Method for inducing immune tolerance using soluble immune checkpoint proteins

Abstract

The present disclosure provides, in part, methods for inducing immune tolerance using truncated membrane-bound and soluble immune checkpoints. Constructs are provided that include an immune checkpoint protein or a portion thereof and are capable of binding to and activating an immune checkpoint protein receptor. A nucleic acid encoding the immune checkpoint protein or a portion thereof is operably linked to a promoter and, optionally, a secretion signal to enable secretion of the immune checkpoint protein or portion thereof. The construct can comprise a viral vector that can be used to deliver or introduce the construct into an implantable object or organ.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 411,298, filed September 29, 2022, the contents of which are incorporated by reference in their entirety. Reference to Electronic Sequence Listing The contents of the electronic sequence listing (155554.00711.xml; size: 18,509 bytes; and creation date: September 29, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0002] The number of patients with heart failure is expected to increase by nearly 46% to approximately 8 million by 2030. Nearly 50% of these patients are predicted to die within five years of diagnosis. For patients with end-stage heart failure, heart transplantation is the gold standard of care. Long-term survival after transplantation is limited by graft dysfunction, rejection, vascular disease, and chronic systemic immunosuppression. There is a significant and unmet need to apply the major advances in gene therapy to the field of transplantation to reduce transplant rejection, decrease the use of immunosuppressants, and expand the donor pool. Therefore, it would be desirable to provide new targets and methods to minimize organ rejection. Summary of the Invention

[0003] The present disclosure provides constructs encoding immune checkpoint proteins, including PDL1, and methods of using the constructs to transduce organs and reduce transplant rejection. One aspect of the present disclosure provides a nucleic acid construct comprising a promoter operably linked to a nucleic acid sequence encoding an immune checkpoint protein or a portion thereof, including an extracellular portion of the immune checkpoint protein, wherein the immune checkpoint protein or a portion thereof, including the extracellular portion of the immune checkpoint protein, is capable of binding to its receptor. In some embodiments, the construct comprises a secretion signal operably linked to the promoter and operably linked to the nucleic acid encoding the immune checkpoint protein or a portion thereof. In some embodiments, the immune checkpoint protein comprises PDL1 or PD1. In some embodiments, the construct comprises a viral vector, e.g., an AAV vector. In some embodiments, the composition comprises a pharmaceutical composition comprising the construct described herein.

[0004] A second aspect of the present disclosure provides a method for expressing an immune checkpoint protein or a portion thereof in a transplantable article. In some embodiments, the method comprises introducing a construct or pharmaceutical composition described herein into the transplantable article. In some embodiments, the method comprises perfusing the construct or pharmaceutical composition into the article ex vivo or in vitro. In some embodiments, the method further comprises transplanting the article into a subject. In some embodiments, the article is selected from the group consisting of an organ, a cell population, skin, and a tissue. Another aspect of the present disclosure provides methods for preventing or reducing rejection of an implanted object in a subject. In some embodiments, the method comprises introducing a construct or pharmaceutical composition described herein into an implantable object prior to implantation of the object into the subject. In some embodiments, the method further comprises implanting the object into the subject. Another aspect of the present disclosure provides a method for introducing a construct into an implantable object, comprising perfusing the construct ex vivo into the object prior to implantation.

[0005] Another aspect of the present disclosure provides methods for preventing or reducing rejection of an implanted object in a subject. In some embodiments, the method comprises introducing a construct or pharmaceutical composition described herein into an implantable object prior to implantation into the subject. In some embodiments, the method further comprises implanting the object into the subject. The present technology can be better understood with reference to the following drawings, which are merely exemplary to illustrate certain features that may be used alone or in combination with other features, and the technology should not be limited to the embodiments shown. [Brief explanation of the drawings]

[0006] [Figure 1] Figure 1 shows PD-L1 mutant-mediated T cell inactivation. PD-L1 mutant constructs include full-length PD-L1, PD-L1 ICD truncated, and soluble PD-L1. [Figure 2-1] Figure 1 shows in vitro expression of PD-L1 full-length, truncated, and secreted isoforms in triple-negative breast cancer cells. [Figure 2-2] Figure 1 shows in vitro expression of PD-L1 full-length, truncated, and secreted isoforms in triple-negative breast cancer cells. [Figure 3] Figure 1 shows elevated PD-L1 (pg / mL) in serum of PD-L1 secreted isoform-bearing tumor-bearing mice. [Figure 4] FIG. 1 shows that expression of PD-L1 isoforms enhances engraftment of immunogenic cell lines in mice. [Figure 5] FIG. 1 shows a construct targeted to the ROSA 26 locus. [Figure 6] Figure 1 shows that in vivo PD-L1 expression is evident in transgenic mice carrying both truncated and secreted mutants, as confirmed by A) histology and B) ELISA. [Figure 7]Figure 1 demonstrates that transgenic mice containing a truncated PD-L1 construct have increased resistance to rejection and prolonged graft survival. [Figure 8] FIG. 1 shows that viral vector delivery of a PDL1 secretion construct into grafts prior to transplantation extended graft survival by more than 80 days. DETAILED DESCRIPTION OF THE INVENTION

[0007] This disclosure is based, in part, on the inventors' discovery of utilizing a gene therapy approach to transduce transplant organs ex vivo or in vitro with immunosuppressive genes. Disclosed herein are constructs encoding immune checkpoint proteins, including PDL1, and methods of using the constructs to transduce organs and reduce transplant rejection. Constructs: In a first aspect, the present invention provides a construct comprising a promoter operably linked to a nucleic acid sequence encoding an immune checkpoint protein or an extracellular portion of an immune checkpoint protein. The immune checkpoint protein or the extracellular portion of the immune checkpoint protein is capable of binding to its receptor and preferably activating the receptor so that immune checkpoint signaling occurs. The construct, or expression vector, is also known as an expression construct and is typically a plasmid or virus designed for gene expression in cells. The construct is used to introduce a specific gene into a target cell, where it can commandeer the cell's protein synthesis machinery to produce the protein encoded by that gene.

[0008] The term "construct" or "polynucleotide construct" refers to a polynucleotide in which an encoded sequence is replicated and / or expressed in a target cell. A construct may include an exogenous promoter operably linked to any one of the polynucleotides described herein. As used herein, a polynucleotide is "operably connected" or "operably linked" when it is placed into a functional relationship with a second polynucleotide sequence. As used herein, the terms "heterologous promoter," "promoter," "promoter region," or "promoter sequence" generally refer to the transcriptional regulatory region of a gene and may be found 5' or 3' to a polynucleotide described herein or within the coding region of said polynucleotide. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. A typical 5' promoter sequence abuts the transcription start site at its 3' end and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0009] In some embodiments, the construct is an expression construct, vector, or viral vector. A vector is any particle used as a vehicle to artificially transport a foreign nucleic acid sequence, typically DNA, into another cell and replicate and / or express it therein. Constructs containing foreign DNA are called recombinant DNA. Four major types of constructs and vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Expression constructs contain a heterologous promoter and a nucleic acid sequence encoding a protein of interest (e.g., an immune checkpoint protein) that can be expressed in the cell into which they are introduced. Expression constructs include constructs that can induce the expression of an exogenous gene to which they are operably linked. Such constructs are referred to herein as "recombinant constructs," "expression constructs," or "recombinant expression vectors" (or simply "expression vectors" or "vectors"), and may be used interchangeably. Suitable constructs are known in the art and contain the elements necessary for a gene encoded within the construct to be expressed as a protein in a host cell. The terms "vector" and "construct" are used interchangeably herein to refer to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments, such as an exogenous DNA segment encoding a mutant α-gal protein, can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Viral vectors are incorporated into viral particles and used to deliver viral polynucleotides encoding proteins of interest to target cells. Some constructs are capable of autonomous replication within the host cell into which they are introduced. Other constructs can integrate into the genome of the host cell upon introduction, thereby replicating along with the host genome (e.g., lentiviral vectors). Furthermore, some vectors can induce the expression of exogenous genes to which they are operatively linked. In general, constructs useful in recombinant DNA technology are often in the form of plasmids.As used herein, the term "vector" includes expression vectors, such as viral vectors (e.g., replication defective retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (AAV)), which serve equivalent functions.

[0010] The construct is a heterologous exogenous construct containing sequences from two or more different sources. Suitable constructs or vectors include, but are not limited to, plasmids, retroviruses, adenoviruses, oncoretroviruses, lentiviruses, spumaviruses, adeno-associated viruses, herpes simplex viruses, and the like, and include constructs capable of expressing a protein of interest. A preferred vector is an adeno-associated vector (AAV). As described herein, suitable methods for producing viral particles that can transduce cells to express a protein of interest are known in the art.

[0011] Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, tissue-specific, and cell-type-specific promoters. Heterologous promoters may be animal, bacterial, fungal, viral, or synthetic promoters. Suitable promoters are known and described in the art. In mammalian cells, typical promoters include, but are not limited to, promoters of Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), SV40 virus, chicken beta-actin, 3-phosphoglycerate kinase, as well as the translation elongation factor EF-lα promoter or the ubiquitin promoter.

[0012] In some embodiments, the constructs described herein may contain a secretion signal. Secretion signals, sometimes called signal peptides, signal sequences, targeting signals, localization signals, localization sequences, transit peptides, leader sequences, or leader peptides, are or encode short peptides (usually 16-30 amino acids long) present at the N-terminus, C-terminus, or internal location of most newly synthesized proteins destined for the secretory pathway. Secretion signals often prompt cells to translocate proteins to the plasma membrane and / or extracellular space. Signal peptides adhere to a common three-domain structure, including a basic N-domain, a hydrophobic H-domain, and a slightly polar C-domain. As a representative example, the mouse follicle-stimulating hormone B signal sequence (5'-atgatgaagttgatccagctttgcatcttattctggtgctggagagcaatctgctgc-3', SEQ ID NO: 13) was used in mouse constructs and experiments.

[0013] The terms "nucleic acid" and "nucleic acid molecule," as used herein, refer to a compound comprising a nucleic acid base and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Nucleic acid generally refers to a polymer comprising nucleotides or nucleotide analogs linked via backbone bonds, such as phosphodiester bonds, but is not limited to such. Nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acids (RNA), such as messenger RNA (mRNA) and transfer RNA (tRNA). Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a chain of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA. A nucleic acid can occur naturally, e.g., in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. Alternatively, a nucleic acid molecule can be a molecule of non-natural origin, e.g., recombinant DNA or RNA, artificial chromosome, engineered genome, or fragment thereof, or synthetic DNA, RNA, DNA / RNA hybrid, or can contain non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having a backbone other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, optionally purified, chemically synthesized, etc.Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs with chemically modified bases or sugars, and backbone modifications. Nucleic acid sequences are shown in the 5' to 3' direction unless otherwise indicated. In some embodiments, nucleic acids include natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methyl ... The bases may be or contain: cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0014] In some embodiments, the constructs described herein encode an immune checkpoint protein or the extracellular portion of an immune checkpoint protein. Immune checkpoints are critical inhibitory regulators of the immune system that maintain self-tolerance, prevent autoimmunity, and control the duration and intensity of immune responses to minimize collateral tissue damage. Binding of checkpoint proteins, which are highly expressed on T cells, to their ligand partner proteins sends an "off" signal to the T cell, reducing the immune response. Immune checkpoint proteins are often overexpressed on tumor cells or on non-transformed cells within the tumor microenvironment, impairing the immune system's ability to mount an effective anti-tumor response and allowing tumor cell proliferation. Therefore, immune checkpoint inhibitors are a standard therapy for the treatment of several cancers. As shown in the Examples, overexpression or inducible expression of immune checkpoint proteins in non-HLA-matched tumors resulted in tumor cell growth in mouse models where the animal's immune response would otherwise suppress non-HLA-typed tumor growth. See Figures 3 and 4. Immune checkpoint proteins lacking transmembrane domains or prepared as secreted forms were significantly better at enabling tumor formation in mouse models, suggesting that these forms may be more suitable for implantation into transplantable objects or organs to reduce the risk of immune-based rejection of the transplanted object or organ. Immune checkpoint proteins and their ligands include, but are not limited to, A2AR, A2BR, B7-H2, B7-H3, B7-H4, 2B4 (CD244), B7.1, B7.2, BTLA, CTLA4, ICOS, IDO, ITL-4, HVEM, KIR, LAG3, gp49B, NOX2, PD1, PDL1, PDL2, PIR-B, TIM-1, TIM-3, TIM-4, TIGIT, VISTA, SIGLEC7, CD47, CD48, CD39, CD73, CD160, CD200, HVEC, CEACAM1, CD155, LAG-3, and HLA-E. In some embodiments, the immune checkpoint protein is CTLA4.

[0015] In some embodiments, the immune checkpoint proteins or extracellular portions of immune checkpoint proteins described herein can bind to their receptors. Ligand-receptor interaction, or binding, results in a molecular response. Binding of an immune checkpoint protein to its receptor can result in changes in immune function, cytokine production, proliferation, cell migration, or signal transduction. Some immune checkpoint proteins can interact with more than one receptor or more than one ligand. For example, CTLA4 is a receptor for CD86 as well as CD80. Truncated forms (portions of) the immune checkpoint proteins provided herein can be the same as the full-length proteins or remain capable of inducing much the same changes in immune function.

[0016] In some embodiments, the construct comprises the immune checkpoint protein programmed death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) or programmed cell death protein 1, and also known as PD-1 and CD279. Engagement of PD-L1 on T cells with its receptor PD-1 results in a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation, thereby downregulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-L1 is a type I transmembrane glycoprotein encoded by the CD274 gene on chromosome 9 in humans. Transcription of this gene can produce multiple PD-L1 splice variants, including PD-L1 lncRNA splice isoforms, truncated PD-L1, and soluble PD-L1. In some embodiments, the constructs described herein may comprise full-length PD-L1 comprising SEQ ID NO:1 (mouse, DNA) or SEQ ID NO:2 (mouse, amino acid), SEQ ID NO:7 (human, DNA) or SEQ ID NO:8 (human, amino acid); PD-L1 truncated after the transmembrane domain comprising SEQ ID NO:3 (mouse, DNA) or SEQ ID NO:4 (mouse, amino acid), SEQ ID NO:9 (human, DNA) or SEQ ID NO:10 (human, amino acid); and / or secreted PD-L1 comprising SEQ ID NO:5 (mouse, DNA) or SEQ ID NO:5 (mouse, amino acid), SEQ ID NO:11 (human, DNA) or SEQ ID NO:12 (human, amino acid).

[0017] In some embodiments of the present disclosure, pharmaceutical compositions are provided that include a construct described herein and a pharmaceutically acceptable carrier, diluent, and / or excipient. As used herein, the term "carrier" refers to a pharmaceutically acceptable solid or liquid filler, diluent, or encapsulating material. Aqueous liquid carriers may contain pharmaceutically acceptable additives such as acidifying agents, alkalinizing agents, antimicrobial preservatives, antioxidants, buffers, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending agents, and / or viscosity-increasing agents, tonicity agents, wetting agents, or other biocompatible materials. A table of ingredients listed by the above categories can be found in the US Pharmacopeia National Formulary, 1857-1859, (1990). Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, polyethylene glycol; esters such as ethyl oleate, ethyl laurate; agar; buffers such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution, ethyl alcohol, and phosphate buffers, as well as other non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, emulsifying agents, lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring and perfuming agents, preservatives, antioxidants, and the like, can also be present in the compositions according to the desire of the formulator.

[0018] Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid. In another embodiment, the formulations may also include other suitable agents, such as stabilizing delivery vehicles, carriers, supports, or complexing species. The coordinated administration methods and combinatorial formulations of the present invention can optionally incorporate effective carriers, processing agents, or delivery vehicles to provide improved formulations for delivery of the constructs or vectors described herein.

[0019] The pharmaceutical formulation may further contain a biologically acceptable buffer to maintain a pH close to neutral (7.0 to 7.3). Such buffers are typically phosphates, carboxylates, and bicarbonates. More preferred buffers are sodium phosphate, potassium phosphate, sodium citrate, calcium lactate, sodium succinate, sodium glutamate, sodium bicarbonate, and potassium bicarbonate. The buffer may comprise approximately 0.0001 to 5% (w / v), more preferably approximately 0.001 to 1% (w / v), of the vaccine formulation. If desired, other excipients may be included as part of the final pharmaceutical formulation. Such compositions may include a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol, etc.; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative.

[0020] method: In a second aspect, the present invention provides methods of introducing one or more of the constructs described herein into an implantable object. In some embodiments, the method comprises introducing any of the constructs described herein or pharmaceutical compositions described herein into the implantable object, and optionally, the introducing comprises ex vivo or in vitro perfusion of the construct or pharmaceutical composition into the object. The constructs described herein can be introduced into an object for the purpose of expressing the immune checkpoint protein of the construct. The construct can be introduced into a cell by any means known in the art. These means can include transfection or transduction. Transfection is the process of introducing nucleic acid into a cell non-virally. Transduction is the process of introducing foreign DNA into another cell via a viral vector. These are common tools for introducing foreign genes into host cells. Other means include, inter alia, transformation, conjugation, etc. Additionally, methods using targeting endonucleases to knock in the construct, such as CRISPR / Cas gene editing, can be used.

[0021] In some embodiments, the constructs are introduced into the object by ex vivo perfusion. Ex vivo perfusion, also known as normothermic perfusion, involves a machine that keeps an organ at body temperature by continuously pumping or perfusing blood or a bloodless solution of nutrients, protein, and oxygen through the organ. Ex vivo perfusion reduces the time of ischemic injury and allows for evaluation of the graft. In some embodiments, the constructs described herein may be included in the solution that is perfused through the object. In some embodiments, the method further comprises analyzing the object or cells of the object for the presence or expression in the object of the construct or an immune checkpoint protein encoded by the construct. Evaluation of the construct or immune checkpoint protein can be performed by any means known in the art. The results of the evaluation may be used to inform the dosage, function, or efficacy of the construct, or may be useful in determining the level or requirement for additional immunosuppressive therapy if the object is to be used as a transplant organ. In some embodiments, the object may comprise an organ, a cell population, or a tissue. The transplant object may include an organ. The organ may include, but is not limited to, the heart, heart valve, lung, kidney, liver, pancreas, skin, spleen, middle ear, connective tissue, intestine, colon, eye, stomach, ovary, testicle, bladder, uterus, and adrenal gland. The cell population may include stem cells, bone marrow, and immune cells. The tissue may include bone, tendon, ligament, skin, heart valve, blood vessel, pancreatic islet, nerve, vein, and limb.

[0022] In some embodiments, the method further includes transplanting the object into the subject. Organ transplantation is a medical procedure in which an organ is removed from one body and placed in a recipient's body to replace a damaged or missing organ. The donor and recipient may be in the same location, or the organ may be transported from the donor's location to another. Allogeneic transplantation is the transplantation of an organ or tissue between two genetically non-identical members of the same species. Genetic differences between the organ and the recipient can cause the recipient's immune system to recognize the organ as foreign and attempt to destroy it, resulting in transplant rejection. Furthermore, in stem cell transplants, bone marrow transplants, and other hematopoietic transplants, immune cells in the graft attack host cells, a condition known as graft-versus-host disease (GvHD). "Graft" refers to the transplanted or donated tissue, and "host" refers to the recipient's tissue. Transplant recipients often undergo preventative treatments to suppress their immune system after the transplant. These treatments continue after the transplant. Immunosuppressant treatments include, but are not limited to, ruxolitinib, belmosudil, ibrutinib, corticosteroids, and photopheresis. Human leukocyte antigen (HLA) typing, or HLA matching, is used to match recipients and donors for transplants. HLA is a protein found on most cells in the body and is used by the immune system to recognize foreign cells. For a successful transplant and to reduce the likelihood of developing GvHD or transplant rejection, the HLA genes of the donor and recipient must be identical or as closely matched as possible. Two major classes of HLA antigens are recognized: HLA class I and HLA class II. HLA class I antigens (A, B, and C in humans) cause cells to be recognized as "self," while HLA class II antigens (DR, DP, and DQ in humans) stimulate the immune system. The methods provided herein can increase the level of HLA type mismatch between donor and recipient, allowing for more widespread use of donated organs and reducing the need for and level of immunosuppressant treatment required to avoid transplant rejection due to GvHD.

[0023] Another aspect of the present invention provides ex vivo methods for preventing or reducing rejection of a transplanted object in a subject. In some embodiments, the methods comprise introducing a construct into the implantable object prior to transplantation into the subject. The construct encodes a promoter operably linked to a nucleic acid sequence encoding an immune checkpoint protein or an extracellular portion of an immune checkpoint protein. The immune checkpoint protein or the extracellular portion of the immune checkpoint protein can bind to its receptor and activate the receptor in a manner similar to that of a native immune checkpoint protein.

[0024] As used herein, a "subject in need thereof" may refer to a subject in need of treatment for a disease or disorder associated with organ transplantation. A subject in need thereof may include a subject having any condition that would benefit from organ, cell, or tissue transplantation. A subject may be experiencing failure or damage to the heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testicle, bladder, uterus, adrenal gland, skin, or any other organ or cell type in the body. The term "subject" may be used interchangeably with the terms "individual" and "patient," and includes human and non-human mammalian subjects.

[0025] In some embodiments, the compositions and methods provided herein may be used in xenotransplantation. Xenotransplantation, or heterologous transplantation, is the transplantation, implantation, or injection of living cells, tissues, or organs from one species to another, e.g., from a non-human animal source to a human recipient. Such cells, tissues, or organs are called xenografts or xenotransplants. Non-human organs, cells, or tissues may be genetically modified by ex vivo viral or non-viral transduction with vectors that enable expression of the PD-l1 gene and variants described herein. Such an approach may reduce the immune system's response to organ rejection, making xenotransplantation more successful.

[0026] Additional definitions The present disclosure is not limited to the specific details of the structures, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein can be manufactured, practiced, used, performed, and / or formed in a variety of ways that will be apparent to those skilled in the art in light of the following disclosure. The phrases and terminology used herein are for descriptive purposes only and should not be construed as limiting the scope of the claims. Ordinal numbers such as first, second, and third used to refer to various structures or method steps in the specification and claims are not intended to be construed as indicating any particular structure or step, or any particular order or configuration for such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to facilitate the disclosure and do not imply a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, or any structure shown in the drawings, should be construed as indicating any unclaimed element as essential to the practice of the disclosed subject matter. As used herein, the use of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as "including," "comprising," or "having" particular elements are also contemplated as "consisting essentially of" and "consisting of" those specific elements.

[0027] Unless otherwise specified or indicated by context, the terms "a," "an," and "the" mean "one or more." For example, "a molecule" should be interpreted as meaning "one or more molecules." As used herein, "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If there are uses of a term that are not clear to a person of ordinary skill in the art from the context in which the term is used, "about" and "approximately" will mean plus or minus 10% or less of the particular term, and "substantially" and "significantly" will mean plus or minus 10% or more of the particular term. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of individually referring to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein. For example, if a concentration range is recited as 1% to 50%, it is intended that values ​​such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly recited herein. These are merely examples of what is specifically intended, and all possible combinations of values ​​between and including the recited minimum and maximum values ​​are considered to be expressly recited in this disclosure. The use of the word "about" to describe a particular recited amount or range of amounts is intended to indicate that the amount includes values ​​that are very close to the recited amount, such as values ​​that can or would reasonably be accounted for due to manufacturing tolerances, instrumental and human error in making measurements, etc. All percentages referring to amounts are by weight unless otherwise specified.

[0028] No admission is made that any document, including non-patent or patent documents, cited herein constitutes prior art. Specifically, unless otherwise stated, it is understood that the citation of any document herein is not an admission that any of such document forms part of the general knowledge in the art in the United States or elsewhere. Any discussion of a reference states what its author asserts, and applicants reserve the right to challenge the accuracy and pertinence of the documents cited herein. All documents cited herein are incorporated by reference in their entirety unless expressly stated otherwise. In the event of a conflict with any definitions and / or explanations found in a cited document, the present disclosure shall control. The following examples are illustrative only and are not intended to limit the scope of the invention or the appended claims. [Example]

[0029] Example 1: There is a significant unmet need to apply major advances in gene therapy to the field of transplantation to reduce transplant rejection, decrease the use of immunosuppressants, and expand the donor pool. Successful development of a gene therapy strategy requires four components: 1) a delivery approach, 2) a vector (usually viral-based), 3) a well-defined disease or injury state and disease model, and 4) a molecular target for which appropriate therapy can be applied to the disease or indication. Previously, the lack of an efficient delivery approach for targeted delivery of transgenes to the donor heart has been a barrier to gene therapy for heart transplantation. Recently, an ex vivo warm blood perfusion system (TransMedics, Organ Care System (OCS)) received FDA approval, allowing for extended donor heart transport time and reduced ischemic injury. This system can also be used opportunistically for viral vector delivery to genetically modify transplant organs before implantation. We successfully demonstrated the ability to homogenously deliver a reporter gene to the entire myocardium of allografts using a viral vector in a porcine transplantation model.2 Viral vector delivery via ex vivo warm blood perfusion into the heart is well established in our laboratory.

[0030] In the following examples, we describe therapeutic targets used in gene therapy to minimize organ rejection. The role of T cells in transplant rejection is well established, as are several costimulatory pathways mediated by T cell activation. Organ rejection is primarily driven by T cells that recognize foreign alloantigens on the donor organ. This recognition triggers subsequent effector responses, ultimately leading to organ damage and rejection. However, several inhibitory pathways exist that limit T cell activity. Among these, the PD-1 / PD-L1 axis is a central pathway for CD8+ T cell suppression. Therapies targeting the PD-1 / PD-L1 pathway with a series of blocking antibodies have revolutionized clinical cancer treatment. Interestingly, the use of PD-1 / PD-L1 blocking antibodies has been reported to be associated with rejection of both cardiac and renal allografts in cancer patients who have undergone heart transplantation. 3,4 In a recent study of biopsies from acutely and chronically rejected human heart transplants, we observed dysregulation of PD-1 and PD-L1 protein expression, supporting the relevance of this pathway in cardiac rejection. 5 Bracamonte-Baran et al. showed that PD-L1 expression on allograft endothelium was inversely correlated with CD8+ T cell infiltration in 23 intramyocardial biopsies. 6 Furthermore, expression of the PD-L1 transgene in a transgenic pig model reduced its ability to stimulate CD4+ T cell proliferation. 7 Preclinical studies by others using donor PD-L1 knockout and recipient PD-1 knockout mice have determined that the absence of PD-L1 significantly accelerates cardiac allograft rejection. 8~10 Conversely, overexpression of PD-1 on mouse T cells before heart transplantation has been shown to improve graft survival and reduce rejection. 11This survival benefit was dependent on the expression of PD-L1 in the donor allograft, and transplants from PD-L1 knockout mice and PD-1-overexpressing T cells in recipient mice did not demonstrate improved survival. However, in clinical settings, organ-specific PD-L1 overexpression may be more beneficial for patients than systemic T cells, minimizing potential systemic side effects. A transgenic islet transplant mouse model demonstrated that immune allograft destruction could be prevented even without immunosuppression for over 50 weeks. 12 Whether cardiac PD-L1 overexpression reverses cardiac allograft rejection remains to be determined.

[0031] result: Molecular targets in transplant rejection. A recent study of cardiac specimens from 18 rejecting and non-rejecting human heart transplants performed at our institution demonstrated dysregulated expression of the PD-1 / PD-L1 pathway, supporting its relevance in cardiac rejection. 5 Acute rejection was associated with a near loss of PD-L1 expression on myocytes. PD-L1 expression was also significantly lower in lymphocyte populations compared with PD-1 expression. A trend toward decreased PD-L1 expression relative to PD-1 expression on cardiomyocytes was observed. However, these studies were performed on rejected allografts that had already been exposed to immunosuppression. A cell-based model to evaluate the resistance ability of PD-L1 mutants to cell rejection. The PD-L1 protein has three major components: an intracellular domain, an extracellular domain, and a transmembrane domain. The intracellular domain (upon binding to PD-1) enables reverse signaling that can alter various cellular behaviors. However, these are not well described. 13 They may also involve a negative feedback loop that could limit the effectiveness of gene therapy aimed at overexpressing PD-L1. Furthermore, a secreted isoform of PD-L1 has also been described in human studies; this soluble form of PD-L1 has been reported to increase during pregnancy. 14, which is associated with poor prognosis in various cancers 15 Plasmid constructs containing three PD-L1 variants—full-length, intracellular domain-truncated, and secreted isoforms—were generated (Figure 1). Lentiviral vectors encoding these constructs under the control of a CMV promoter were generated and used to transduce various tumor models, confirming robust expression in vitro (Figure 2) and in vivo (Figure 3). To evaluate the ability of these genes to resist immune-mediated T cell attack, they were transfected into breast (E0771) or colon (MC38) cancer cells, which also express the immunogenic form of ovalbumin (membrane-bound), which can potentially trigger rejection. These cells were then implanted, and the ability to form tumors in mice was used to determine which genes could resist immune attack against syngeneic C57Bl6 / J cells (Figure 4). We found that control cells infected with an empty vector resulted in rejection in 80% of cases, a rate remarkably comparable to that observed in cells containing the full-length version of PDL1 in both colon and mammary cells. Notably, tumor cells containing truncated and secreted forms of PDL1 resisted rejection in the majority of mice, likely mediated by the absence of specific feedback signaling loops, suggesting that the lack of reverse signaling is essential for establishing local immunosuppression.

[0032] References: TIFF2025532986000002.tif188168 TIFF2025532986000003.tif109166

[0033] Example 2: Targeted allograft-specific PD-L1 overexpression prolongs time to rejection after heterotopic heart transplantation in a transgenic mouse model After demonstrating the utility of PD-L1 mutants in suppressing cellular immune responses more effectively than full-length PD-L1, we decided to test their potential in mouse animal models by generating transgenic mice as well as vector-based transduction studies. To generate mice, PD-L1 (full-length), PD-L1-TM (ICD truncated), and PD-L1-SS (soluble version) were cloned into a ROSA targeting vector with a CAG promoter that is activated after CRE recombination (Figure 5). After constructing and sequence-verifying these plasmids, they were electroporated into embryonic stem cells and selected with G418. After selection, positive clones were screened by multiplex PCR to verify intact targeted clones. These ES cells were used to generate chimeric mice, which were then bred to establish germline transmission of these transgenes. After transgenesis, the mice were crossed with Myh6CRE mice. After multiple offspring were generated, we crossed PDL1-FL mice with non-transgenic C57BL6 / J mice. In contrast to the Mendelian ratio observed, we determined that PD-L1 × Myh6-cre mice caused embryonic lethality without producing viable double-positive mice. In contrast, crossing with Myh6-CRE resulted in PD-L1-TM and PD-L1-SS mice, suggesting a potentially non-lethal, more beneficial cardiac phenotype. To determine whether this was related to CRE expression in the developing heart (or other tissues), we further crossed Myh6-CRE / ER mice, which allow for temporal regulation of CRE expression after tamoxifen administration. In this setting, we were able to generate transgenic mice with Mendelian ratios, demonstrating that lack of expression results in embryonic lethality for PD-L1-FL. To examine the temporal regulation of PD-L1 expression, tamoxifen (75 mg / kg) was administered for 5 days and cardiac tissue was evaluated 1 week after the final tamoxifen injection (12 days after the first injection). These studies demonstrated that PD-L1-TM exhibited robust PDL1 expression on cardiomyocytes by IHC compared with PDL1-FL and PDL1-SS (Figure 6).Using PD-L1 ELISA, we detected significant levels of PD-L1 in the serum of mice with PD-L1-SS, which showed some increase from PD-L1-TM, but no detectable expression in PD-L1-FL or control non-transgenic mice. Therefore, we conclude that a secondary mechanism of PD-L1 suppression via signaling from the intracellular domain (ICD) may be responsible for suppressing PD-L1 expression, as has been shown to affect several different signaling pathways in cancer models (Jalali et al., Blood Cancer J 2019; Tamburini, Cell Reports 2020). Thus, the demonstration of a lack of tissue-specific PD-L1 expression was unexpected but essential for pursuing this approach, indicating a potential barrier to the success of this strategy via ICD-regulated protein expression.

[0034] To determine whether transgenic hearts could suppress immune rejection, we next initiated studies performing major mismatch transplants between transgenic and non-transgenic hearts. Experimental design: Donor Myh6-Cre / PD-L1 transgenic mice were on a C57BL6 background, and recipient mice were wild-type BALB / c mice. There were three experimental groups: one for each transgenic PD-L1 mutant donor and one for an HLA-mismatched wild-type positive control (C57BL6 donor hearts transplanted into BALB / c recipients). Heterotopic heart transplantation was performed as follows: donor mice were anesthetized and prepared. After sternotomy, the hearts were aseptically removed. The hearts were arrested by infusing cold buffer into the aortic root. The hearts were transplanted into HLA-mismatched recipients. The donor ascending aorta was connected end-to-end to the recipient abdominal aorta, and the donor pulmonary artery was connected end-to-end to the recipient inferior vena cava. The grafts were then depilated. The abdomen was closed, and the recipient mice were harvested.

[0035] Assessment of Rejection: Assessment of cardiac rejection is primarily performed in three ways: 1) Routine physical examination. Graft function can be easily assessed by simple palpation of the animal's abdomen and is graded on the standard 4-point International Society for Heart and Lung Transplantation scale. 16 2) Weekly echocardiography. 3) A subset of animals (n=5 per time point) will be euthanized weekly for blood and tissue collection. Peripheral blood mononuclear cells (PBMCs) will be isolated and stored for immune profiling. Flow cytometric immune profiling will be performed to characterize the cellular composition of the immune response during rejection in PD-L1 overexpressed compared to control animals. Hearts will be harvested for sectioning and H&E staining. Mice will be continuously examined for intimal hyperplasia or other signs of cardiac allograft occlusive vasculopathy / rejection.

[0036] In the first experiment, we preferentially used PD-L1-TM transgenic hearts due to their high surface expression of PD-L1. In these experiments, C57BL6 hearts were transplanted into BALB / c recipients and treated with a single dose of abatacept (250 μg / mouse on the day of transplantation). In this setting, control hearts were rejected 50% by 21 days post-transplant, while PD-L1-TM transgenic hearts were not rejected (Figure 7). To validate the vector-mediated delivery potential of this approach, we generated a PD-L1-SS-expressing AAV vector and used it to transduce mouse hearts in vivo (2 × 10E12 vg of AAV per mouse injected intravenously 14 days prior to transplantation). These mouse hearts were then excised and used in major mismatch experiments, along with a single dose of belatacept (a less potent immunosuppressant in mice) (250 μg / mouse on the day of transplantation) to induce immunosuppression. These experiments demonstrated that PD-L1-SS transduced hearts survived significantly longer than control hearts, all of which were rejected by day 14 after transplantation (Figure 8). Some of these hearts survived for over 80 days before being sacrificed for cardiac tissue evaluation. These experiments demonstrate the potential of PD-L1-TM and PD-L1-SS to prevent solid organ rejection from genetically engineered transgenic organs, and also demonstrate this potential using viral vectors capable of expressing these genes after ex vivo tissue transduction. Although these experiments were performed on mouse cardiac tissue, the inventors believe this approach could be used with different human organs and may also enable xenotransplantation using organs from different species.

[0037] References: TIFF2025532986000004.tif173159 TIFF2025532986000005.tif121161

[0038] Example 3: The present disclosure provides constructs and methods that can be used to transduce immunosuppressive genes into organs ex vivo. While this method of gene therapy is described, the implanted object can be preserved by any method known in the art, including, but not limited to, preservation methods that maintain normothermic and aerobic metabolism of the object. The preserved object can be exposed ex vivo to one or more of the constructs described herein. These constructs may include a promoter operably linked to an immune checkpoint protein, such as PDL1, CTLA4, or Galaectin-3, or the extracellular portion of an immune checkpoint protein. These constructs may further include a secretion signal operably linked to the promoter and operably linked to a nucleic acid encoding the immune checkpoint protein or the extracellular portion of the immune checkpoint protein, such that the immune checkpoint protein is secreted. For example, the heart may undergo normothermic ex vivo perfusion, which may include an AAV vector. The AAV vector can encode a cellular promoter, such as a cardiac promoter, operably linked to a secretion signal, and a nucleic acid encoding secreted PDL1. After perfusion of the organ or object, expression of the vector or immunosuppressive gene may be assessed. The vector-perfused organ can then be transplanted into a recipient. Organs perfused ex vivo with constructs encoding immunosuppressive genes are expected to improve transplant outcomes, including reduced rejection of the transplanted organ, reduced immune responses to the transplant, and reduced ischemic injury. Ex vivo perfusion with constructs encoding immunosuppressive genes may also reduce the amount, duration, or frequency of post-transplant immunosuppressive therapy. The methods and constructs described herein may also enable transplantation of organs with a higher degree of HLA mismatch. Without wishing to be bound by theory, organs perfused ex vivo with constructs encoding immunosuppressive genes, such as PDL1 or a variant thereof, may exhibit a lower incidence of rejection and a higher incidence of HLA mismatch than organs not perfused with the construct.By suppressing the immune mechanisms responsible for organ rejection in non-human organs, this approach may also be useful for genetically modifying animal organs and improving the success of xenotransplantation procedures by using ex vivo viral (or non-viral) transduction with vectors that enable the expression of these PD-L1 genes.

Claims

1. A nucleic acid construct comprising a promoter operably linked to a nucleic acid sequence encoding an immune checkpoint protein or an extracellular portion of an immune checkpoint protein, wherein the immune checkpoint protein or the extracellular portion of the immune checkpoint protein is capable of binding to its receptor.

2. 2. The construct of claim 1, further comprising a secretion signal operably connected to a promoter and functionally linked to a nucleic acid encoding an immune checkpoint protein or an extracellular portion of an immune checkpoint protein.

3. The construct of claim 2 , wherein the secretory signal encodes a signal peptide that enables the extracellular portion of the immune checkpoint protein to be secreted.

4. 4. The construct of any one of claims 1 to 3, wherein the immune checkpoint protein is selected from the group consisting of A2AR, A2BR, B7-H2, B7-H3, B7-H4, 2B4 (CD244), B7.1, B7.2, BTLA, CTLA4, ICOS, IDO, ITL-4, HVEM, KIR, LAG3, gp49B, NOX2, PD1, PDL1, PDL2, PIR-B, TIM-1, TIM-3, TIM-4, TIGIT, VISTA, SIGLEC7, CD47, CD48, CD39, CD73, CD160, CD200, HVEC, CEACAM1, CD155, LAG-3, HLA-E, and combinations thereof.

5. The construct of claim 1 or 2, wherein the immune checkpoint protein comprises PDL1.

6. The construct of claim 1 or 2, wherein the immune checkpoint protein comprises PD1.

7. 2. The construct of claim 1, wherein the immune checkpoint protein comprises PDL1 or an extracellular portion thereof and has a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, a sequence having 95% identity to SEQ ID NO:8, a sequence having 95% identity to SEQ ID NO:10, a sequence having 95% identity to SEQ ID NO:12, and any fragment or variant thereof capable of binding to and activating PD1.

8. The construct of any one of claims 1 to 7, wherein the immune checkpoint protein comprises CTLA4.

9. 9. The construct of any one of claims 1 to 8, wherein the promoter sequence encodes a constitutively active promoter, an inducible promoter, a tissue-specific promoter, a cell-type specific promoter, or a temporally restricted promoter.

10. The construct of any one of claims 1 to 9, comprising a viral vector.

11. The construct of claim 10, wherein the viral vector is selected from the group consisting of retrovirus, adenovirus, oncoretrovirus, lentivirus, spumavirus, adeno-associated virus, herpes simplex virus, and combinations thereof.

12. The construct of claim 11 , wherein the viral vector comprises an adeno-associated (AAV) vector.

13. A pharmaceutical composition comprising the construct of any one of claims 1 to 12 and a pharmaceutically acceptable carrier, diluent and / or excipient.

14. 14. An ex vivo or in vitro method for expressing an immune checkpoint protein or part thereof in an implantable object, comprising introducing a construct according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 into the implantable object, wherein introducing may comprise ex vivo or in vitro perfusion of the construct or pharmaceutical composition into the object.

15. 15. The method of claim 14, further comprising analyzing the object or cells of the object for the presence or expression in the object of the construct or an immune checkpoint protein encoded by the construct.

16. 16. The method of claim 15, further comprising implanting the object into the subject.

17. 17. The method of claim 16, further comprising administering to the subject a therapeutically effective amount of one or more additional therapeutic agents.

18. 20. The method of claim 17, wherein one or more additional therapeutic agents are administered before, during, or after transplantation of the object into the subject.

19. The method according to any one of claims 14 to 18, wherein the object is selected from the group consisting of an organ, a cell population, skin, and tissue.

20. 20. The method of claim 19, wherein the organ is selected from the group consisting of heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testicle, bladder, uterus, adrenal gland, and combinations thereof.

21. An ex vivo method for preventing or reducing rejection of a transplanted object in a subject, the method comprising introducing a construct according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 into an implantable object before the object is transplanted into a subject.

22. 22. The method of claim 21, further comprising analyzing the object or cells of the object for the presence or expression in the object of the construct or an immune checkpoint protein encoded by the construct.

23. 23. The method of claim 22, further comprising implanting the object into the subject.

24. 24. The method of claim 23, wherein the one or more additional therapeutic agents are administered before, during, or after transplantation of the object into the subject.

25. The method according to any one of claims 21 to 24, wherein the object for transplantation is selected from the group consisting of an organ, a cell population, skin, and tissue.

26. 26. The method of claim 25, wherein the organ is selected from the group consisting of heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testicle, bladder, uterus, adrenal gland, and combinations thereof.

27. 1. An ex vivo method for introducing a construct into an implantable object, comprising perfusing the construct ex vivo into the object prior to implantation, wherein the construct comprises a promoter operably linked to a nucleic acid sequence encoding an immune checkpoint protein or an extracellular portion of an immune checkpoint protein, and wherein the immune checkpoint protein or the extracellular portion of the immune checkpoint protein is capable of binding to its receptor.

28. 28. The method of claim 27, further comprising implanting the object into the subject.

29. 29. The method of claim 27 or 28, wherein the object is selected from the group consisting of an organ, a cell population, skin, and tissue.

30. 30. The method of claim 29, wherein the organ is selected from the group consisting of heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testicle, bladder, uterus, adrenal gland, and combinations thereof.

31. 28. The method of claim 27, wherein the construct further comprises a secretion signal operably connected to the promoter and functionally linked to the nucleic acid encoding the immune checkpoint protein or the extracellular portion of the immune checkpoint protein, wherein the secretion signal encodes a signal peptide enabling the immune checkpoint protein or the extracellular portion of the immune checkpoint protein to be secreted.

32. 32. The method of claim 27 or 31, wherein the immune checkpoint protein is selected from the group consisting of A2AR, A2BR, B7-H2, B7-H3, B7-H4, 2B4 (CD244), B7.1, B7.2, BTLA, CTLA4, ICOS, IDO, ITL-4, HVEM, KIR, LAG3, gp49B, NOX2, PD1, PDL1, PDL2, PIR-B, TIM-1, TIM-3, TIM-4, TIGIT, VISTA, SIGLEC7, CD47, CD48, CD39, CD73, CD160, CD200, HVEC, CEACAM1, CD155, LAG-3, HLA-E, and combinations thereof.

33. 33. The method of claim 32, wherein the immune checkpoint protein comprises PDL1 or an extracellular portion thereof and has a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, a sequence having 95% identity to SEQ ID NO:8, a sequence having 95% identity to SEQ ID NO:10, a sequence having 95% identity to SEQ ID NO:12, and any fragment or variant thereof that is capable of binding to and activating PD1.

34. 28. The method of claim 27, wherein the construct is a viral vector, and the viral vector is selected from the group consisting of retrovirus, adenovirus, oncoretrovirus, lentivirus, spumavirus, adeno-associated virus, herpes simplex virus, and combinations thereof.

35. 1. An ex vivo method for preventing or reducing rejection of a transplanted object in a subject, comprising introducing a construct into the transplantable object prior to transplantation into the subject, wherein the construct encodes a promoter operably linked to a nucleic acid sequence encoding an immune checkpoint protein or an extracellular portion of an immune checkpoint protein, wherein the immune checkpoint protein or the extracellular portion of the immune checkpoint protein is capable of binding to its receptor.

36. 36. The method of claim 35, further comprising implanting the object into the subject.

37. 37. The method of claim 35 or 36, wherein the object is selected from the group consisting of an organ, a cell population, skin, and tissue.

38. 38. The method of claim 37, wherein the organ is selected from the group consisting of heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testicle, bladder, uterus, adrenal gland, and combinations thereof.

39. 36. The method of claim 35, wherein the construct further comprises a secretion signal operably connected to the promoter and functionally linked to the nucleic acid encoding the immune checkpoint protein or the extracellular portion of the immune checkpoint protein, wherein the secretion signal encodes a signal peptide enabling the immune checkpoint protein or the extracellular portion of the immune checkpoint protein to be secreted.

40. 40. The method of any one of claims 35 to 39, wherein the immune checkpoint protein is selected from the group consisting of A2AR, A2BR, B7-H2, B7-H3, B7-H4, 2B4 (CD244), B7.1, B7.2, BTLA, CTLA4, ICOS, IDO, ITL-4, HVEM, KIR, LAG3, gp49B, NOX2, PD1, PDL1, PDL2, PIR-B, TIM-1, TIM-3, TIM-4, TIGIT, VISTA, SIGLEC7, CD47, CD48, CD39, CD73, CD160, CD200, HVEC, CEACAM1, CD155, LAG-3, HLA-E, and combinations thereof.

41. 41. The method of claim 40, wherein the immune checkpoint protein comprises PD1 or an extracellular portion thereof and has a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, a sequence having 95% identity to SEQ ID NO:8, a sequence having 95% identity to SEQ ID NO:10, a sequence having 95% identity to SEQ ID NO:12, and any fragment or variant thereof that is capable of binding to and activating PD1.

42. 42. The method of claim 41, wherein the construct is a viral vector, and the viral vector is selected from the group consisting of retrovirus, adenovirus, oncoretrovirus, lentivirus, spumavirus, adeno-associated virus, herpes simplex virus, and combinations thereof.

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