WO2026064255A1 - Chimeric antigen receptor (car) tregs against pan class i swine leukocyte antigen and uses thereof - Google Patents

Abstract

Compositions and methods for suppressing rejection of xenotransplants are provided herein, including compositions comprising modified Treg cells that comprise a chimeric antigen receptor (CAR), wherein the CAR comprises a class-I swine leukocyte antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and methods for generating and using such modified Treg cells.

Description

Dkt No. 93597/7374 92441-A-PCT

CHIMERIC ANTIGEN RECEPTOR (CAR) TREGS AGAINST PAN CLASS I SWINE LEUKOCYTE ANTIGEN AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/695,455, filed September 17, 2024, the content of which is hereby incorporated by reference.

[0002] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.

STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under grant number AI045897 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

[0004] This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named '‘93597-7374_92441 -A-PCT _Sequence_Listing_AWG.xml”, which is 20,146 bytes in size, and which was created on August 31, 2025 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the XML file filed September 16, 2025 as part of this application.

BACKGROUND OF THE INVENTION

[0005] The disclosures of all publications, patents, patent application publications and books referred to in this application are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains. [0006] Over the past few decades, the waiting lists for transplants have continued to grow, while the number of available organs has not increased at the same rate. Finding alternative sources of donor organs is of great importance. Transplantation of organs from other animals

4924-6564-8233v.1 to humans, which is called xenotransplantation, has emerged as a promising source. Major advancements in generating transgenic pigs in order to make them more compatible with human immune systems have occurred during the last two decades. Despite all advances in developing immunosuppressive medications, immune responses in xenotransplantation are difficult to control without excessive toxicity \ Regulatory⁷ T cell (Treg) therapy has been widely used for control of immune responses in different models of autoimmunity and transplantation² . It is well known that compared to non-specific Tregs, antigen-specific Tregs are more potent in suppressing immune responses and harbor a lower risk of general immunosuppression^6,7. Chimeric antigen receptor (CAR) Tregs against HLA-A2 have been shown to be effective in preventing xeno-GVHD caused by HLA-A2 -restricted T cells in a human PBMC -transferred NSG mouse model⁸. HLA-A2-CAR Tregs were also effective in controlling allogeneic responses in a skin transplantation model in humanized mice⁹. CAR Tregs were shown to be effective in controlling immune reactions in some autoimmune disease models ^{10 l2}.

[0007] Despite promising results in using CAR Tregs for tolerance induction, there are still major considerations regarding the plasticity of CAR Tregs¹³ and the effect of T celldepleting conditioning regimens^14,15 on these cells that need to be addressed before their clinical application. Notably, inflammation drives Treg conversion¹⁶. CAR Tregs could quickly⁷ reject target organs if they⁷ lose their regulatory phenoty pe and convert to effector T cells (Teffs).

BRIEF SUMMARY OF THE INVENTION

[0008] The invention provides a modified Treg cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a class-I swine leukocyte antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory and/or co-stimulatory molecule signaling domain.

[0009] The invention also provides a pharmaceutical composition comprising a plurality of modified Treg cells described herein and a pharmaceutically acceptable carrier.

[0010] Also provided is a method for inducing immune tolerance against one or more pig antigens in a non-pig mammalian subject, comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the modified Treg cell described herein.

4924-6564-8233v.1 [0011] The invention also provides a method for suppressing in a human an immune response against one or more pig antigens, or for inhibiting rejection of a pig organ, pig tissue or pig cell infusion in a non-pig subject, comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the modified Treg cell described herein.

[0012] Additionally, the invention also provides a method of reducing the likelihood of, or extent of, graft versus host disease and/or host versus graft disease in a human subject who is to receive, is receiving, or who has received a transplanted tissue or organ comprising a pig antigen, the method comprising administenng to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified Treg cell described herein. [0013] The invention also provides a method of generating a modified Treg cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a class-I swine leukocyte antigen-binding domain, the method comprising providing Treg cells from a sample obtained from a human, and transducing the Treg cells with a nucleic acid encoding the CAR.

[0014] A modified Treg cell generated by the method described herein is also provided.

[0015] Also provided is a polynucleotide encoding a chimeric antigen receptor (CAR), the CAR comprising a class-I swine leukocyte antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory and/or co-stimulatory molecule signaling domain.

[0016] A cell comprising or genetically modified by any one of the polynucleotides or vectors is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 : Xeno CAR Tregs: a new CAR Treg for xenotransplantation.

[0018] FIGS. 2A-2D: Development of SLA CARs based on antibody sequences from two

SLA-I hybridoma lines. Figs. 2A-2D show a process of development of SLA CAR constructs (Fig. 2A, Fig. 2B) and testing their expression and functionality using transfected TCRB KO Jurkat (JRT3.3) cells non-stimulated and stimulated with irradiated pig PBMCs (Fig. 2C, Fig. 2D).

[0019] FIG. 3: Selection and validation of SLA CARs based on their capacity to induce T cell activation upon their interaction with SLA-I molecule. SLA CAR lentiviruses are generated and titrated. Human J76 cells (a human T cell tumor line) were transduced with

4924-6564-8233v.1 different SLA CARs for testing their capacity to react to pig PBMCs. J76 cells contain 3 reporter genes under the control of 3 promoters downstream of TCR signaling: NF AT, NFKB and AP-1. All of these 3 signaling pathways were shown get activated upon interaction of SLA CARs # 1 and #2 with pig PBMCs. Additionally, these T cells where shown to upregulate CD69, which is a T cell activation marker, upon this interaction. It seems that SLA CAR #1 induces higher levels of T cell activation compared to SLA CAR #2. Therefore, this SLA CAR was chosen for other in vitro and in vivo experiments.

[0020] FIG. 4: In vitro testing of SLA CAR Treg. SLA CAR (#1) Tregs showed a higher capacity in suppressing anti-pig immune responses at different Treg : Teff ratios compared to polyclonal (CAR-) Tregs.

[0021] FIG. 5: SLA CAR Tregs prevent the destruction of fetal pig lung grafts by human PBMCs (PBMC-NSG mouse model).

[0022] FIG. 6: Development of a human immune system (HIS) mouse model to evaluate SLA CAR Tregs in prevention of fetal pig lung transplantation rejection.

[0023] FIG. 7: SLA CAR Tregs prevent the destruction of fetal pig lung grafts by human immune system (HIS) mouse T cells.

[0024] FIG. 8: Knockout CD2: a solution to protect Tregs from siplizumab-containing conditioning regimens. (CD2 provides co-stimulatory signals and forms immunological synapse).

[0025] FIG. 9: CD2-KO Tregs are resistant to siplizumab-containing conditioning regimens.

[0026] FIG. 10: FOXP3 expression in CD2-KO Tregs is similar to WT Tregs.

[0027] FIG. 11 : CD2-KO Tregs exhibit comparable suppressive capacity as WT Tregs in vitro.

[0028] FIG. 12: CD2-KO Tregs exhibit decreased glycolysis but enhanced oxidative phosphorylation.

[0029] FIG. 13: CD2-KO Tregs show superior mitochondrial function with lower ROS production. CD2-KO Tregs also exhibit decreased TCR signaling, proinflammatory and senescence gene signatures (data not shown).

[0030] FIG. 14: CD2-KO Tregs are comparable to WT Tregs in preventing GVHD in mice.

[0031] FIG. 15: CD2-KO Tregs show a less activated phenotype than WT Tregs in vivo.

4924-6564-8233v.1 [0032] FIG. 16: CAR-Tregs experience more exhaustion and senescence than untransduced Tregs due to tonic signaling.

[0033] FIG. 17: CAR-transduced CD2-KO Tregs are more stable and functional compared to WT counterparts.

[0034] FIG. 18: Map of an exemplary SLA CAR construct.

[0035] FIG. 19: Map of an exemplary SLA CAR construct.

[0036] FIG. 20: Map of an exemplary⁷ SLA CAR construct.

[0037] FIG. 21: Map of an exemplary SLA CAR construct.

[0038] FIG. 22: Map of an exemplary SLA CAR construct.

[0039] FIG. 23: Map of an exemplary SLA CAR construct.

[0040] FIGS. 24A-24B: SLA-1 CAR+ J76 cells undergo activation upon interaction with pig PBMCs. SLA-I CAR constructs containing NGFR (truncated cytoplasmic tail) as a reporter were lentivirally transduced into TCR-KO J76 cells that were further co-cultured with pig PBMCs from the haplotypes SLA-CC, SLA-DD, and SLA-HH. Cell activation markers i.e., PD-1 and CD25, were measured 48h post co-culture (Fig. 24A). J76 TCR-KO cells expressing the new (adapted from anti-SLA-I antibody secreted by hybridoma cells) vs original SLA-I CAR (framework regions adapted from HLA-A2 CAR) were co-cultured with Pig PBMCs and analyzed for expression of activation markers CD69, CD25, and HLA-DR 48h post co-culture (Fig. 24B).

[0041] Fig. 25: CAR constructs designed to study CAR conversion.

[0042] Fig. 26: Schematic presentation of events in generation of humanized mouse models of T1D and xenotransplantation. TBI: total body irradiation.

[0043] Fig. 27: Kinetics of human and pig immune cell reconstitution in mixed chimeric (MC) vs non-MC humanized mice. Mixed chimeras were generated by co-transplantation of enriched HSCs from pig bone marrow and human fetal liver-derived CD34+ HSCs. Human cells have an advantage for immune reconstitution and by week 16 after transplantation, the majority of pig cells have disappeared.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Disclosed are CAR constructs that can recognize all pig cells. In some embodiments, the constructs were generated by performing B cell receptor (BCR) sequencing on hybridoma lines that generate pan anti class-I swine leukocyte antigen (SLA) antibodies. In some embodiments, the CAR constructs recognize all pig cells and are used to generate

4924-6564-8233v.1 CAR Treg cells, which can be deployed in order to control the immune responses. This allows for implementation of tolerogenic strategies to prevent immune rejection of pig organs in xenotransplantation such that a limitless source of donor organs may be provided in order to overcome the challenge of donor organ shortage for transplantation.

[0045] The invention provides a modified Treg cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a class-I swine leukocyte antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory and/or co-stimulatory molecule signaling domain.

[0046] In some embodiments, the modified Treg cell is a human T cell transduced with a nucleic acid encoding the CAR. In some embodiments, the nucleic acid encoding the CAR is introduced to the cell by lentiviral transduction. In some embodiments, the nucleic acid encoding the CAR also encodes an inducible suicide gene.

[0047] In some embodiments, the CAR is under control of a Treg-specific promoter. In some embodiments, the promoter is a Foxp3 promoter. In some embodiments, the promoter is a minimal sequence Foxp3 promoter.

[0048] In some embodiments, the modified Treg cell further comprises an inducible suicide gene. In some embodiments, the inducible suicide gene comprises a CD8/caspase8 sequence, optionally under the control of a promoter of an IL-7 receptor (CD 127), or a promoter which induces expression that inversely correlates with Foxp3 expression, e.g., to ensure that CAR Tregs undergo apoptosis if they acquire an effector phenotype. In some embodiments, the inducible suicide gene is encoded on the same nucleic acid molecule that encodes the CAR, optionally on a strand opposite to the strand that encodes the CAR. In some embodiments, the inducible suicide gene is encoded on a different nucleic acid molecule than the nucleic acid molecule that encodes the CAR.

[0049] In some embodiments, the antigen binding domain of the CAR comprises a singlechain variable fragment (scFv), an antibody, or an antigen-binding fragment of an antibody. In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, and a single domain antibody. In some embodiments, the antigen-binding domain of the CAR comprises a single-chain variable fragment (scFv).

[0050] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises three VH CDRs chosen from: SEQ ID NOs: 1, 2, 3, 7, 8, and 9, and three VL CDRs chosen from SEQ ID NOs: 4, 5, 6, 10, 11, 12, 13, 14, and 15.

4924-6564-8233v.1 [0051] In some embodiments, the transmembrane domain of the CAR comprises a CD8 transmembrane domain and/or a CD28 transmembrane domain.

[0052] In some embodiments, the Treg cell has been treated so as to reduce or remove endogenous TCR a and (3 chains.

[0053] In some embodiments, the Treg cell has been treated so as to reduce or remove their endogenous beta-2 microglobulin (B2M), class II major histocompatibility complex, transactivator (CIITA) and/or the Treg cell has had introduced therein HLA-G and/or HLA- E molecules, and/or has been modified so as not to express CD2 or express CD2 at a reduced amount relative to its non-modified form.

[0054] In some embodiments, the Treg is a modified allogeneic Treg.

[0055] In some embodiments, the Treg cell has been treated to overexpress PD-L1, TGF- beta, and/or CTLA-4Ig. In some embodiments, the PD-L1, TGF-beta, and CTLA-4Ig encoding sequences are under the control of a NFAT6 promoter.

[0056] In some embodiments, the Treg cell has been treated to express SCF, FLT3L, thrombopoietin, and/or CXCL12. In some embodiments, the PD-L1, TGF-beta, and/or CTLA-4Ig encoding sequences are under the control of aNFAT6 promoter.

[0057] In some embodiments, the intracellular signaling domain of the CAR comprises a CD28 intracellular domain.

[0058] In some embodiments, the intracellular domain of the CAR comprises a y (gamma) subunit of an immunoglobulin Fc receptor (FcRy) signaling domain. In some embodiments, the Fc receptor (FcRy) signaling domain is a human Fc receptor (FcRy) signaling domain.

[0059] In some embodiments, the intracellular signaling domain of the CAR comprises a CD3zeta (CD3Q subunit. In some embodiments, the intracellular signaling domain of the CAR comprises a CD3zeta (CD3Q subunit or a human Megfl 0 cytoplasmic domain. In some embodiments, the intracellular signaling domain of the CAR comprises a BAI1. In some embodiments, the intracellular signaling domain of the CAR comprises a MERTK. In some embodiments, the intracellular signaling domain of the CAR comprises a TIME In some embodiments, the intracellular signaling domain of the CAR comprises a TIM4. In some embodiments, the intracellular signaling domain of the CAR comprises a 4- IBB. In some embodiments, the intracellular signaling domain of the CAR comprises more than one type of the intracellular signaling domains listed herein.

[0060] In some embodiments, the transmembrane domain of the CAR comprises a human CD8 transmembrane domain. In some embodiments, the transmembrane domain of the CAR

4924-6564-8233v.1 comprises a human CD28 transmembrane domain. In some embodiments, the transmembrane domain of the CAR further comprises a hinge region between the binding domain and the transmembrane domain. In some embodiments, the hinge region is a CD8 hinge region. In some embodiments, the CAR comprises a CD8 hinge region between the class-I swine leukocyte antigen-binding domain and the transmembrane domain.

[0061]

[0062] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH compnsing the following:

CDR1

GYTFTNYG (SEQ ID NO: 1),

CDR2

INTYTGEP (SEQ ID NO: 2), and

CDR3

ARRGDGYY (SEQ ID NO: 3), and/or a VL comprising the following:

CDR1

QNVGTN (SEQ ID NO:4),

CDR2

SAS (SEQ ID NO:5), and

CDR3

QQYNSYPLT (SEQ ID NO:6).

[0063] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises

(a) a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NOV), and/or

4924-6564-8233v.1 (b) a VL comprising the following:

CDR1

ENVVTY (SEQ ID NO: 10),

CDR2

GAS (SEQ ID NO: 11), and

CDR3

GQGYSYPYT (SEQ ID NO: 12) or

CDR1

ENIYSN (SEQ ID NO.13),

CDR2

AAT (SEQ ID NO: 14). and

CDR3

QHFWGTPRT (SEQ ID NO: 15).

[0064] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NO: 9), and a VL comprising the following:

CDR1

ENVVTY (SEQ ID NOTO),

CDR2

GAS (SEQ ID NO: 11), and

CDR3

GQGYSYPYT (SEQ ID NO: 12).

[0065] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises

4924-6564-8233v.1 a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NOV), and a VL comprising the following:

CDR1

ENIYSN (SEQ ID NO: 13),

CDR2

AAT (SEQ ID NO: 14). and

CDR3

QHFWGTPRT (SEQ ID NO: 15).

[0066] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

GYTFTNYG (SEQ ID NO: 1),

CDR2

INTYTGEP (SEQ ID NO: 2). and

CDR3

ARRGDGYY (SEQ ID NO:3).

[0067] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NOV).

4924-6564-8233v.1 [0068] In some embodiments, the class-T swine leukocyte antigen-binding domain comprises a VL comprising the following:

CDR1

ENVVTY (SEQ ID NO: 10),

CDR2

GAS (SEQ ID NO: 11), and

CDR3

GQGYSYPYT (SEQ ID NO: 12).

[0069] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VL comprising the following:

CDR1

ENIYSN (SEQ ID NO: 13),

CDR2

AAT (SEQ ID NO: 14). and

CDR3

QHFWGTPRT (SEQ ID NO: 15).

[0070] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VL comprising the following:

CDR1

QNVGTN (SEQ ID NO:4),

CDR2

SAS (SEQ ID NO:5), and

CDR3

QQYNSYPLT (SEQ ID NO:6).

[0071] In some embodiments, in N-terminal to C-terminal order, the class-I swine leukocyte antigen-binding domain comprises a VL and then a VH.

[0072] In some embodiments, in N-terminal to C-terminal order, the class-I swine leukocyte antigen-binding domain comprises a VH and then a VL.

[0073] In some embodiments, the VH and VL are connected via a linker peptide.

4924-6564-8233v.1 [0074] In some embodiments, one, more than one, or all framework region(s) of the scFv have a sequence of human framework region(s).

[0075] The invention also provides a pharmaceutical composition comprising a plurality of modified Treg cells described herein and a pharmaceutically acceptable carrier. In some embodiments, at least 33% of the cells in the pharmaceutical composition express the CAR.

[0076] The invention provides a method for inducing immune tolerance against one or more pig antigens in a non-pig mammalian subject, comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition compnsing the modified Treg cell described herein. In some embodiments, an additional tolerance-inducing agent is administered to the subject in combination with the pharmaceutical composition comprising the modified Treg cell described herein. In some embodiments, the subject is human.

[0077] Additionally, the invention provides a method for suppressing in a human an immune response against one or more pig antigens, or for inhibiting rejection of a pig organ, pig tissue or pig cell infusion in a non-pig subject, comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the modified Treg cell described herein. In some embodiments, an additional toleranceinducing agent is administered to the subject in combination with the pharmaceutical composition. In some embodiments, the non-pig subject is human.

[0078] The invention also provides a method for suppressing in a human an immune response against one or more pig antigens, comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition described herein. [0079] The invention provides a method of reducing the likelihood of, or extent of, graft versus host disease and/or host versus graft disease in a human subject who is to receive, is receiving, or who has received a transplanted tissue or organ comprising a pig antigen, or a pig cell infusion, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified Treg cell described herein. In some embodiments, the method reduces the likelihood of, or extent of, rejection of the transplanted tissue, organ, or cells.

[0080] In some embodiments, the effective amount of a pharmaceutical composition comprising the modified Treg cell is administered to the subject prior to or during transplant surgery. In some embodiments, the effective amount of a pharmaceutical composition

4924-6564-8233v.1 comprising the modified Treg cell is administered to the subject prior to or during cell infusion.

[0081] In some embodiments, the effective amount of a pharmaceutical composition comprising the modified Treg cell is administered to the subject subsequent to transplant surgery. In some embodiments, the effective amount of a pharmaceutical composition comprising the modified Treg cell is administered to the subject subsequent to cell infusion. [0082] In some embodiments, the method further comprises administering a T celldepleting conditioning regimen to the subject.

[0083] In some embodiments, the T cell-depleting conditioning regimen comprises an anti-CD2 antibody and wherein the modified Treg cells have been treated to remove or deplete cell-surface CD2. In some embodiments, CRISPR is used to remove or deplete CD2 from the modified Treg cells.

[0084] In some embodiments, the modified Treg cells have been modified from Treg cells obtained from a human subject.

[0085] In some embodiments, the Treg cells obtained from a human subject are obtained from the same human being subjected to the method, or from the same human who is to receive, is receiving, or who has received a transplanted tissue.

[0086] In some embodiments, the Treg cells obtained from a human subject are obtained from a different human than the one being subjected to the method, or from a different human from the one who is to receive, is receiving, or who has received a transplanted tissue.

[0087] In some embodiments, the Treg cells are obtained from cord blood or from a PBMC sample.

[0088] In some embodiments, the pig cell infusion comprises a pig bone marrow or pig islet cell transplantation.

[0089] The invention also provides a method of generating a modified Treg cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a class-I swine leukocyte antigen-binding domain, the method comprising providing Treg cells from a sample obtained from a human, and transducing the Treg cells with a nucleic acid encoding the CAR.

[0090] In some embodiments, the Treg cells are transduced with a nucleic acid encoding the CAR, preferably via a lentivirus encoding the CAR. In some embodiments, a nucleic acid molecule encoding the CAR is introduced to a Treg cell by other known delivery methods, for example, transfection or lipid nanoparticle delivery' systems.

4924-6564-8233v.1 [0091] In some embodiments, the Treg cells transduced with or which have otherwise been delivered a nucleic acid encoding the CAR are expanded subsequent to transduction.

[0092] In some embodiments, the Treg cells transduced with or which have otherwise been delivered a nucleic acid encoding the CAR are expanded by contact with IL-2.

[0093] In some embodiments, the Treg cells are CD4⁺ CD25⁺ CD 127" cells sorted from human donor PBMCs or cord blood cells.

[0094] In some embodiments, the method further comprises activating the Treg cells. In some embodiments, the method further comprises activating the Treg cells with anti-CD3/28 beads.

[0095] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

GYTFTNYG (SEQ ID NO: 1 ),

CDR2

INTYTGEP (SEQ ID NO: 2), and

CDR3

ARRGDGYY (SEQ ID NO: 3), and/or a VL comprising the following:

CDR1

QNVGTN (SEQ ID NO:4),

CDR2

SAS (SEQ ID NO:5), and

CDR3

QQYNSYPLT (SEQ ID NO:6).

[0096] In embodiments, the class-I swine leukocyte antigen-binding domain comprises

(a) a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7).

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

4924-6564-8233v.1 ARWGNYPHYAMDY (SEQ ID NO: 9) and/or

(b) a VL comprising the following:

CDR1

ENVVTY (SEQ ID NO: 10),

CDR2

GAS (SEQ ID NO: 11), and

CDR3GQGYSYPYT (SEQ ID NO: 12), or

CDR1

ENIYSN (SEQ ID NO: 13),

CDR2AAT (SEQ ID NO: 14), and

CDR3

QHFWGTPRT (SEQ ID NO: 15).

[0097] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NON), and a VL comprising the following:

CDR1

ENVVTY (SEQ ID NOTO),

CDR2

GAS (SEQ ID NO: 11), and

CDR3

GQGYSYPYT (SEQ ID NO: 12).

[0098] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises

4924-6564-8233v.1 a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NO:9), and a VL comprising the following:

CDR1

ENIYSN (SEQ ID NO: 13),

CDR2

AAT (SEQ ID NO: 14). and

CDR3

QHFWGTPRT (SEQ ID NO: 15).

[0099] Additionally, a modified Treg cell generated by the method described herein is provided.

[00100] In some embodiments, the transmembrane domain of the CAR comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain of the CAR comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain of the CAR comprises a human CD8 transmembrane domain. In some embodiments, the transmembrane domain of the CAR comprises a human CD28 transmembrane domain. In some embodiments, the transmembrane domain of the CAR further comprises a hinge region between the binding domain and the transmembrane domain. In some embodiments, the hinge region is a CD8 hinge region.

[00101] In some embodiments, the intracellular signaling domain of the CAR comprises a y (gamma) subunit of an immunoglobulin Fc receptor (FcRy) signaling domain. In some embodiments, the Fc receptor (FcRy) signaling domain is a human Fc receptor (FcRy) signaling domain.

[00102] In some embodiments, the intracellular signaling domain of the CAR comprises a CD3zeta (CD3Q subunit or a human Megfl 0 cytoplasmic domain. In some embodiments, the intracellular signaling domain of the CAR comprises a BAI1. In some embodiments, the intracellular signaling domain of the CAR comprises a MERTK. In some embodiments, the

4924-6564-8233v.1 intracellular signaling domain of the CAR comprises a TIM1. In some embodiments, the intracellular signaling domain of the CAR comprises a TIM4. In some embodiments, the intracellular signaling domain of the CAR comprises a 4-1BB. In some embodiments, the intracellular signaling domain of the CAR comprises more than one type of the intracellular signaling domains listed herein.

[00103] Anti-SLA scFvs with CDRs substantially equivalent to those set forth in the present application are also disclosed. Within this embodiment, the term "substantially equivalent to" is understood to comprise amino acid sequences homologous to any of SEQ ID NOs: 1-15, or by at least 90%. based on a comparison of primary amino acid sequence. Such degrees of homology may be determined by standard sequence alignment programs such as Vector NTI (InforMax™, Maryland, USA). Such programs compare aligned sequences on an amino acid-by -amino acid basis, and can be set to various levels of stringency for the comparison (e.g. identical amino acid, conservative amino acid substitution, etc.). Within the meaning of this embodiment, two amino acids in question are considered as being "homologous" when they are either identical to one another or conservative substitutions of one another. By way of non-limiting example, tw o different amino acids belonging to the class of lipophilic amino acids would be considered homologous in the sense of this embodiment, even if these two amino acids were not identical, whereas a lipophilic amino acid on the one hand and a charged acidic amino acid oh the other hand would not be considered homologous.

[00104] Single chain fragment variables (scFv) are known generally in the art. They are single polypeptides that contain a variable light chain (VL) and a variable heavy chain (VH) of an antibody. They are often ~25 kDa. The two chains are connected by a flexible linker peptide, often 15-20 amino acids long and usually made up of glycine and serine, optionally with dispersed hydrophilic residues for increased solubility. The linker generally keeps the C-terminus of one variable domain and the N-terminus of the other domain at a distance that favors proper folding and formation of the antigen-binding site while also minimizing oligomerization of the scFv. The variable domains order of an scFv can mirror that of an antibody (VL-linker-VH), or be VL-linker-VH or have a VH-linker-VL configuration.

[00105] In some embodiments, the SLA-CAR comprises the polypeptide sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20 or 21. In some embodiments, the SLA-CAR comprises

4924-6564-8233v.1 a polypeptide sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20 or 21, preferably wherein the CDRs are unaltered.

[00106] In some embodiments, at least 50% of the cells in the pharmaceutical composition express a CAR. In some embodiments, at least 33% of the cells in the pharmaceutical composition express a CAR. In some embodiments, at least 25% of the cells in the pharmaceutical composition express a CAR.

[00107] In some embodiments, PBMCs are obtained from the subject 1, 2, 3, 4, or 5 days after administration of an agent to the subject which elicits mobilization of PBMCs. In some embodiments, the agent comprises G-CSF. In some embodiments, cells are obtained from the subject by apheresis. In some embodiments, cells are obtained from the subject to be treated. In some embodiments, cells are obtained from a different subject than the subject to be treated.

[00108] In some embodiments, transduction with a nucleic acid encoding the CAR is effected using a viral vector. In some embodiments, transduction with anucleic acid encoding the CAR is effected using a lentiviral vector. In some embodiments, transduction with a nucleic acid encoding the CAR is effected using a gamma-retroviral vector. Viral vectors for transducing CAR into cells are known in the art, for example see Irving et al., Human Gene Therapy. Oct 2021.1044-1058 on the world wide web at doi.org/10. 1089/hum.2021. 173, and hereby incorporated by reference in its entirety.

[00109] Conventional viral and non-viral based gene transfer methods can be used to introduce polynucleotide molecules to target cells or tissues. In certain embodiments, polynucleotide molecules are administered in vivo or ex vivo. Non-viral vector delivery systems include naked nucleic acid, and nucleic acid complexed with a deliver}⁷ vehicle such as a liposome, lipid nanoparticle, or poloxamer.

[00110] In certain embodiments, a viral system may be used to deliver a polynucleotide molecule to a target a cell or tissue. The use of RNA or DNA viral -based systems for viral- mediated deliver}' of polynucleotide molecules takes advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to those cells. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[00111] Vectors suitable for introduction of transgenes into immune cells include nonintegrating lentivirus vectors. See, e.g., U.S. Patent Application Publication No. 2009/0117617. A polynucleotide molecule may be delivered to a target cell using a non-

4924-6564-8233v.1 integrating lentiviral particle method. Such a method may be used to deliver RNAs into the target cell, such that delivery⁷ of the RNAs to the target cell results in assembly of the compositions described herein inside of the target cell.

[00112] Viral vectors can be administered directly to a patient or they can be used to treat cells and the modified cells are administered to patients. Conventional viral-based systems for the delivery of polynucleotide molecules include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors. An RNA virus may be utilized for delivery of polynucleotide molecules to cells. Also, a polynucleotide molecule may be delivered to a cell by non-integrating lentivirus.

[00113] Vectors may be delivered in vivo by administration to an individual patient, for example by systemic administration (e.g., intravitreal, intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application. Specifically, vectors (e.g., retroviruses, liposomes, lipid nanoparticles, etc.) containing therapeutic nucleic acid compositions can be administered directly to an organism for transduction of cells in vivo. Administration may be by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application (e.g.. eye drops and cream) and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and more than one route can be used to administer a particular composition.

[00114] Alternatively, vectors may be delivered to cells ex vivo, such as to cells explanted from an individual patient (e g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, optionally after selection for cells which have incorporated the vector. A non-limiting exemplary ex vivo approach may involve removal of tissue (e.g., peripheral blood, bone marrow, and spleen) from a patient for culture, nucleic acid transfer to the cultured cells (e.g., hematopoietic stem cells), followed by grafting the cells to a target tissue (e.g., bone marrow, and spleen) of the patient. Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In an embodiment, cells are isolated from the subject organism, transfected with a nucleic acid composition, and re-infused back into the subject organism (e.g., patient).

[00115] Also provided is a polynucleotide encoding a chimeric antigen receptor (CAR), the CAR comprising a class-I swine leukocyte antigen-binding domain, a transmembrane

4924-6564-8233v.1 domain, and an intracellular signaling domain comprising a stimulatory and/or co-stimulatory molecule signaling domain.

[00116] In some embodiments, the polynucleotide encodes a Treg-specific promoter that controls expression of the CAR. In some embodiments, the promoter is a Foxp3 promoter. In some embodiments, the promoter is a minimal sequence Foxp3 promoter.

[00117] In some embodiments, the polynucleotide further encodes an inducible suicide gene. In some embodiments, the inducible suicide gene comprises a CD8/caspase8 sequence, optionally wherein the expression of the suicide gene is under the control of a promoter of an IL-7 receptor (CD127). or a promoter which induces expression that inversely correlates with Foxp3 expression. In some embodiments, the inducible suicide gene is encoded on the same nucleic acid molecule that encodes the CAR, optionally on a strand opposite to the strand that encodes the CAR.

[00118] In some embodiments, the antigen binding domain of the CAR comprises a singlechain variable fragment (scFv), an antibody, or an antigen-binding fragment of an antibody. [00119] In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, and a single domain antibody.

[00120] In some embodiments, the transmembrane domain of the CAR comprises a CD8 transmembrane domain or CD28 transmembrane domain, and/or wherein the intracellular domain of the CAR comprises a y (gamma) subunit of an immunoglobulin Fc receptor (FcRy) signaling domain, and/or wherein the intracellular domain of the CAR comprises a CD3zeta (CD3Q subunit.

[00121] In some embodiments, the intracellular signaling domain of the CAR comprises a

CD28 intracellular domain.

[00122] In some embodiments, the intracellular domain of the CAR comprises a y (gamma) subunit of an immunoglobulin Fc receptor (FcRy) signaling domain. In some embodiments, the Fc receptor (FcRy) signaling domain is a human Fc receptor (FcRy) signaling domain.

[00123] In some embodiments, the intracellular signaling domain of the CAR comprises a CD3zeta (CD3Q subunit. In some embodiments, the intracellular signaling domain of the CAR comprises a CD3zeta (CD3Q subunit or a human Megfl 0 cytoplasmic domain. In some embodiments, the intracellular signaling domain of the CAR comprises a BAIL In some embodiments, the intracellular signaling domain of the CAR comprises a MERTK. In some embodiments, the intracellular signaling domain of the CAR comprises a TIM1. In some

4924-6564-8233v.1 embodiments, the intracellular signaling domain of the CAR comprises a TIM4. In some embodiments, the intracellular signaling domain of the CAR comprises a 4- IBB. In some embodiments, the intracellular signaling domain of the CAR comprises more than one type of the intracellular signaling domains listed herein.

[00124] In some embodiments, the transmembrane domain of the CAR comprises a human CD8 transmembrane domain. In some embodiments, the transmembrane domain of the CAR comprises a human CD28 transmembrane domain. In some embodiments, the transmembrane domain of the CAR further comprises a hinge region between the binding domain and the transmembrane domain. In some embodiments, the hinge region is a CD8 hinge region. In some embodiments, the CAR comprises a CD8 hinge region between the class-I swine leukocyte antigen-binding domain and the transmembrane domain.

[00125] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises: a VH comprising the following:

CDR1

GYTFTNYG (SEQ ID NO: 1),

CDR2

INTYTGEP (SEQ ID NO: 2), and

CDR3

ARRGDGYY (SEQ ID NO: 3), and/or a VL comprising the following:

CDR1

QNVGTN (SEQ ID NO:4),

CDR2

SAS (SEQ ID NO:5), and

CDR3

QQYNSYPLT (SEQ ID NO: 6).

[00126] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises:

(a) a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

4924-6564-8233v.1 CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NOV), and/or

(b) a VL comprising the following:

CDR1

ENVVTY (SEQ ID NO: 10),

CDR2

GAS (SEQ ID NO: 11), and

CDR3

GQGYSYPYT (SEQ ID NO: 12) or

CDR1

ENIYSN (SEQ ID NO: 13),

CDR2

AAT (SEQ ID NO: 14). and

CDR3

QHFWGTPRT (SEQ ID NO: 15).

[00127] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NO: 9), and a VL comprising the following:

CDR1

ENVVTY (SEQ ID NOTO),

CDR2

4924-6564-8233v.1 GAS (SEQ ID NO: 11), and

CDR3

GQGYSYPYT (SEQ ID NO: 12).

[00128] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7).

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NO:9), and a VL comprising the following:

CDR1

ENIYSN (SEQ ID NO: 13),

CDR2

AAT (SEQ ID NO: 14), and

CDR3

QHFWGTPRT (SEQ ID NO: 15).

[00129] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

GYTFTNYG (SEQ ID NO: 1),

CDR2

INTYTGEP (SEQ ID NO .2), and

CDR3

ARRGDGYY (SEQ ID NO:3).

[00130] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

4924-6564-8233v.1 GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO: 8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NOV).

[00131] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VL comprising the following:

CDR1

ENVVTY (SEQ ID NO: 10),

CDR2

GAS (SEQ ID NO: 11), and

CDR3

GQGYSYPYT (SEQ ID NO: 12).

[00132] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VL comprising the following:

CDR1

ENIYSN (SEQ ID NO: 13),

CDR2

AAT (SEQ ID NO: 14). and

CDR3

QHFWGTPRT (SEQ ID NO: 15).

[00133] In some embodiments, the class-I swine leukocyte antigen-binding domain comprises a VL comprising the following:

CDR1

QNVGTN (SEQ ID NO:4),

CDR2

SAS (SEQ ID NO:5), and

CDR3

QQYNSYPLT (SEQ ID NO: 6).

4924-6564-8233v.1 [00134] In some embodiments, the CAR further comprises a CD8 hinge region between the class-I swine leukocyte antigen-binding domain and the transmembrane domain.

[00135] In some embodiments, the polynucleotide molecule encodes a sequence having at least 85% sequence identity to any one of SEQ ID NO: 16, 17, 18, 19, 20, or 21, preferably wherein the CDRs are unaltered.

[00136] Also provided is a vector comprising any one of the polynucleotides described above.

[00137] In some embodiments, the vector is a lentiviral vector.

[00138] In some embodiments, the vector is an RNA vector.

[00139] In some embodiments, the vector comprises an inducible promoter operably linked to the polynucleotide sequence encoding the CAR.

[00140] Also provided is a cell comprising or genetically modified by any one of the polynucleotides or vectors described above.

[00141] In some embodiments, a nucleic acid herein comprises a cDNA. In some embodiments, a nucleic acid herein comprises a DNA. In some embodiments, a nucleic acid herein comprises an RNA. In some embodiments, a nucleic acid herein comprises an mRNA. [00142] Administration in an embodiment of the methods is intravenous. Administration in an embodiment of the methods is via infusion. Administration can also be auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrabdominal, intraamniotic, intra-arterial, intraarticular, intrabiliaiy, intrabronchial, intrabursal. intracardiac, intracartilaginous, intracaudal, intracavemous. intracavitary, intracerebral, intracistemal. intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary. intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intraventricular, intravesical, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, rectal, inhalationally, retrobulbar, subarachnoid, subconjuctivaL sublingual, submucosal, topically, transdermal, transmucosaL transplacental, transtracheal, ureteral, uretheral, and vaginal.

4924-6564-8233v.1 [00143] The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anyw here in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

[00144] The "variable region" or "variable domain" of an scFv or antigen binding domain of a CAR refers to regions derived from the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites. The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. How ever, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) (or CDRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity byFR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda. Md. (1991)).

[00145] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

4924-6564-8233v.1 [00146] The term "complementarity-determining region" or "CDR" when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six CDRs; three in the VH (Hl, H2, H3) and three in the VL (LI, L2, L3). CDRS 1-3 of the VH or VL as referred to herein may alternatively be named with the H# or L# designation. In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e g., Xu et al., Immunity 13:37- 45 (2000); Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press. Totowa. N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e g., Hamers- Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Sendee, National Institutes of Health, Bethesda, Md. (1991) hereby incorporated by reference in its entirety). There are CDRs 1, 2, and 3 for each of the heavy and light chains. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of the available complex crystal structures. HVRs may comprise "extended HVRs" as follows: 24-36 or 24- 34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (Hl ). 50-65 or 49- 65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[00147] In some embodiments, the variable regions disclosed herein are not modified. In some embodiments, the invention encompasses modifications to the variable regions disclosed herein. For example, the invention includes antibodies comprising functionally equivalent variable regions and CDRs which do not significantly affect their properties as well as variants which have enhanced or decreased activity and/or affinity. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or which mature (enhance) the affinity' of the polypeptide for its ligand or use of chemical analogs.

4924-6564-8233v.1 [00148] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Substitution variants have at least one amino acid residue in the amino acid sequence removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but framework alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "conservative substitutions." If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplar}' substitutions" in Table 1, or as further described below' in reference to amino acid classes, may be introduced and the products screened.

[00149] Table 1: Amino Acid Substitutions

4924-6564-8233v.1

[00150] Substantial modifications in the biological properties of an antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining

(а) the structure of the polypeptide backbone in the area of the substitution, for example, as a P-sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) Non-polar: Norleucine. Met, Ala. Vai, Leu, He;

(2) Polar without charge: Cys, Ser, Thr, Asn, Gin;

(3) Acidic (negatively charged): Asp, Glu;

(4) Basic (positively charged): Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro; and

(б) Aromatic: Trp, Tyr, Phe, His.

[00151] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[00152] One type of substitution, for example, that may be made is to change one or more cysteines, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine. For example, there can be a substitution of a non-canonical cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant region of an antibody. In some embodiments, the cysteine is canonical. Any cysteine residue not involved in maintaining the proper conformation of an antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability.

4924-6564-8233v.1 [00153] According to the invention, a single scFv derived from variable regions of an antibody may have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant region.

[00154] The term "‘subject” as used in this application means a mammal, preferably a non- porcine mammal. Mammals include canines, felines, rodents, bovine, equines, ovines, and primates including humans. Thus, the invention can be used in human medicine or also in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications. In a preferred embodiment the subject is a human.

[00155] The term “patient” as used in this application means a human subject.

[00156] The terms “treat”, “treatment” of a disease, and the like refer to slowing down, relieving, ameliorating or alleviating at least one of the symptoms of the disease, or reversing the disease after its onset.

[00157] The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or minimize the extent of the disease or disorder or slow its course of development.

[00158] The terms “therapeutically effective amount” or "amount effective to" encompasses an amount sufficient to ameliorate or prevent a symptom or sign of the medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular subject may vary' depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects. An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects.

[00159] Any of the compositions described herein may be for use in any of the methods described herein. As non-limiting examples, the any one of the pharmaceutical compositions described herein may be used for treating, preventing, or reducing the likelihood or extent of a disease, or for inducing immune tolerance or suppressing an immune response in a subject. [00160] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably

4924-6564-8233v.1 up to 10%, more preferably up to 5%, and more preferably still up to 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[00161] “And/or” as used herein, for example with option A and/or option B, encompasses the separate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[00162] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context. [00163] This invention will be better understood from the Experimental Details, which follow; However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXPERIMENTAL DETAILS

Example 1

[00164] In order to generate the CAR construct with anti-SLA class-I antibody (sometimes referred to hereafter as “SLA CAR”), we performed BCR sequencing on two hybridoma lines that generate pan class-I SLA antibodies to identify the antibody sequences needed for designing the CAR constructs. In order to select the best SLA CAR construct, first CAR constructs are synthesized with the sequences from the two hybridoma lines using a ubiquitous promoter (murine stem cell virus (MSCV)). Primary human T cells are then transduced with lentiviral vectors containing these two CARs, the transduced T cells are sorted and a killing assay is run against swine PBMCs. The CAR with a higher killing efficiency is selected. Further, novel genetic engineering approaches to induce apoptotic cell death and/or block the CAR expression in CAR Tregs that convert to Teffs are employed to achieve a conversion resistant and conditioning resistant cell product.

[00165] The SLA CAR sequence comprising the single-chain variable fragments (scFv), followed by the CD28 and CD3-zeta sequences will be placed under a ubiquitous promoter (MSCV)¹⁷ (construct #1. Fig. 25) or a Treg-specific promoter (Foxp3)¹⁸ (construct #2, Fig. 25). Introduction of the CARs under the Foxp3 promoter will ensure their silencing as soon as the Treg converts to a Foxp3 -negative Teff, thereby preventing the Teff from killing the

4924-6564-8233v.1 target cells. Foxp3 might be transiently expressed in some activated T cells¹⁹. Therefore, to provide another layer of protection, an inducible suicide gene is implemented. A chimeric CD8/caspase8 sequence²⁰ is designed under the promoter of the IL-7 receptor (CD127)²¹, which inversely correlates with Foxp3 expression²², to ensure that CAR Tregs undergo apoptosis as soon as they acquire an effector phenotype. The transmembrane domain of CD8 in this chimeric molecule allows for the oligomerization of caspase 8 in the membrane, which is an essential step in initiation of the cascade of events that leads to apoptosis²³. This sequence will be placed on opposite strands of the same vectors as constructs #1 and #2, thereby generating constructs #3 and #4. respectively (Fig. 25). The essential regions of both Foxp3¹⁸ and CD127²¹, which are examples of the minimal promoters that can be used in the CAR construct.

[00166] In vitro model. Human Tregs (CD4+ CD25+ CD127-) will be sorted from donor PBMCs, activated with anti- CD3/28 beads for two days and then transduced with SLA CAR constructs with different designs (e.g., those shown in Fig. 25). Transduced Tregs will be expanded in the presence of human IL-2 and sorted based on the reporters. Sorted CAR Tregs will be stained with violet cell proliferation dye 450 and co-cultured with CFSE-labeled autologous human T cells (as responder cells) and swine PBMC-derived dendritic cells (as stimulators) at different responder/Treg ratios. After one week, the suppressive capacity of CAR Tregs in different groups will be measured by analyzing the % of proliferation of responder cells using flow cytometry. Additionally, the % of Foxp3 -negative reporter-i- cells will be determined, as a measure of CAR Treg conversion. In some experimental conditions, TGF-p. IL-6, IL- 113 and IL-12 (16.24-26) will be added to the culture, in order to force Treg conversion. We propose that CAR Tregs with construct #4 will demonstrate the lowest levels of Treg conversion and the highest suppressive capacity.

[00167] In vivo model. We will use human cord blood T cells from the same tissue that was used as the source of HSCs. In order to prevent graft versus host disease (GVHD) caused by cord blood T cells, we will use NSG- (Kb Db)null (IA)null mice [referred to hereafter as NSG MHC ko mice], which are deficient for both MHC class I (H2-K and D) and MHC class II (IA) molecules and have been shown to be resistant to GVHD²⁷.

[00168] We will inject human cord blood HSCs into thymectomized and irradiated recipient NSG MHC ko mice and freeze the HSC-depleted cord blood cells, which include T cells. Adult swine skin will be transplanted onto the back of recipient mice and swine fetal lung will be transplanted subcutaneously. Later, when the mice are reconstituted with human

4924-6564-8233v.1 cord blood-derived APCs, we will inject them with autologous cord blood T cells. These T cells will expand through lymphopenia-induced proliferation and contribute to rejection of transplanted pig lung/skin tissues. At the same time, SLA CAR Tregs will be generated with the four construct designs and their efficiency in prevention of destruction of swine tissues will be evaluated. The skin graft in xeno model will be evaluated every 2-3 days for signs of rejection ²⁸. The grafted swine fetal lung will be removed at the end of the study and evaluated for rejection with hematoxylin and eosin (H&E) and immunofl uorescent (IF) staining. The best condition will be selected based on graft survival, level of CAR-Treg conversion resistance and gene expression profile. Fig. 26 shows a schematic schedule of events in this model.

[00169] Generation of conditioning-resistant CAR Tregs. While lymphodepleting conditioning regimens may be needed to open some space for adoptively -transferred CAR Tregs, they could potentially be toxic to these cells^14,15. In order to generate conditioningresistant CAR Tregs, we propose to use CRISPR to remove the CD2 molecule in these cells in order to make them resistant to a conditioning regimen consisting of anti-CD2 antibody, which has been successfully used in induction of mixed chimerism in patients receiving combined kidney and bone marrow transplantation²⁹. This would allow CAR Tregs to persist and expand in a lymphopenic environment after anti-CD2 injection. In order to generate conditioning-resistant CAR Tregs, a guide RNA (gRNA) will be designed to specifically silence human CD2 molecules using the electroporation-based system for introduction of Cas9 and gRNA (Cas9-gRNA ribonucleoprotein). This ensures that CAR Tregs are not deleted during conditioning with anti-CD2 antibody, which we will use to deplete the recipient T cells. The best construct design selected from the above experiments will be used to generate SLA CAR Tregs. Similar in vivo studies with the xeno model will be performed to compare the functionality' of conditioning-resistant vs nonmodified CAR Tregs. Humanized mice will be generated with human cord blood HSCs. A pool of autologous cord blood T cells containing one portion of T cells that undergo CRISPR-removal of CD2 and three portions of non-modified T cells will be injected at a later time point when the mice are reconstituted with human APCs. This will ensure that after injection of anti-CD2 antibody, a T cell lymphopenic environment is created that allows for expansion of adoptively-transferred CAR Tregs, while the remaining non-modified T cells will also expand to maintain the pool of Teffs for rejection of swine grafts. Swine skin and fetal lung will be transplanted to the recipient mice, as explained above. Conditioning-resistant vs non-modified CAR Tregs will

4924-6564-8233v.1 be injected to the recipient mice in different groups. One dose of the anti-CD2 antibody³⁰ will also be injected into the mice in the conditioning-resistant CAR Treg group. Graft survival and the level of infiltration of CAR Tregs in target organs will be evaluated as the readouts of this study.

[00170] Generation of SLA CAR Tregs carrying immunomodulatory molecules (enhanced CAR Tregs). Once we determine the best design for the generation of conversion/conditioning-resistant CAR Tregs, we will aim to improve the functionality of these Tregs by overexpressing the immunomodulatory molecules PD-L1 (176 amino acids³¹), TGF-J3 (112 amino acids³²), and CTLA-4Ig (445 amino acids³³) under the control of the NFAT6 minimal promoter^34,35. Transcription of these factors will begin upon CAR-redirected T cell activation in order to target local T cells, B cells, macrophage/monocytes, and DCs within the grafts. SLA CAR constructs with the lowest degree of CAR Treg conversion and the best graft survival (selected from the previous studies) will be used in these experiments. New constructs will be produced with each of the 3 different immunomodulatory genes, each with a separate reporter, under the control of the NFAT6 minimal promoter^34,35. Human Tregs will be transduced with a CAR construct in addition to one of these immunomodulatory molecules. These enhanced SLA CAR Tregs will be tested in the in vivo model of xenotransplantation. We have identified IL-10 (178 amino acids³⁶), IDO1 (403 amino acids³⁷) and blocking antibodies for CD40 and IFN-g as other potential immunomodulatory molecules for use with this system.

[00171] Generation of CAR Tregs carrying molecules that improve engraftment of swine HSCs for induction of mixed human/swine chimerism. Mixed hematopoietic chimerism is the only approach that has successfully induced immune tolerance in a clinical setting ^29,38. However, it is more difficult to achieve for xenografts than for allografts¹. Tregs enhance mixed chimerism³⁹ by improving the engraftment of hematopoietic stem cells (HSCs) through providing immune privilege to the HSCs niche^40,41 and possibly by preventing the immune rejection of donor HSC-derived hematopoietic cells^39,42. Factors like SCF⁴³, FLT3L⁴⁴, thrombopoietin⁴⁵ and CXCL12⁴⁶ are showm to be essential in HSC engraftment, maintenance and self-renew al. We have shown that induction of durable mixed human/swine chimerism in NSG mice is difficult (Fig. 27). However, durable porcine HSC engraftment and chimerism can be achieved in a strain of immunodeficient NOD/SCID-Tg mice expressing porcine cytokine transgenes (IL-3, GM-CSF, and SCF)^47,48. It is undersood that local introduction of factors that improve swine HSC engraftment and self-renewal via

4924-6564-8233v.1 enhanced SLA CAR Tregs would not only protect the swine HSCs from immune attack, but also provide a niche in which swine HSCs survive and self-replicate. To achieve this, constructs will be generated that express the four mentioned swine factors (SCF, FLT3L, thrombopoietin, and CXCL12) under the control of the NFAT6 minimal promoter. Human Tregs will be transduced with the selected SLA CAR construct in addition to each of these four factors. Similar to the above studies, in vivo xeno models will be generated by injection of cord blood HSCs into NSG MHC ko mice, followed by adoptive transfer of polyclonal T cells. When the mice are fully reconstituted with human APCs and T cells, mice will receive a subl ethal irradiation, followed by injection of swine enriched bone marrow cells and enhanced SLA CAR Tregs. The recipient mice will be bled every other week to measure the level of human and swine hematopoietic cells. In different groups, introduction of SLA CAR Tregs expressing one of these factors and a combination of all of them will be investigated.

[00172] Generation of “off-the-shelf’ SLA CAR Tregs. Initial experiments are performed with autologous Tregs to generate SLA CAR Tregs. As it will be easier, more consistent, and less costly to use allogeneic Tregs, the above-mentioned engineering strategies are combined with these strategies to generate “off-the-shelf' CAR Tregs: 1) CRISPR-based removal of endogenous TCR a and chains. 2) CRISPR-based removal of beta-2 microglobulin (B2M) and class II, major histocompatibility complex, transactivator (CIITA), and 3) lenti viral introduction of HLA-G and HLA-E molecules.

[00173] Removal of endogenous TCRs will prevent graft-versus-host disease (GVHD) and/or host-versus-graft disease. Removal of B2M and CIITA will prevent expression of HLA-I and II molecules on CAR Tregs and hence prevents their rejection by the recipient T cells. Lentiviral introduction of HLA-E and G will prevent rejection of CAR Tregs by the recipient NK and T cells.

Example 2

[00174] A schematic showing a new CAR Treg for xenotransplantation and their application is provided in Fig. 1. As proof-of-concept. SLA CARs were developed based on antibody sequences from two SLA-I hybridoma lines. Specifically, a process of development of SLA CAR constructs is shown in Fig. 2A, Fig. 2B, and testing their expression and functionality using transfected TCRB KO Jurkat (JRT3.3) cells non-stimulated and stimulated with irradiated pig PBMCs is shown in Fig. 2C, Fig. 2D.

[00175] SLA CARs were then selected and validated based on their capacity to induce T cell activation upon their interaction with SLA-I molecule (Fig. 3). SLA CAR lentiviruses

4924-6564-8233v.1 were generated and titrated, and human J76 cells (a human T cell tumor line) were transduced with different SLA CARs for testing their capacity to react to pig PBMCs. J76 cells contain three (3) reporter genes under the control of three (3) promoters downstream of TCR signaling: NFAT, NFKB and AP-1. All three (3) signaling pathways were shown to get activated upon interaction of SLA CARs #1 and #2 with pig PBMCs. Additionally, these T cells were shown upregulate CD69, which is a T cell activation marker, upon this interaction. SLA CAR #1 induces higher levels of T cell activation compared to SLA CAR #2 and therefore this SLA CAR was chosen for other in vitro and in vivo experiments.

[00176] Additionally, in vitro testing of SLA CAR Treg was performed, demonstrating a higher capacity of SLA CAR (#1) Tregs, compared to polyclonal (CAR-) Tregs, in suppressing anti-pig immune responses at different Treg : Teff ratios (Fig. 4). SLA CAR Tregs also prevented the destruction of fetal pig lung grafts by human PBMCs in a PBMC- NSG mouse model (Fig. 5).

[00177] A human immune system (HIS) mouse model to evaluate SLA CAR Tregs in prevention of fetal pig lung transplantation rejection was developed (Fig. 6). SLA CAR Tregs were shown to prevent the destruction of fetal pig lung grafts by human immune system (HIS) mouse T cells (Fig. 7).

[00178] Importantly, knockout CD2 was proposed as a solution to protect Tregs from siplizumab-containing conditioning regimens, as CD2 provides co-stimulatory signals and forms immunological synapse (Fig. 8). As shown in Fig. 9, CD2-KO Tregs are resistant to siplizumab-containing conditioning regimens. Also, FOXP3 expression in CD2-KO Tregs is similar to WT Tregs (Fig. 10). CD2-KO Tregs exhibited comparable suppressive capacity to WT Tregs in vitro (Fig. 11), and also exhibited decreased glycolysis but enhanced oxidative phosphorylation (Fig. 12). CD2-KO Tregs also displayed superior mitochondrial function with lower ROS production (Fig. 13) and exhibited decreased TCR signaling, and proinflammatory and senescence gene signatures (data not shown). As shown in Fig. 14, CD2-KO Tregs w ere comparable to WT Tregs in preventing GVHD in mice. CD2-KO Tregs also displayed a less activated phenotype than WT Tregs in vivo (Fig. 15).

[00179] As shown in Fig. 16, CAR-Tregs w ere shown to experience more exhaustion and senescence than untransduced Tregs due to tonic signaling. CAR-transduced CD2-KO Tregs were also more stable and functional compared to WT counterparts (Fig. 17).

[00180] Non-limiting examples of specific SLA CAR constructs are shown in Figs. 18-23.

4924-6564-8233v.1 REFERENCES

1. Sachs, D. H. Transplantation tolerance through mixed chimerism: From allo to xeno. Xenotransplantation (2018). doi: 10. 1111/xen. 12420

2. Bluestone, J. A., Bour-Jordan, H., Cheng, M. & Anderson, M. T cells in the control of organ- specific autoimmunity. Journal of Clinical Investigation (2015). doi: 10.1172/JCI78089

3. Romano, M., Fanelli, G., Albany, C. J., Giganti, G. & Lombardi, G. Past, present, and future of regulatory T cell therapy in transplantation and autoimmunity. Frontiers in Immunology (2019). doi: 10.3389/fimmu.2019.00043

4. Tang, Q. & Bluestone, J. A. Regulatory T-cell therapy in transplantation: Moving to the clinic. Cold Spring Harbor Perspectives in Medicine (2013). doi:10.1101/cshperspect.a015552

5. Sharabi, A. et al. Regulatory T cells in the treatment of disease. Nature Reviews Drug Discovery (2018). doi: 10.1038/nrd.2018.148

6. Haddadi, M. H. et al. Autoimmunity as a target for chimeric immune receptor therapy: A new vision to therapeutic potential. Blood Reviews (2020). doi: 10.1016/j.blre.2019.100645

7. Adair, P. R., Kim, Y. C., Zhang, A. H., Yoon, J. & Scott, D. W. Human tregs made antigen specific by gene modification: The power to treat autoimmunity and antidrug antibodies with precision. Frontiers in Immunology (2017). doi: 10.3389/fimmu.2017.011 17

8. MacDonald, K. G. et al. Alloantigen-specific regulator}' T cells generated with a chimeric antigen receptor. Journal of Clinical Investigation (2016). doi: 10.1172/JCI82771

9. Dawson, N. A. J. et al. Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells. JCI Insight (2019). doi: 10.1172/j ci . insight.123672

10. Elinav, E., Waks, T. & Eshhar, Z. Redirection of Regulatory T Cells With Predetermined Specificity for the Treatment of Experimental Colitis in Mice. Gastroenterology (2008). doi: 10.1053/j .gastro.2008.02.060

11. Skuljec, J. et al. Chimeric antigen receptor-redirected regulatory' T cells suppress experimental allergic airway inflammation, a model of asthma. Frontiers in Immunology' (2017). doi: 10.3389/fimmu.2017.01125

12. Fransson. M. et al. CAR/FoxP3 -engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery⁷. Journal of Neuroinflammation (2012). doi: 10.1 186/1742- 2094-9-112

4924-6564-8233v.1 13. Koenen, H. J. P. M. et al. Human CD25highFoxp3pos regulator) T cells differentiate into IL-17 producing cells. Blood (2008). doi: 10.1182/blood-2008-01-133967

14. Neelapu, S. S. CAR-T efficacy: Is conditioning the key? Blood (2019). doi: 10. 1182/blood-2019-03- 900928

15. Mancusi, A., Piccinelli, S., Velardi, A. & Pierini, A. CD4+FOXP3+ Regulatory T Cell Therapies in HLA Haploidentical Hematopoietic Transplantation. Frontiers in Immunology (2019). doi:10.3389/fimmu.2019.02901

16. Hua, J. et al. Pathological conversion of regulatory T cells is associated with loss of allotolerance. Scientific Reports (2018). doi: 10.1038/s41598-018-25384-x

17. Li, Y. et al. Humanized mice reveal new insights into the thymic selection of human autoreactive CD8 + T cells. Frontiers in Immunology⁷ (2019). doi: 10.3389/fimmu.2019.00063

18. Mantel, P.-Y. et al. Molecular Mechanisms Underlying FOXP3 Induction in Human T Cells. The Journal of Immunology (2006). doi: 10.4049/jimmunol. 176.6.3593

19. Kmieciak, M. et al. Human T cells express CD25 and Foxp3 upon activation and exhibit effector/memory phenoty pes without any regulatory/suppressor function. Journal of Translational Medicine (2009). doi:10.1186/1479-5876-7-89

20. Carlotti, F. et al. Development of an inducible suicide gene system based on human caspase 8. Cancer Gene Therapy (2005). doi: 10.1038/sj.cgt.7700825

21. DeKoter, R. P. et al. Regulation of the interleukin-7 receptor a promoter by the Ets transcription factors PU. l and GA-binding protein in developing B cells. Journal of Biological Chemistry (2007). doi: 10.1074/jbc.M700377200

22. Liu. W. et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. Journal of Experimental Medicine (2006). doi: 10.1084/j em.20060772

23. Martin, D. A., Siegel. R. M., Zheng, L. & Lenardo, M. J. Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACHal) death signal. Journal of Biological Chemistry (1998). doi:10.1074/jbc.273.8.4345

24. Tejon, G. et al. Vitamin A Impairs the Reprogramming of Tregs into IL-17-Producing Cells during Intestinal Inflammation. BioMed Research International (2015). doi: 10.1155/2015/137893

25. Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature (2006). doi: 10.1038/nature04753

4924-6564-8233v.1 26. Feng, T., Cao, A. T., Weaver, C. T., Elson, C. O. & Cong, Y. Interleukin-12 converts Foxp3+ regulator}' T cells to interferony-producing Foxp3+ T cells that inhibit colitis. Gastroenterology (2011). doi: 10.1053/j.gastro.2011.03.009

27. Brehm, M. A. et al. Lack of acute xenogeneic graft-versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression. FASEB Journal (2019). doi: 10.1096/fj .201800636R

28. Kalscheuer, H. et al. Xenograft Tolerance and Immune Function of Human T Cells Developing in Pig Thymus Xenografts. The Journal of Immunology (2014). doi: 10.4049/jimmunol. 1302886

29. Kawai, T. et al. HLA-mismatched renal transplantation without maintenance immunosuppression. New England Journal of Medicine (2008). doi: 10. 1056/NEJMoa071074

30. Kalscheuer, H. et al. A model for personalized in vivo analysis of human immune responsiveness. Science Translational Medicine (2012). doi: 10.1126/scitranslmed.3003481

31. Dong, H., Zhu, G., Tamada, K. & Chen, L. B7-H1, a third member of the B7 family, costimulates T-cell proliferation and interleukin- 10 secretion. Nature Medicine (1999). doi: 10. 1038/70932

32. Poniatowski, L. A., Wojdasiewicz, P., Gasik, R. & Szukiewicz, D. Transforming growth factor beta family: Insight into the role of growth factors in regulation of fracture healing biology and potential clinical applications. Mediators of Inflammation (2015). doi: 10. 1155/2015/137823

33. Yazdanpanah-Samani, M._ Maymand, E. M._ Jahangeerfam, T. & Ghaderi, A. Construction of CTLA-4-ig fusion gene in pBudCE4. 1 expression vector. Avicenna Journal of Medical Biotechnology (2015).

34. Chmielewski, M., Kopecky, C., Hornbach, A. A. & Abken, H. IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression. Cancer Research (2011). doi:10.1158/0008-5472.CAN-l l-0103

35. Uchibori, R. et al. Functional Analy sis of an Inducible Promoter Driven by Activation Signals from a Chimeric Antigen Receptor. Molecular Therapy - Oncolytics (2019). doi: 10.1016/j.omto.2018. 11.003

4924-6564-8233v.1 36. Gesser, B. et al. Identification of functional domains on human interleukin 10. Proceedings of the National Academy of Sciences of the United States of America (1997). doi: 10.1073/pnas.94.26.14620

37. Dai, W. & Gupta, S. L. Molecular cloning, sequencing and expression of human interferon-y-inducible indoleamine 2,3-dioxygenase cDNA. Biochemical and Biophysical Research Communications (1990). doi: 10.1016/0006-291X(90)91666-G

38. Yamada, K., Sykes, M. & Sachs, D. H. Tolerance in xenotransplantation. Current Opinion in Organ Transplantation (2017). doi: 10.1097/MOT.0000000000000466

39. Duran- Struuck. R. et al. Effect of Ex Vivo-Expanded Recipient Regulatory T Cells on Hematopoietic Chimerism and Kidney Allograft Tolerance Across MHC Barriers in Cynomolgus Macaques. Transplantation (2017). doi: 10.1097/TP.0000000000001559

40. Fujisaki, J. et al. In vivo imaging of T reg cells providing immune privilege to the haematopoietic stem-cell niche. Nature (2011). doi: 10. 1038/naturel0160

41. Hirata, Y. et al. CD150 high Bone Marrow Tregs Maintain Hematopoietic Stem Cell Quiescence and Immune Privilege via Adenosine. Cell Stem Cell (2018). doi:10.1016/j.stem.2018.01.017

42. Mancusi, A., Piccinelli, S., Velardi, A. & Pierini. A. CD4+FOXP3+ Regulatory T Cell Therapies in HLA Haploidentical Hematopoietic Transplantation. Frontiers in Immunology (2019). doi : 10.3389/fimmu.2019.02901

43. Bernstein, A., Forrester, L., Reith, A. D., Dubreuil, P. & Rottapel, R. The murine W/c-kit and steel loci and the control of hematopoiesis. Seminars in Hematology (1991).

44. McKenna, H. J. et al. Mice lacking Ht3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood (2000). doi: 10.1182/blood. v95.11.3489.01 lk45_3489_3497

45. Fox, N., Priestley, G., Papayannopoulou, T. & Kaushansky, K. Thrombopoietin expands hematopoietic stem cells after transplantation. Journal of Clinical Investigation (2002). doi: 10.1172/JCI0215430

46. Greenbaum, A. et al. CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance. Nature (2013). doi: 10.1038/naturel 1926

47. Lan, P. et al. Induction of human T-cell tolerance to porcine xenoantigens through mixed hematopoietic chimerism. Blood (2004). doi: 10. 1182/blood-2003-l 0-3697

4924-6564-8233v.1 48. Chen, A. M. et al. Porcine stem cell engraftment and seeding of murine thymus with class 11+ cells in mice expressing porcine cytokines: Toward tolerance induction across discordant xenogeneic barriers. Transplantation (2000). doi: 10. 1097/00007890-200006270-00005

4924-6564-8233v.1

Claims

CLAIMS What is claimed is:

1. A modified Treg cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a class-I swine leukocyte antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

2. The modified Treg cell of Claim 1, which is a human Treg cell transduced with a nucleic acid encoding the CAR, optionally wherein the CAR is under control of a Treg-specific promoter; wherein the nucleic acid encoding the CAR further encodes an inducible suicide gene; wherein the class-I swine leukocyte antigen-binding domain of the CAR comprises a single-chain variable fragment (scFv), an antibody, or an antigen-binding fragment of an antibody; optionally wherein the class-I swine leukocyte antigen-binding domain comprises three VH CDRs chosen from: SEQ ID NOs: l, 2, 3, 7, 8 and 9, and three VL CDRs chosen from SEQ ID NOs: 4, 5, 6, 10, 11, 12, 13, 14 and 15; wherein the transmembrane domain of the CAR comprises a CD8 transmembrane domain and/or CD28 transmembrane domain; wherein the intracellular signaling domain of the CAR comprises a CD28 intracellular domain and/or a y (gamma) subunit of an immunoglobulin Fc receptor (FcRy) signaling domain; wherein the intracellular signaling domain of the CAR comprises a CD3zeta (CD3Q subunit; wherein in N-terminal to C-terminal order, the class-I swine leukocyte antigenbinding domain comprises a VL and then a VH, or in N-terminal to C-terminal order, the class-I swine leukocyte antigen-binding domain comprises a VH and then a VL. optionally wherein the VH and VL are connected via a linker peptide;

4924-6564-8233v.1 wherein the Treg cell has been treated so as to reduce or remove endogenous TCR a and P chains, and/or wherein the CAR further comprises a CD8 hinge region between the class-I swine leukocyte antigen-binding domain and the transmembrane domain; wherein the Treg cell has been treated so as to reduce or remove their endogenous beta-2 microglobulin (B2M), class II major histocompatibility complex, transactivator (CIITA), and/or wherein the Treg cell has had introduced therein HLA-G and/or HLA-E molecules, and/or has been modified so as not to express CD2 or express CD2 at a reduced amount relative to its non-modified form.

3. The modified Treg cell of Claim 1 or 2, wherein the class-I swine leukocyte antigenbinding domain comprises a VH comprising the following:

CDR1

GYTFTNYG (SEQ ID NO:1),

CDR2

INTYTGEP (SEQ ID NO: 2), and

CDR3

ARRGDGYY (SEQ ID NO:3), and/or a VL comprising the following:

CDR1

QNVGTN (SEQ ID NO:4).

CDR2

SAS (SEQ ID NO: 5), and

CDR3

QQYNSYPLT (SEQ ID NO:6); or wherein the class-I swine leukocyte antigen-binding domain comprises

4924-6564-8233v.1 (a) a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO:8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NO: 9), and/or

(b) a VL comprising the following:

CDR1

ENVVTY (SEQ ID NO: 10),

CDR2

GAS (SEQ ID NO: 11), and

CDR3

GQGYSYPYT (SEQ ID NO: 12); or

CDR1

ENIYSN (SEQ ID NO: 13),

CDR2

AAT (SEQ ID NO: 14), and

CDR3

QHFWGTPRT (SEQ ID NO: 15)

4. A pharmaceutical composition comprising a plurality of modified Treg cells of any one of Claims 1-3 and a pharmaceutically acceptable carrier.

5. A method for inducing immune tolerance against one or more pig antigens in a nonpig mammalian subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the modified Treg cell of any of Claims 1-3.

6. A method for suppressing in a human an immune response against one or more pig antigens, or for inhibiting rejection of a pig organ, pig tissue or pig cell infusion in a

4924-6564-8233v.1 non-pig subject, comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the modified Treg cell of any of Claims 1-3.

7. A method of reducing the likelihood of, or extent of, graft versus host disease and/or host versus graft disease in a human subject who is to receive, is receiving, or who has received a transplanted tissue or organ or cell infusion comprising a pig antigen, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified Treg cell of any of Claims 1-3.

8. The method of Claim 7, wherein the effective amount of a pharmaceutical composition comprising the modified Treg cell is administered to the subject prior to or during transplant surgery or cell infusion; or wherein the effective amount of a pharmaceutical composition comprising the modified Treg cell is administered to the subject subsequent to transplant surgery or cell infusion.

9. The method of anyone of claims 5-8, further comprising administering a T celldepleting conditioning regimen to the subject, optionally wherein the T cell-depleting conditioning regimen comprises an anti-CD2 antibody and wherein the modified Treg cells have been treated to remove or deplete cell-surface CD2.

10. The method of anyone of claims 5-9. wherein the modified Treg cells have been modified from Treg cells obtained from a human subject, optionally wherein the Treg cells obtained from a human subject are obtained from the same human being subjected to the method, or from the same human who is to receive, is receiving, or who has received a transplanted tissue, or wherein the Treg cells obtained from a human subject are obtained from a different human than the one being subjected to the method, or from a different human from the one who is to receive, is receiving, or who has received a transplanted tissue; wherein the Treg cells are obtained from cord blood or from a PBMC sample.

4924-6564-8233v.1

11. A method of generating a modified Treg cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a class-I swine leukocyte antigen-binding domain, the method comprising providing Treg cells from a sample obtained from a human, and transducing the Treg cells with a nucleic acid encoding the CAR.

12. The method of Claim 11, wherein the Treg cells are transduced with a lentivirus encoding the CAR, optionally wherein the Treg cells transduced with a nucleic acid encoding the CAR are expanded subsequent to transduction, wherein the Treg cells transduced with a nucleic acid encoding the CAR are expanded by contact with IL-2; and/or wherein the Treg cells are CD4+ CD25+ CD 127- cells sorted from human donor PBMCs or cord blood cells; optionally further comprising activating the Treg cells, optionally comprising activating the Treg cells with anti-CD3/28 beads.

13. The method of Claim 11 or 12, wherein the class-I swine leukocyte antigen-binding domain comprises a VH comprising the following:

CDR1

GYTFTNYG (SEQ ID NOT),

CDR2

INTYTGEP (SEQ ID NO:2), and

CDR3

ARRGDGYY (SEQ ID NO: 3), and/or a VL comprising the following:

CDR1

QNVGTN (SEQ ID NO: 4),

4924-6564-8233v.1 CDR2

SAS (SEQ ID NO: 5), and

CDR3

QQYNSYPLT (SEQ ID NO:6).

14. The method of Claim 11 or 12, wherein the class-I swine leukocyte antigen-binding domain comprises

(a) a VH comprising the following:

CDR1

GFTFSSFG (SEQ ID NO: 7),

CDR2

ISSGSSTL (SEQ ID NO:8), and

CDR3

ARWGNYPHYAMDY (SEQ ID NO: 9), and/or

(b) a VL comprising the following:

CDR1

ENVVTY (SEQ ID NO: 10),

CDR2

GAS (SEQ ID NO: 11), and

CDR3

GQGYSYPYT (SEQ ID NO: 12); or

CDR1

ENIYSN (SEQ ID NO: 13).

CDR2

AAT (SEQ ID NO: 14), and

CDR3

QHFWGTPRT (SEQ ID NO: 15).

15. A modified Treg cell generated by the method of any of Claims 11-14.

4924-6564-8233v.1