid sequence) atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccagcccagg ccagggcacccagtctgagaacagctgcacccacttcccag caacctgcctaacatgcttc gagatctccgagatgccttcagcagagtgaagactttctttcaaatgaaggatcagctggac aacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagcctt gtct agatgatccagttttacctggaggaggtgatgccccaagctgagaaccaagacccag acatcaaggcgcatgtgaactccctggggga aacctgaagaccctca gctgaggctacgg cgct tcatcgatttcttccctgtgaaaacaagagcaaggccgt gagcaggtgaagaatgc ctttaataagctccaagagaaag catctacaaagccatgagtgagtttgacatcttcatca actacatagaagcctacatgacaatgaagatacgaaactgaSEQ ID NO: 3 (ANGFR amino acid sequence) MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQT VCEPCLDSVTFS DWSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEA CRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADA ECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTT DNLIPVYCSILAAWVGLVAYIAFKRWNRGILSEQ ID NO: 4 (ANGFR exemplary7nucleic acid sequence )85IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOatgggggcaggtgccaccggccgcgccatggacgggccgcgcctgctgctgttgctgcttct gggggtgtcccttggaggtgccaaggaggcatgccccacaggcctgtacacacacagcggtg agtgctgcaaagcctgcaacctgggcgagggtgtggcccagccttgtggagccaaccagacc gtgt tgagccctgcctggacagcgtgacgttctccgacgtggt agcgcgaccgagccgtg caagccgtgcaccgagtgcgtggggctccagagcatgtcggcgccgtgcgtggaggccgacg acgccgtgtgccgctgcgcctacggctactaccaggatgagacgactgggcgctgcgaggcg tgccgcgtgtgcgaggcgggctcgggcctcgtgttctcctgccaggacaagcagaacaccgt gtgcgaggagtgccccgacggcacgtattccgacgaggccaaccacgt gacccgtgcctgc cctgcaccgtgtgcgaggacaccgagcgccagctccgcgagtgcacacgctgggccgacgcc gagtgcgaggagatccctggccgttggattacacggtccacaccccc gagggctcggacag caca cccccagcacccaggagcctgaggcacctccagaacaagacctcatagccagcacgg tggcaggtgtggtgaccacagtgatgggcagctcccagcccgtggtgacccgaggcaccacc gacaacctcatccctgtctattgctccatcctggctgctgtggttgtgggccttgtggccta catagccttcaagaggtggaacagggggatcctctagSEQ ID NO: 5 (nucleotide sequence of pLVIL-10) tggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaac attaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcat tagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggc tgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgcc aatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcag tacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggccc gcct gcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgt att gtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagc ggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgg caccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatggg cggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagt aaccggggtctct ctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagc ctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggt aactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaac agggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggctt ctga agcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcg gaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcg cgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagt atgggcaagcagggagctagaacg ttcgcagttaatcctggcctgttagaaacatcagaag gctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttaga tcattatataatacagtagcaaccctctattgtgtgcatcaaaggat gagataaaagacac caaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaag cggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattata taaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagag tggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagca gcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtc tggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgc aactcacagtctggggcatcaagcagctcca gcaagaatcctggctgtggaaagataccta aaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgt gccttggaatgctagttggagtaataaatctctggaacagattt gaatcacacgacctgga tggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcg caaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtg86IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOgaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggag gcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcaggga tattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaagg aatagaa aagaaggtggagagagagacagagacagatccattcgattagtgaacggatctc gacggtatcggttaacttttaaaagaaaaggggggattggggggtacagtgcaggggaaaga atagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaat tcaaaattttatcgatcacgagactagcctcgagagatctgatcataatcagccataccaca tttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataa aatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaggca atagcatcacaaatttcacaaataaggcatttttttcactgcattctagttttggtttgtcc aaactcatcaat tatcttatcat tctggatctcaaatccctcggaagctgcgcctgtctt aggttggagtgatacatttttatcacttttacccgtctttggattaggcagtagctctgacg gccctcctgtcttaggttagtgaaaaatgtcactctcttacccgtcattggctgtccagctt agctcgcaggggaggtggtctggatccaccatgtctagaggatccccctgttccacctcttg aag ctatgtaggccacaaggcccacaaccacagcagccaggatgga caatagacagggat gaggttgtcggtggtgcctcgggtcaccacgggctgggagctgcccatcactgtggt caeca cacctgccaccgtgct gctatga gtcttgttctggaggtgcctcaggctcctgggtgctg ggggctgtgctgtccgagccctctgggggtgtggaccgtgtaatccaacggccagggatctc ctc cactcggcgtcggcccagcgtgtgcactcgcggagctggcgctcggtgtcctcgcaca cggtgcagggcaggcacgggtccacgtggttggcctcgtcggaatacgtgccgtcggggcac tcctcgcacacggtgttctgcttgtcctggcaggagaacacgaggcccgagcccgcctcgca cac cggcacgcctcgcagcgcccagtcgtctcatcctggta tagccgtaggcgcagcggc acacggcgtcgtcggcctccacgcacggcgccgacatgctctggagccccacgcactcggtg cacggcttgcacggctcggtcgcgctcaccacgtcggagaacgtcac ctgtccaggcaggg ctcacacacggtctggttggctccacaaggctgggccacaccctcgcccaggttgcaggctt tgcagcactcaccgct tgtgtgtacaggcctgtggggcatgcctccttggcacctccaagg gacacccccagaagcagcaacagcagcaggcgcggcccgtccatggcgcggccggtggcacc tgcccccatcgcccgcctcccgcggcagcgctcgacttccagctcggtccgctttgcggact gatggggctgcgctgcgctgcgctccagcgccccccctgcccgccggagctggccgcggccc gaattcctgcaggaattcgatgga gctggatcggtcccggt tcttctatggaggtcaaaa cagcgtggatggcgtctccaggcgatctgacggttcactaaacgagctctgcttatataggc ctcccaccgtacacgcctaccctcgagaagcttgatatcgaattcccacggggttggggttg cgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgg gaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtc ccc ggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgg gaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactag taccctcgcagacggacagcgcca ggagcaat gca cgcgccgaccgcgatgggctgtgg ccaatagcggctgctcagcggggcgcgccgagagcagcggccgggaaggggcggtgcgggag gcggggt tggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaa gcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctcccc agggggatccccggtctgcaggaattcatgcacagctcagcactgctctgttgcctggtcct cctgactggggtgagggccagcccaggccagggcacccagtctgagaacagctgcacccact tcccag caacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagact ttctttcaaatgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactt taagggttacct ggttgccaagccttgtctgagatgatcca ttttacctggaggaggtga tgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaac ctgaagaccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacaagag caaggccgtggagcaggtgaagaatgcctttaataagctccaagagaaaggcatctacaaag ccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacga aactgagtcgagaatcaacctctggattacaaaattt tgaaagattgactggtattcttaa 87IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOctatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattg cttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgag gagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccc cactggttggg cattgccaccacctgtcagctcctttccgggactttcgctttccccctcc ctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctg ttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggct ctc c ctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatc cagcggaccttccttcccgcggcct ctgccggctctgcggcctcttccgcgtcttcgcctt cgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggta cctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggg gggactggaagggctaattcactcccaacgaagacaa atct ctttttgcttgtactgggt ctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctt aagcctcaataaagcttgccttga tgcttcaagtagtgtgt cccgtctgttgtgtgactc tggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagtt catgtcatcttattattcagtatttataacttgcaaagaaat aatatcagagagtgagagg aacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaa taaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatc atgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagtt ccgcccattctccgccccatggct actaattttttttatttatgcagaggccgaggccgcc tcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcgt cgagacgtacccaattcgccctatagtgagtcgtattacgcgcgctc ctggccgtcgtttt acaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatcccc ctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaagcggccgca cgctcagtggaacgaaaactcacgttaagggattttggtcat aacaataaaactgtct ct tacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctctag gcc cgattaaattccaacatggat ctgatttatat ggtataaatgggctcgcgataatg tcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgttt ctgaaacatggcaaaggtagcgtt ccaatgat ttacagat agatggtcagactaaactg gctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcat ggtt act caeca ctgcgatccccgggaaaacagcattccaggtattagaagaatatcctgat tcaggtgaaaacattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgt ttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatga ataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaa gtctggaaagaaatgcataaacttttgccattctcaccggattcagtcgtcactcatggtga tttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggac gagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttt tctccttcattacagaaacggctttttcaaaaatatggtatt ataatcctgatatgaataa attgcagtttcatttgatgctcgatgagtttttctaagaattaattcatgagcggatacata tttgaat tatttagaaaaataaacaaataggggttccgcgactatgtctttgataatctca tgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatc aaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacc accgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaa ctggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccac cacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggc tgctgccagtg cgataagtcgtgtcttaccgggttggactcaagacgatagttaccggata aggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacc tac ccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggag aaaggcggacaggtatccggtaagcggcagggtc gaacaggagagc cacgagggagcttc cagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt cgatttttgtgatgctcgtcaggggggcggagcctat gaaaaacgccagcaacgcggcctt 88IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOtttacggttcctggccttttgctggcgttatcccctgattctgtggataaccgtattaccgc ctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcg aggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttgtatgcttccggct cgtatgttgtgt gaattgtgagcggataacaatttcacacaggaaacagctatgaccatga ttacgccaagccgaattaaccctcactaaagggaacagctagcSEQ ID NO: 6 (Viral interleukin- 10 homolog aka interleukin- 10 BCRF1 aka IL10H EBVB9) Protein Sequence: Ref: P03180 MERRLWTLQCLVLLYLAPECGGTDQCDNFPQMLRDLRDAFSRVKTFFQTKDEVDNLLLKES LLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPEAKDHVNSLGENLKTLRLRLRRCHRFLP CENKSKAVEQIKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTIKAR*SEQ ID NO: 7 (Viral interleukin- 10 homolog cDNA sequence)Nucleotide sequence (cDNA): Ref: NC_007605.15' _atggagcgaaggttagtggtcactctgcagtgcctggtgctgctttacctggcacctga gtgtggaggtacagaccaatgtgacaattttccccaaatgttgagggacctaagagatgcct tcagtcgtgttaaaacctttttccagacaaaggacgaggtagataaccttttgctcaaggag tctctgctagaggactttaagggctaccttggatgccaggccctgtcagaaatgatccaatt ctacct gaggaagtcatgccaca gctgaaaaccaggaccctgaagccaaagaccatgtca attctttgggtgaaaatctaaagaccctacggctccgcctgcgcaggtgccacaggttcctg ccgtgtgagaacaagagtaaagct tggaacagataaaaaat cctttaacaagctgcagga aaaaggaatttacaaagccatgagtgaatttgacatttttattaactacatagaagcataca tgacaattaaa ccaggtga_3 'SEQ ID NO: 8 (exemplary human IL-10 variant with amino acid substitutions based on viral IL- 10) MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQTKDEVD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPEAKDHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQIKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 9 (exemplary human IL-10 variant with amino acid substitutions based on viral IL- 10) MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDAKAHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 10 (Mus musculus: '" MOUSE" ) MPGSALLCCLLLLTGMRISRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLD NILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLR RCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS SEQ ID NO: 11 (Rattus norvegicus; “RAT")89IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOMPGSALLCCLLLLAGVKTSKGHSIRGDNNCTHFPVSQTHMLRELRAAFSQVKTFFQKKDQLD NILLTDSLLQDFKGYLGCQALSEMIKFYLVEVMPQAENHGPEIKEHLNSLGEKLKTLWIQLR RCHRFLPCENKSKAVEQVKNDFNKLQDKGVYKAMNEFDIFINCIEAYVTLKMKN SEQ ID NO: 12 (Macaca mulatta; “MACMU”) MHSSALLCCLVLLTGVRASPGQGTQSENSCTRFPGNLPHMLRDLRDAFSRVKTFFQMKDQLD NILLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENHDPDIKEHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFSKLQEKGVYKAMSEFDIFINYIEAYMTMKIQN SEQ ID NO: 13 (Gorilla gorilla; “GORILLA”) MHSSALLCCLVLLTGVRASPGHGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFNKLQEKGVYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 14 (Macaca fasciculans; “CYNO”) MHSSALLCCLVLLTGVRASPGQGTQSENSCTRFPGNLPHMLRDLRDAFSRVKTFFQMKDQLD NILLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENHDPDIKEHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFSKLQEKGVYKAMSEFDIFINYIEAYMTMKIQN SEQ ID NO: 15 (Papio Anubis; “OLIVE BABOON”) MHSSALLCCLWLTGVRASPGQGTQSENSCTRFPGNLPHMLRDLRDAFSRVKTFFQMKDQLD NILLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENHDPDIKEHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFSKLQEKGVYKAMSEFDIFINYIEAYMTMKIQN SEQ ID NO: 16 (Pan paniscus; “BONOBO”) MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKVHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 17 (Pan troglodytes; “CHIMP”) MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALXEMIQFYLEEVMPQAENQDPDIKVHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFNKLQEKGIVKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 18 (EBVB9) MERRLWTLQCLVLLYLAPECGGTDQCDNFPQMLRDLRDAFSRVKTFFQTKDEVDNLLLKES LLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPEAKDHVNSLGENLKTLRLRLRRCHRFLP CENKSKAVEQIKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTIKAR SEQ ID NO: 19 huIL-10 HYBRID#!90IPTS / 200307516.4Attorney Docket Ref: TR1X-012WOMHSSALLCCLVLLTGRASPGQGTQSENSCTHFPGNIPNMLRDIRDAFSRVKTEFQTKDEVDN LLLKESLLEDEKGYLGCQALSEMIQFYLEEVMPQAENQDPEAKDHVNSLGENLKTLRLRIRR CHRFLPCENKSKAVEQIKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 20 huIL-10 HYBRID# 2 MHSSALLCCLVLLTGRASPGQGTQSENSCTHFPGNIPNMLRDIRDAFSRVKTEFQMKDQLDN LLLKESLLEDEKGYLGCQALSEMIQFYLEEVMPQAENQDPDAKAHVNSLGENLKTLRLRIRR CHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNIPTS / 200307516.4

Claims

Attorney Docket Ref: TR1X-012WOWHAT IS CLAIMED IS:

1. A method of treating a patient with a hematological malignancy, comprising:(a) treating the patient with a conditioning regimen;(b) administering hematopoietic stem cells (HSCs) of an HSC donor to the patient; and(c) administering a therapeutically effective amount of poly donor CD4IL 10cells to the patient, wherein the polydonor CD4IL-10cells are CD4+T cells that have been genetically modified to comprise an exogenous polynucleotide encoding IL-10, wherein the CD4+T cells were obtained from at least two different T cell donors.

2. The method of claim 1, wherein the therapeutically effective amount is at least 25 x 106[± 10%] polydonor CD4IL-10cells.

3. The method of claim 2, wherein the therapeutically effective amount is at least 75 x 106|± 10%] polydonor CD4IL’10cells.

4. The method of claim 3, wherein the therapeutically effective amount is at least 225 x 106[± 10%] poly donor CD4IL’10cells.

5. The method of claim 4, wherein the therapeutically effective amount is at least 625 x 106[± 10%] polydonor CD4IL-10cells.

6. The method of any one of claims 1-5, wherein the therapeutically effective amount is no more than 625 x 106[± 10%] polydonor CD4IL-10cells.

7. The method of claim 6, wherein the therapeutically effective amount is no more than 225 x 106[± 10%] polydonor CD4IL’10cells.

8. The method of claim 7, wherein the therapeutically effective amount is no more than 75 x 106[± 10%] polydonor CD4IL’10cells.

9. The method of any one of claims 1-8, wherein the therapeutically effective amount comprises 25 x 106, 75 x 106, 225 x 106, or 625 x 106[± 10%] polydonor CD4IL-10cells.92IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO10. The method of any one of claims 1-9 wherein the therapeutically effective amount is between 25 x 106[± 10%] and 625 x 106[± 10%] polydonor CD4IL-10cells.

11. The method of any one of claims 1-9, wherein the therapeutically effective amount is between 25 x 106[± 10%] and 225 x 106[± 10%] polydonor CD4IL-10cells.

12. The method of any one of claims 1-9 wherein the therapeutically effective amount is between 75 x 106[± 10%] and 625 x 106[± 10%] polydonor CD4IL-10cells.

13. The method of any one of claims 1-9 wherein the therapeutically effective amount is between 225 x 106[± 10%] and 625 x 106[± 10%] polydonor CD4IL’10cells.

414. The method of any one of claims 1-13, wherein the effective amount of polydonor CD4IL-10cells is sufficient to suppress or prevent GvHD without suppressing graft versus leukemia (GvL) or graft versus tumor (GvT) efficacy of the HSCs.

15. The method of any one of claims 1-14, wherein the effective amount is sufficient to provide presence of the poly donor CD4IL’10cells in the peripheral blood of the patient for at least 14 days or at least 21 days after administration of the polydonor CD4IL’10cells.

16. The method of any one of claims 1-15, wherein the HSCs are administered on day 0; and the patient is treated with the conditioning regimen before day 0.

17. The method of claim 16, wherein the conditioning regimen is MAC-1 comprising administration of fludarabine and fractionated total body irradiation (FTBI).

18. The method of claim 17, wherein the fractionated total body irradiation (FTBI) is provided at 150 cGy x 2 doses.

19. The method of claim 17 or 18, wherein the MAC-1 comprises (i) 30 mg / m2of fludarabine on day -7, day -6, and day -5; and (ii) fractionated total body irradiation (FTBI) on day -4, day -3, day -2, and day -1.

20. The method of claim 16, wherein the conditioning regimen is RIC-3 comprising administration of fludarabine and melphalan.93IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO21. The method of claim 20, wherein the RIC-3 further comprises fractionated total body irradiation (FTBI).

22. The method of claim 21, wherein the FTBI is provided if the HSC donor is haploidentical to the patient.

23. The method of any one of claims 20-22, wherein the RIC-3 comprises administration of fludarabine at 25, 30 or 40 mg / m2IV qd on day -5, day -4, day -3 and day -2, and optionally on day -6.

24. The method of any one of claims 20-23, wherein the RIC-3 comprises administration of melphalan at 140 mg / m2on day -2 if the patient is not older than 60 and administration of melphalan at no more than 100 mg / m2on day -2 if the patient is older than 60.

25. The method of any one of claims 20-24, wherein the RIC-3 comprises administration of fractionated total body irradiation (FTBI) at 200 cGy on day -1 if the HSC donor is haploidentical to the patient.

26. The method of any one of claims 1-25, wherein the HSC donor is HLA-mismatched or haploidentical to the patient.

27. The method of any one of claims 1-26, wherein each of the at least two different T cell donors is allogeneic to the HSC donor and the patient.

28. The method of any one of claims 1-27, wherein the HSCs are administered at 105-108cells / kg, 105-107cells / kg, or 106-108cells / kg.

29. The method of claim 27, wherein the HSCs are administered at 106-107cells / kg.

30. The method of any one of claims 1-29, wherein the hematological malignancy is Acute Lymphoblastic Leukemia (B- or T-ALL), Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS) or Chronic myelomonocytic leukemia (CMML).

31. The method of any one of claims 1-30, wherein the HSCs are administered on day 0; and the polydonor CD4IL-10cells are administered after day 0.94IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO32. The method of claim 31, wherein the polydonor CD4IL-10cells are administered on day +1, day +2. day +3, day +4, day +5, day +6, day +7. day +8, day +9 or day +10.

33. The method of claim 31, wherein the polydonor CD4IL-10cells are administered on day +6 by infusion.

34. The method of any one of claims 1-33, further comprising administration of sirolimus.

35. The method of claim 34, wherein sirolimus is administered daily starting on day +5, tapered from day +60 to +90 and discontinued on day +100 to +150.

36. The method of any one of claims 1-35, further comprising administration of mycophenolate mofetil.

37. The method of claim 36, wherein the mycophenolate mofetil is administered daily starting on day +5 and discontinued on or after day +28 and no later than day +35.

38. The method of any one of claims 1-37, further comprising administration of posttransplant cyclophosphamide.

39. The method of claim 38, wherein the cyclophosphamide is administered on day +3 and day +4.

40. The method of any one of claims 1-39, wherein the polydonor CD4IL-10cells prevents or reduces severity' of GvHD by the HSCs.

41. The method of any one of claims 1-40, wherein the patient does not have grade III or IV GvHD after administration of the HSCs.

42. The method of any one of claims 1-41, wherein the polydonor CD4IL-10cells prevents or reduces severity of pathogenic response of lymphoid cells from the transplanted HSCs.

43. The method of any one of claims 1-42, wherein none of the donors of the CD4IL-10cells in the poly donor CD4n'l(1cells is the donor of the HSCs.

44. The method of any' one of claims 1-43, wherein administering a therapeutically' effective amount of polydonor CD4IL-10cells increases the number of natural Tri cells.95IPTS / 200307516.4Attorney Docket Ref: TR1X-012WO45. The method of any one of claims 1-43, wherein administering a therapeutically effective amount of polydonor CD4IL-10cells increases the number of DC-10 cells.

46. The method of any one of claims 1-43, wherein administering a therapeutically effective amount of poly donor CD4IL-10cells increases the number of FOXP3+ T cells.

47. The method of any one of claims 1-43, wherein administering a therapeutically effective amount of polydonor CD4IL-10cells increases the number of at least one of DC-10, natural Tri, and FOXP3+ T cells.

48. The method of any one of claims 1-47, wherein administering a therapeutically effective amount of polydonor CD4IL-10cells does not substantially increase nonspecific cytotoxicity.96IPTS / 200307516.4

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